precaution step in transport across membrane experiment

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Chapter 8. Membrane Transport

Chapter Outline

• 8.1 Membrane Components and Structure
• 8.2 Passive Transport
• 8.3 Active Transport
• 8.4 Bulk Transport

Introduction

The plasma membrane, which is also called the cell membrane, has many functions, but the most basic one is to define the borders of the cell and keep the cell functional. The plasma membrane is selectively permeable. This means that the membrane allows some materials to freely enter or leave the cell, while other materials cannot move freely, but require the use of a specialized structure, and occasionally, even energy investment for crossing.

8.1 | Membrane Components and Structure

Learning Objectives

By the end of this section, you will be able to:

• Understand the fluid mosaic model of cell membranes.
• Describe the functions of phospholipids, proteins, and carbohydrates in membranes.
• Discuss membrane fluidity.

A cell’s plasma membrane defines the cell, outlines its borders, and determines the nature of its interaction with its environment (see Table 8.1 for a summary). Cells exclude some substances, take in others, and excrete still others, all in controlled quantities. The plasma membrane must be very flexible to allow certain cells, such as red blood cells and white blood cells, to change shape as they pass through narrow capillaries. These are the more obvious functions of a plasma membrane. In addition, the surface of the plasma membrane carries markers that allow cells to recognize one another, which is vital for tissue and organ formation during early development, and which later plays a role in the “self” versus “non-self” distinction of the immune response.

Among the most sophisticated functions of the plasma membrane is the ability to transmit signals by means of complex, integral proteins known as receptors. These proteins act both as receivers of extracellular inputs and as activators of intracellular processes. These membrane receptors provide extracellular attachment sites for effectors like hormones and growth factors, and they activate intracellular response cascades when their effectors are bound. Occasionally, receptors are hijacked by viruses (HIV, human immunodeficiency virus, is one example) that use them to gain entry into cells, and at times, the genes encoding receptors become mutated, causing the process of signal transduction to malfunction with disastrous consequences.

8.1.1 Fluid Mosaic Model

The existence of the plasma membrane was identified in the 1890s, and its chemical components were identified in 1915. The principal components identified at that time were lipids and proteins. The first widely accepted model of the plasma membrane’s structure was proposed in 1935 by Hugh Davson and James Danielli; it was based on the “railroad track” appearance of the plasma membrane in early electron micrographs. They theorized that the structure of the plasma membrane resembles a sandwich, with protein being analogous to the bread, and lipids being analogous to the filling. In the 1950s, advances in microscopy, notably transmission electron microscopy (TEM), allowed researchers to see that the core of the plasma membrane consisted of a double, rather than a single, layer. A new model that better explains both the microscopic observations and the function of that plasma membrane was proposed by S.J. Singer and Garth L. Nicolson in 1972.

The explanation proposed by Singer and Nicolson is called the fluid mosaic model . The model has evolved somewhat over time, but it still best accounts for the structure and functions of the plasma membrane as we now understand them. The fluid mosaic model describes the structure of the plasma membrane as a mosaic of components—including phospholipids, cholesterol, proteins, and carbohydrates—that gives the membrane a fluid character. Plasma membranes range from 5 to 10 nm in thickness. For comparison, human red blood cells, visible via light microscopy, are approximately 8 µm wide, or approximately 1,000 times wider than a plasma membrane. The membrane does look a bit like a sandwich ( Figure 8.2 ).

The principal components of a plasma membrane are lipids, proteins, and carbohydrates. The lipids include phospholipids and cholesterol Proteins either float in the bilayer or are attached to one side or the other of it. Carbohydrate chains are attached to the proteins and lipids on the outside surface of the membrane. The proportions of proteins, lipids, and carbohydrates in the plasma membrane vary with cell type, but for a typical human cell, protein accounts for about 50 percent of the composition by mass, lipids account for about 40 percent of the composition by mass, with the remaining 10 percent of the composition by mass being carbohydrates.

Phospholipids

The main fabric of the membrane is composed of amphiphilic phospholipid molecules. Recall from chapter 4 that a phospholipid is a molecule consisting of glycerol, two fatty acids, and a phosphate-linked head group ( Figure 8.3 ) . The hydrophilic “head” of these molecules are in contact with the aqueous fluid both inside and outside the cell. The hydrophobic “tails” face each other in the inside of the bilayer. Therefore, phospholipids form an excellent two-layer cell membrane that separates fluid within the cell from the fluid outside of the cell ( Figure 8.2 ).

The amphipathic nature of phospholipids is vital to the structure of a plasma membrane because, in water, phospholipids automatically become arranged with their hydrophobic tails facing each other and their hydrophilic heads facing out. In this way, they form a lipid bilayer—a barrier composed of a double layer of phospholipids that separates the water and other materials on one side of the barrier from the water and other materials on the other side ( Figure 8 .4 top ). In fact, phospholipids heated in an aqueous solution tend to spontaneously form small spheres or droplets called micelles, with their hydrophilic heads forming the exterior and their hydrophobic tails on the inside ( Figure 8 .4 bottom ).

Proteins make up the second major component of plasma membranes. Integral proteins are, as their name suggests, integrated completely into the membrane structure, and their hydrophobic membrane-spanning regions interact with the hydrophobic region of the the phospholipid bilayer ( Figure 8.2 ). Single-pass integral membrane proteins usually have a hydrophobic transmembrane segment that consists of 20–25 amino acids. Some span only part of the membrane— associating with a single layer—while others stretch from one side of the membrane to the other, and are exposed on either side. Since they cross the membrane, these are often called transmembrane proteins .

Some complex integral proteins are composed of up to 12 segments, which are extensively folded and embedded in the membrane ( Figure 8.5 ). This type of protein has a hydrophilic region or regions, and several hydrophobic regions. This arrangement of regions of the protein tends to orient the protein alongside the phospholipids, with the hydrophobic region of the protein adjacent to the tails of the phospholipids and the hydrophilic region or regions of the protein protruding from the membrane and in contact with the cytosol or extracellular fluid.

Peripheral proteins are found on the exterior and interior surfaces of membranes, attached either to integral proteins or to phospholipids. Peripheral proteins, along with integral proteins, may serve as enzymes, as structural attachments for the fibers of the cytoskeleton, or as part of the cell’s recognition sites. These are sometimes referred to as “cell-specific” proteins. The body recognizes its own proteins and attacks foreign proteins associated with invasive pathogens.

Carbohydrates

Carbohydrates are the third major component of plasma membranes. They are always found on the exterior surface of cells and are bound either to proteins (forming glycoproteins ) or to lipids (forming glycolipids ) ( Figure 8.2 ). These carbohydrate chains may consist of 2–60 monosaccharide units and can be either straight or branched. Along with peripheral proteins, carbohydrates form specialized sites on the cell surface that allow cells to recognize each other. These sites have unique patterns that allow the cell to be recognized, much the way that the facial features unique to each person allow him or her to be recognized. This recognition function is very important to cells, as it allows the immune system to differentiate between body cells (called “self”) and foreign cells or tissues (called “non-self”). Similar types of glycoproteins and glycolipids are found on the surfaces of viruses and may change frequently, preventing immune cells from recognizing and attacking them.

These carbohydrates on the exterior surface of the cell—the carbohydrate components of both glycoproteins and glycolipids—are collectively referred to as the glycocalyx (meaning “sugar coating”). The glycocalyx is highly hydrophilic and attracts large amounts of water to the surface of the cell. This aids in the interaction of the cell with its watery environment and in the cell’s ability to obtain substances dissolved in the water. As discussed above, the glycocalyx is also important for cell identification, self/non-self determination, and embryonic development, and is used in cell-cell attachments to form tissues.

How Viruses Infect Specific Organs

Glycoprotein and glycolipid patterns on the surfaces of cells give many viruses an opportunity for infection. HIV and hepatitis viruses infect only specific organs or cells in the human body. HIV is able to penetrate the plasma membranes of a subtype of lymphocytes called T-helper cells, as well as some monocytes and central nervous system cells. The hepatitis virus attacks liver cells.

These viruses are able to invade these cells, because the cells have binding sites on their surfaces that are specific to and compatible with certain viruses ( Figure 8 .6 ). Other recognition sites on the virus’s surface interact with the human immune system, prompting the body to produce antibodies. Antibodies are made in response to the antigens or proteins associated with invasive pathogens, or in response to foreign cells, such as might occur with an organ transplant. These same sites serve as places for antibodies to attach and either destroy or inhibit the activity of the virus. Unfortunately, these recognition sites on HIV change at a rapid rate because of mutations, making the production of an effective vaccine against the virus very difficult, as the virus evolves and adapts. A person infected with HIV will quickly develop different populations, or variants, of the virus that are distinguished by differences in these recognition sites. This rapid change of surface markers decreases the effectiveness of the person’s immune system in attacking the virus, because the antibodies will not recognize the new variations of the surface patterns. In the case of HIV, the problem is compounded by the fact that the virus specifically infects and destroys cells involved in the immune response, further incapacitating the host.

8.1.2 Membrane Fluidity

The mosaic characteristic of the membrane, described in the fluid mosaic model, helps to illustrate its nature. The integral proteins and lipids exist in the membrane as separate but loosely attached molecules. These resemble the separate, multicolored tiles of a mosaic picture, and they float, moving somewhat with respect to one another. The membrane is not like a balloon, however, that can expand and contract; rather, it is fairly rigid and can burst if penetrated or if a cell takes in too much water. However, because of its mosaic nature, a very fine needle can easily penetrate a plasma membrane without causing it to burst, and the membrane will flow and self-seal when the needle is extracted.

The mosaic characteristics of the membrane explain some but not all of its fluidity. There are two other factors that help maintain this fluid characteristic. One factor is the nature of the phospholipids themselves. In their saturated form, the fatty acids in phospholipid tails are saturated with bound hydrogen atoms. There are no double bonds between adjacent carbon atoms. This results in tails that are relatively straight. In contrast, unsaturated fatty acids do not contain a maximal number of hydrogen atoms, but they do contain some double bonds between adjacent carbon atoms; a double bond results in a bend in the string of carbons of approximately 30 degrees ( Figure 8.3 ).

Thus, if saturated fatty acids, with their straight tails, are compressed by decreasing temperatures, they press in on each other, making a dense and fairly rigid membrane. If unsaturated fatty acids are compressed, the “kinks” in their tails elbow adjacent phospholipid molecules away, maintaining some space between the phospholipid molecules. This “elbow room” helps to maintain fluidity in the membrane at temperatures at which membranes with saturated fatty acid tails in their phospholipids would “freeze” or solidify. The relative fluidity of the membrane is particularly important in a cold environment. A cold environment tends to compress membranes composed largely of saturated fatty acids, making them less fluid and more susceptible to rupturing. Many organisms (fish are one example) are capable of adapting to cold environments by changing the proportion of unsaturated fatty acids in their membranes in response to the lowering of the temperature.

Animals have an additional membrane constituent that assists in maintaining fluidity. Cholesterol, which lies alongside the phospholipids in the membrane, tends to dampen the effects of temperature on the membrane. Thus, this lipid functions as a buffer, preventing lower temperatures from inhibiting fluidity and preventing increased temperatures from increasing fluidity too much. Thus, cholesterol extends, in both directions, the range of temperature in which the membrane is appropriately fluid and consequently functional. Cholesterol also serves other functions, such as organizing clusters of transmembrane proteins into lipid rafts.

Table 8.1 The components and functions of the plasma membrane.

Immunologist

The variations in peripheral proteins and carbohydrates that affect a cell’s recognition sites are of prime interest in immunology. These changes are taken into consideration in vaccine development. Many infectious diseases, such as smallpox, polio, diphtheria, and tetanus, were conquered by the use of vaccines.

dImmunologists are the physicians and scientists who research and develop vaccines, as well as treat and study allergies or other immune problems. Some immunologists study and treat autoimmune problems (diseases in which a person’s immune system attacks his or her own cells or tissues, such as lupus) and immunodeficiencies, whether acquired (such as acquired immunodeficiency syndrome, or AIDS) or hereditary (such as severe combined immunodeficiency, or SCID). Immunologists are called in to help treat organ transplantation patients, who must have their immune systems suppressed so that their bodies will not reject a transplanted organ. Some immunologists work to understand natural immunity and the effects of a person’s environment on it. Others work on questions about how the immune system affects diseases such as cancer. In the past, the importance of having a healthy immune system in preventing cancer was not at all understood.

To work as an immunologist, a PhD or MD is required. In addition, immunologists undertake at least 2–3 years of training in an accredited program and must pass an examination given by the American Board of Allergy and Immunology. Immunologists must possess knowledge of the functions of the human body as they relate to issues beyond immunization, and knowledge of pharmacology and medical technology, such as medications, therapies, test materials, and surgical procedures.

8.2 | Passive Transport

• Explain why and how passive transport occurs.
• Understand the processes of osmosis and diffusion.
• Define tonicity and describe its relevance to passive transport.

Plasma membranes must allow certain substances to enter and leave a cell, and prevent some harmful materials from entering and some essential materials from leaving. In other words, plasma membranes are selectively permeable —they allow some substances to pass through, but not others. If they were to lose this selectivity, the cell would no longer be able to sustain itself, and it would be destroyed. Some cells require larger amounts of specific substances than do other cells; they must have a way of obtaining these materials from extracellular fluids. This may happen passively, as certain materials move back and forth, or the cell may have special mechanisms that facilitate transport. Some materials are so important to a cell that it spends some of its energy, hydrolyzing adenosine triphosphate (ATP), to obtain these materials. All cells spend the majority of their energy to maintain an imbalance of sodium and potassium ions between the interior and exterior of the cell.

The most direct forms of membrane transport are passive. Passive transport is a naturally occurring phenomenon and does not require the cell to exert any of its energy to accomplish the movement. In passive transport, substances move from an area of higher concentration to an area of lower concentration. A physical space in which there is a range of concentrations of a single substance is said to have a concentration gradient .

8.2.1 Selective Permeability

Plasma membranes are asymmetric: the interior of the membrane is not identical to the exterior of the membrane. In fact, there is a considerable difference between the array of phospholipids and proteins between the two leaflets that form a membrane. On the interior of the membrane, some proteins serve to anchor the membrane to fibers of the cytoskeleton. There are peripheral proteins on the exterior of the membrane that bind elements of the extracellular matrix. Carbohydrates,

attached to lipids or proteins, are also found on the exterior surface of the plasma membrane. These carbohydrate complexes help the cell bind substances that the cell needs in the extracellular fluid. This adds considerably to the selective nature of plasma membranes ( Figure 8.7 ).

Recall that plasma membranes are amphipathic: They have hydrophilic and hydrophobic regions. This characteristic helps the movement of some materials through the membrane and hinders the movement of others. Lipid-soluble material with a low molecular weight can easily slip through the hydrophobic lipid core of the membrane. Substances such as the fat- soluble vitamins A, D, E, and K readily pass through the plasma membranes in the digestive tract and other tissues. Fat-soluble drugs and hormones also gain easy entry into cells and are readily transported into the body’s tissues and organs. Molecules of oxygen and carbon dioxide have no charge and so pass through membranes by simple diffusion.

Polar substances present problems for the membrane. While some polar molecules connect easily with the outside of a cell, they cannot readily pass through the lipid core of the plasma membrane. Additionally, while small ions could easily slip through the spaces in the mosaic of the membrane, their charge prevents them from doing so. Ions such as sodium, potassium, calcium, and chloride must have special means of penetrating plasma membranes. Larger polar molecules, such as simple sugars and amino acids also need help with transport across plasma membranes.

8.2.2 Diffusion

Diffusion is a passive process of transport. A single substance tends to move from an area of high concentration to an area of low concentration until the concentration is equal across a space. You are familiar with diffusion of substances through the air. For example, think about someone opening a bottle of ammonia in a room filled with people. The ammonia gas is at its highest concentration in the bottle; its lowest concentration is at the edges of the room. The ammonia vapor will diffuse, or spread away, from the bottle, and gradually, more and more people will smell the ammonia as it spreads. Materials move within the cell’s cytosol by diffusion, and certain materials move through the plasma membrane by diffusion ( Figure 8.8 ). Diffusion expends no energy. On the contrary, concentration gradients are a form of potential energy, dissipated as the gradient is eliminated.

Each separate substance in a medium, such as the extracellular fluid, has its own concentration gradient, independent of the concentration gradients of other materials. In addition, each substance will diffuse according to that gradient. Within a system, there will be different rates of diffusion of the different substances in the medium.

Factors That Affect Diffusion

Molecules move constantly in a random manner, at a rate that depends on their mass, their environment, and the amount of thermal energy they possess, which in turn is a function of temperature. This movement accounts for the diffusion of molecules through whatever medium in which they are localized. A substance will tend to move into any space available to it until it is evenly distributed throughout it. After a substance has diffused completely through a space, removing its concentration gradient, molecules will still move around in the space, but there will be no net movement of the number of molecules from one area to another. This lack of a concentration gradient in which there is no net movement of a substance is known as dynamicequilibrium . While diffusion will go forward in the presence of a concentration gradient of a substance, several factors affect the rate of diffusion.

“Steepness” of the concentration gradient: The greater the difference in concentration, the more rapid the diffusion. The closer the distribution of the material gets to equilibrium, the slower the rate of diffusion becomes.

Mass of the molecules diffusing: Heavier molecules move more slowly; therefore, they diffuse more slowly.

Temperature: Higher temperatures increase the energy and therefore the movement of the molecules, increasing the rate of diffusion.

Solvent density: As the density of a solvent increases, the rate of diffusion decreases. The molecules slow down because they have a more difficult time getting through the denser medium. If the medium is less dense, diffusion increases. Because cells primarily use diffusion to move materials within the cytoplasm, any increase in the cytoplasm’s density will inhibit the movement of the materials. An example of this is a person experiencing dehydration. As the body’s cells lose water, the rate of diffusion decreases in the cytoplasm, and the cells’ functions deteriorate. Neurons tend to be very sensitive to this effect. Dehydration frequently leads to unconsciousness and possibly coma because of the decrease in diffusion rate within the cells.

8.2.3 Facilitated diffusion

In facilitated diffusion , materials diffuse across the plasma membrane with the help of membrane proteins. A concentration gradient exists that would allow these materials to diffuse into the cell without expending cellular energy. However, these materials are ions or polar molecules that are repelled by the hydrophobic parts of the cell membrane. Facilitated diffusion proteins shield these materials from the repulsive force of the membrane, allowing them to diffuse into the cell. These proteins are called transportproteins and can be channels or carrier proteins.

Channelproteins are transmembrane proteins that fold in such as way as to form a channel or pore through the membrane. Each channel is specific for one particular substance. Channel proteins have hydrophilic domains exposed to the intracellular and extracellular fluids. In addition, they have a hydrophilic channel through their core that provides a hydrated opening through the membrane layers ( Figure8.9 ). Passage through the channel allows polar compounds to avoid the nonpolar central layer of the plasma membrane that would otherwise slow or prevent their entry into the cell. Aquaporins are channel proteins that allow water to pass through the membrane at a very high rate.

Some channel proteins are always open but many are “gated,” meaning that they can be opened and closed. If a channel is ligand-gated, the attachment of a particular molecule to the channel protein may cause it to open. Other channels are voltage-gated, requiring a change in voltage across the membrane to open them. Cells involved in the transmission of electrical impulses, such as nerve and muscle cells, have voltage-gated ion channels in their membranes.

Carrier Proteins

Another type of transmembrane transporter protein is a  carrierprotein . Like channels, carrier proteins are usually specific for particular molecules. A carrier proteins binds a substance and, in doing so, triggers a change of its own shape, moving the bound molecule across the membrane ( Figure8.10 ). Carrier proteins are used to transport molecules that are too large to pass through channels, such as amino acids and glucose.

There are a finite number of each type of carrier proteins in any membrane. This can cause problems in transporting enough of the material for the cell to function properly. When all of the proteins are bound to their ligands, they are saturated and the rate of transport is at its maximum. Increasing the concentration gradient at this point will not result in an increased rate of transport.

An example of this process occurs in the kidney. Glucose, water, salts, ions, and amino acids needed by the body are filtered out of the blood in one part of the kidney. This filtrate, which includes glucose, is then reabsorbed in another part of the kidney. Because there are only a finite number of carrier proteins for glucose, if more glucose is present than the proteins can handle, the excess is not transported and it is excreted from the body in the urine. In a diabetic individual, this is described as “spilling glucose into the urine.”

A different group of carrier proteins called glucose transport proteins, or GLUTs, are involved in transporting glucose and other hexose sugars into cells within the body. The hormone insulin, increases the number of GLUTs on cells, causing them to take glucose from the blood when its levels are high. It is this process that is compromised in diabetic individuals.

Channel proteins transport much more quickly than do carrier proteins. Channel proteins facilitate diffusion at a rate of tens of millions of molecules/second, whereas carrier proteins work at a rate of a thousand to a million molecules/second.

8.2.4 Osmosis

Osmosis is the diffusion of water across a semipermeable membrane. Since it is diffusion, it depends on the concentration gradient, or the amount of water on each side of the membrane. The amount of water in a solute is inversely proportional to the concentration of solutes. In other words, the higher the concentration of water, the lower the concentration of solutes, and vice versa. Water can move readily across most membranes, due in part to the presence of aquaporins; however, the membrane limits the diffusion of solutes in the water.

Mechanism of Osmosis

Osmosis is a special case of diffusion. Water, like other substances, moves from an area of high concentration to one of low concentration. An obvious question is what makes water move at all? Imagine a beaker with a semipermeable membrane separating the two sides or halves ( Figure8.11 ). On both sides of the membrane the water level is the same, but there are different concentrations of a dissolved substance, or solute , that cannot cross the membrane (otherwise the concentrations on each side would be balanced by the solute crossing the membrane). If the volume of the solution on both sides of the membrane is the same, but the concentrations of solute are different, then there are different amounts of water, the solvent, on either side of the membrane.

To illustrate this, imagine two full glasses of water. One has a single teaspoon of sugar in it, whereas the second one contains one-quarter cup of sugar. If the total volume of the solutions in both cups is the same, which cup contains more water? Because the large amount of sugar in the second cup takes up much more space than the teaspoon of sugar in the first cup, the first cup has more water in it.

Returning to the beaker example, recall that it has a mixture of solutes on either side of the membrane. A principle of diffusion is that the molecules move around and will spread evenly throughout the medium if they can. However, only the material capable of getting through the membrane will diffuse through it. In this example, the solute cannot diffuse through the membrane, but the water can. Water has a concentration gradient in this system. Thus, water will diffuse down its concentration gradient, crossing the membrane to the side where it is less concentrated. This diffusion of water through the membrane—osmosis—will continue until the concentration gradient of water goes to zero or until the hydrostatic pressure of the water balances the osmotic pressure. Osmosis proceeds constantly in living systems.

8.2.5 Tonicity

Tonicity describes how an extracellular solution can change the volume of a cell by affecting osmosis. A solution’s tonicity often directly correlates with the osmolarity of the solution. Osmolarity describes the total solute concentration of the solution. A solution with low osmolarity has a greater number of water molecules relative to the number of solute particles; a solution with high osmolarity has fewer water molecules with respect to solute particles. In a situation in which solutions of two different osmolarities are separated by a membrane permeable to water, though not to the solute, water will move from the side of the membrane with lower osmolarity (and more water) to the side with higher osmolarity (and less water). This effect makes sense if you remember that the solute cannot move across the membrane, and thus the only component in the system that can move—the water—moves along its own concentration gradient.

Three terms—hypotonic, isotonic, and hypertonic—are used to relate the osmolarity of a cell to the osmolarity of the extracellular fluid. In living systems, the point of reference is always the cytoplasm, so the prefix hypo- (“lower”) means that the extracellular fluid has a lower concentration of solutes, or a lower osmolarity, than the cell cytoplasm. Blood cells and plant cells in hypertonic, isotonic, and hypotonic solutions take on characteristic appearances ( Figure 8.12 ).

Hypotonic Solutions

In a hypotonic situation, the extracellular fluid has lower osmolarity than the fluid inside the cell. The extracellular fluid has a higher concentration of water than does the cell and water will move down its concentration gradient and enter the cell.

Hypertonic Solutions

In a hypertonic solution (hyper- = “more”), the extracellular fluid has a higher osmolarity than the cell’s cytoplasm. The fluid contains less water than the cell does, so water will leave the cell.

Isotonic Solutions

In an isotonic solution, the extracellular fluid has the same osmolarity as the cell. There is no net movement of water into or out of the cell (although water will still move in and out).

Concept Check

A doctor injects a patient with what the doctor thinks is an isotonic saline solution. The patient dies, and an autopsy reveals that many red blood cells have been destroyed. Do you think the solution the doctor injected was really isotonic?

8.2.6 Tonicity in Living Systems

A red blood cell will burst, or lyse, when it swells beyond the plasma membrane’s capability to expand. In contrast, when excessive amounts of water leave a red blood cell, the cell shrinks, or crenates. Crenation has the effect of concentrating the solutes left in the cell, making the cytosol denser and interfering with diffusion within the cell. The cell’s ability to function will be compromised and it may die. ( Figure 8.12 ).

Living things have ways of controlling the effects of osmosis—a mechanism called osmoregulation. Some organisms, such as plants, fungi, bacteria, and some protists, have cell walls that surround the plasma membrane and prevent cells from lysing. In fact, the cytoplasm in plants is always slightly hypertonic to the cellular environment, and water will always enter a cell if water is available. This inflow of water produces turgor pressure, which stiffens the cell walls of the plant ( Figure 8.13 ). In nonwoody plants, turgor pressure supports the plant. If the plant is not watered, the extracellular fluid will become hypertonic, causing water to leave the cell. In this condition, the cell membrane detaches from the cell wall and constricts the cytoplasm. This process, called plasmolysis , causes plants to lose turgor pressure ( Figure 8.14 ).

Tonicity is a concern for all living things. For example, paramecia and amoebas, which are protists that lack cell walls, have contractile vacuoles. This vesicle collects excess water from the cell and pumps it out, keeping the cell from lysing as it takes on water from its environment ( Figure 8.15 ).

Many marine invertebrates have internal salt levels matched to their environments, making them isotonic with the water in which they live. Fish, however, must spend approximately five percent of their metabolic energy maintaining osmotic homeostasis. Freshwater fish live in an environment that is hypotonic to their cells. These fish actively take in salt through their gills and excrete diluted urine to rid themselves of excess water. Saltwater fish live in the reverse environment, which is hypertonic to their cells, and they secrete salt through their gills and excrete highly concentrated urine.

In vertebrates, the kidneys regulate the amount of water in the body. Osmoreceptors are specialized cells in the brain that monitor the concentration of solutes in the blood. If the levels of solutes increase beyond a certain range, a hormone is released that retards water loss through the kidney and dilutes the blood to safer levels. Animals also have high concentrations of albumin, which is produced by the liver, in their blood. This protein is too large to pass easily through plasma membranes and is a major factor in controlling the osmotic pressures applied to tissues.

8.3 | Active Transport

By the end of this section, you will be able to:Understand how electrochemical gradients affect ionsDistinguish between primary active transport and secondary active transport

Active transport mechanisms require the use of the cell’s energy, usually in the form of adenosine triphosphate (ATP). If a substance must move into the cell against its concentration gradient—that is, if the concentration of the substance inside the cell is greater than its concentration in the extracellular fluid (and vice versa)—the cell must use energy to move the substance. Some active transport mechanisms move small-molecular weight materials, such as ions, through the membrane. Other mechanisms transport much larger molecules.

8.3.1 Electrochemical Gradient

We have discussed simple concentration gradients—different concentrations of a substance across a space or a membrane—but in living systems, gradients are more complex. Because ions move into and out of cells and because cells contain proteins that do not move across the membrane and are mostly negatively charged, there is also an electrical gradient, a difference of charge, across the plasma membrane.

The interior of living cells is electrically negative with respect to the extracellular fluid surrounding them. At the same time, cells have a lower concentration of (Na+) than does the extracellular fluid. Therefore, both the concentration

gradient and the electrical gradient tend to drive Na+ into the cell. Conversely, cells have a higher concentration of K+ than the extracellular fluid does. Therefore, the concentration gradient tends to drive K+ out of the cell, while the electrical gradient tends to drive it inside the cell. The combined gradient of concentration and electrical charge that affects an ion is called its electrochemical gradient  ( Figure 8.16 ).

Injection of a potassium solution into a person’s blood is lethal; this is used in capital punishment and euthanasia. Why do you think a potassium solution injection is lethal?

Moving Against a Gradient

To move substances against a concentration or electrochemical gradient, the cell must use energy, usually in the form of ATP. Active transport proteins, called pumps , work against electrochemical gradients. Small substances constantly pass through plasma membranes. Active transport maintains concentrations of ions and other substances needed by living cells in the face of these passive movements. Much of a cell’s supply of metabolic energy may be spent maintaining these processes.

Proteins for Active Transport

The specific proteins that facilitate active transport are called transporters . There are three types of transporters ( Figure 8.17 ). A uniporter carries one specific ion or molecule. A symporter carries two different ions or molecules, both in the same direction. An antiporter carries two different ions or molecules in different directions. All of these transporters can transport small, uncharged organic molecules such as glucose.

Two mechanisms exist for the transport of small-molecular weight material and small molecules. Primary active transport is directly dependent on ATP. Secondary active transport does not directly require ATP, because it uses electrochemical gradients established by primary active transport for fuel. Primary active transport must occur first to in order to allow secondary active transport to occur. Although it does not use ATP, secondary active transport is still considered active because it requires energy.

8.3.2 Primary Active Transport

One of the most important pumps in animals cells is the sodium-potassium pump (Na+-K+ ATPase), which maintains the electrochemical gradient and the correct concentrations of Na+ and K+ in living cells. The sodium-potassium pump moves two K+ into the cell while moving three Na+ out of the cell ( Figure 8.18 ).

The sodium-potassium pump works in the following six steps:

• Three sodium ions bind to the protein.
• ATP is hydrolyzed by the protein carrier and a low-energy phosphate group attaches to it.
• The carrier changes shape and opens towards the exterior of the membrane. The three sodium ions are released.
• Two potassium ions attach to the protein, causing the low-energy phosphate group to detach.
• The carrier protein changes shape so that is open towards the interior of the cell.
• The two potassium ions are released into the cytoplasm and the process begins again.

Several things have happened as a result of this process. First, there are now more sodium ions outside of the cell than inside and more potassium ions inside than out. Second, since three sodium ions moved out for each two potassium ions that moved in, the interior is slightly more negative relative to the exterior. This difference in charge is important in creating the conditions necessary for secondary active transport. The sodium-potassium pump is, therefore, an electrogenic pump (a pump that creates a charge imbalance), creating an electrical imbalance across the membrane and contributing to the membrane potential.

The sodium-potassium pump (Na+/K+ pump) is one example of energy coupling. Each cycle of the Na+/K+ pump moves three sodium out of the cell and brings two potassium into the cell. For each cycle, one ATP is hydrolyzed and its free phosphate group is transferred to the pump protein. This process of a phosphate group binding to a molecule is called phosphorylation . Phosphorylation of the pump protein causes it to change shape, moving ions across the membrane. ATP performs cellular work using this basic form of energy coupling through phosphorylation. Here, the exergonic (energy-releasing) process of ATP breakdown “pays for” the endergonic (energy-requiring) process of moving ions against their concentration gradients.

8.3.3 Secondary Active Transport (Co-transport)

Secondary active transport moves a solute against its concentration gradient, an endergonic process, by moving another solute down its concentration gradient, an exergonic process. For instance, as sodium ion concentrations build outside of the plasma membrane because of the action of the sodium-potassium pump, an electrochemical gradient is created. If a channel protein exists and is open, the sodium ions will be pulled through the membrane, down their concentration gradient. This exergonic movement is used to transport other substances that can attach themselves to the transport protein through the membrane ( Figure 8.20 ). Many amino acids, as well as glucose, enter a cell this way.

8.4 | Bulk Transport

• Describe endocytosis, including phagocytosis, pinocytosis, and receptor-mediated endocytosis.
• Understand the process of exocytosis.

In addition to moving small ions and molecules through the membrane, cells also need to remove and take in larger molecules and particles (see Table 8.2 for examples). Some cells are even capable of engulfing entire unicellular microorganisms. You might have correctly hypothesized that the uptake and release of large particles by the cell requires energy. A large particle, however, cannot pass through the membrane, even with energy supplied by the cell.

8.4.1 Endocytosis

Endocytosis is a type of active transport that moves particles, such as large molecules, parts of cells, and even whole cells, into a cell. There are different variations of endocytosis, but all share a common characteristic: The plasma membrane of the cell invaginates, forming a pocket around the target particle. The pocket pinches off, resulting in the particle being contained in a newly created intracellular vesicle formed from the plasma membrane. The three types of endocytosis are phagocytosis, pinocytosis, and receptor-mediated endocytosis.

Phagocytosis

Phagocytosis (“cell eating”) is the process by which large particles, such as other cells or relatively large particles, are taken in by a cell. For example, when microorganisms invade the human body, a type of white blood cell called a neutrophil will “eat” the invaders through phagocytosis, surrounding and engulfing the microorganism, which is then destroyed by lysosomes inside the neutrophil ( Figure 8.21 ).

In preparation for phagocytosis, a portion of the inward-facing surface of the plasma membrane becomes coated with a protein called clathrin , which stabilizes this section of the membrane. The coated portion of the membrane then extends from the body of the cell and surrounds the particle, eventually enclosing it. Once the vesicle containing the particle is enclosed within the cell, the clathrin disengages from the membrane and the vesicle merges with a lysosome for the breakdown of the material in the newly formed compartment. When accessible nutrients from the degradation of the vesicular contents have been extracted, the newly formed endosome merges with the plasma membrane and releases its contents into the extracellular fluid. The endosomal membrane again becomes part of the plasma membrane.

Pinocytosis

Through pinocytosis (“cell drinking”), cells take in molecules, including water, which the cell needs from the extracellular fluid. Pinocytosis results in a much smaller vesicle than does phagocytosis, and the vesicle does not need to merge with a lysosome ( Figure 8.22 ).

Receptor-mediated Endocytosis

Receptor-mediated endocytosis is a targeted variation of endocytosis that employs receptor proteins in the plasma membrane that have a specific binding affinity for certain substances ( Figure 8.23 ).

Receptor-mediated endocytosis, as in phagocytosis, uses clathrin protein attached to the cytoplasmic side of the plasma membrane. Some human diseases are caused by the failure of receptor-mediated endocytosis. For example, the form of cholesterol termed low-density lipoprotein or LDL (also referred to as “bad” cholesterol) is removed from the blood by receptor-mediated endocytosis. In the human genetic disease familial hypercholesterolemia, the LDL receptors are defective or missing entirely. People with this condition have life-threatening levels of cholesterol in their blood, because their cells cannot clear LDL particles from their blood.

Although receptor-mediated endocytosis is designed to bring specific substances that are normally found in the extracellular fluid into the cell, other substances may gain entry into the cell at the same site. Flu viruses, diphtheria, and cholera toxin all have sites that cross-react with normal receptor-binding sites and gain entry into cells.

8.4.2 Exocytosis

The reverse process of moving material into a cell is the process of exocytosis . The purpose of exocytosis is to expel material from the cell into the extracellular fluid. Waste material is enveloped in vesicle, which fuses with the interior of the plasma membrane, expelling the waste material into the extracellular space ( Figure 8.24 ). Cells also use exocytosis to secrete proteins such as hormones, neurotransmitters, or parts of the extracellular matrix.

Table 8.2 Methods of transport, energy requirements, and types of material transported

Introduction to Molecular and Cell Biology Copyright © 2020 by Katherine R. Mattaini is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License , except where otherwise noted.

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Membrane Transport

William stillwell.

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Issue date 2016.

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Life depends on a membrane's ability to precisely control the level of solutes in the aqueous compartments, inside and outside, bathing the membrane. The membrane determines what solutes enter and leave a cell. Transmembrane transport is controlled by complex interactions between membrane lipids, proteins, and carbohydrates. How the membrane accomplishes these tasks is the topic of this chapter.

Keywords: Biological membrane, Cells, Fick's laws, Membrane, Membrane transport, Semipermeability, Solutes

1. Introduction

Life depends on a membrane's ability to precisely control the level of solutes in the aqueous compartments, inside and outside, bathing the membrane. The membrane determines what solutes enter and leave a cell. Transmembrane transport is controlled by complex interactions between membrane lipids, proteins, and carbohydrates. How the membrane accomplishes these tasks is the topic of Chapter 19.

A biological membrane is semipermeable, meaning it is permeable to some molecules, most notably water, while being very impermeable to most solutes (various biochemicals and salts) found in the bathing solution. This very important concept of unequal transmembrane distribution and, hence, permeability between water and other solutes came out of the pioneering work of Charles Overton in the 1890s (see Chapter 2). How does a biological membrane accomplish semipermeability? The barrier to solute movement is largely provided by the membrane's hydrophobic core, a very thin (∼40 Å thick), oily layer. The inherent permeability of this core varies from membrane to membrane. Generally, the more tightly packed the lipids comprising the bilayer, the lower its permeability will be. Lipid bilayers are very impermeable to most solutes because of their tight packing. Fig. 19.1 depicts the membrane permeability of a variety of common solutes [1] . Note the data are presented as a log scale of solute permeability ( P in cm/s) and ranges from Na +  = 10 −12  cm/s to water = 0.2 × 10 −2  cm/s, spanning almost 10 orders of magnitude!

Figure 19.1.

Log of the permeability ( P in cm/s) across lipid bilayer membranes for common solutes ranging from Na + (10 −12  cm/s) to water (0.2 × 10 −2  cm/s). This range spans almost 10 orders of magnitude [1] .

Lipid bilayer permeability is not a constant but instead is affected by environmental factors. For example, LUVs (large unilamellar veicles) made from DPPC (16:0, 16:0 PC) have a sharp phase transition temperature, T m , of 41.3°C. At temperatures well below T m , the LUVs are in the tightly packed gel state and permeability is extremely low. At temperatures well above T m , the LUVs are in the loosely packed liquid disordered state ( l d , also called the liquid crystalline state) and permeability is high. However, maximum permeability is not found in the l d state, but rather at the T m [2] . As the LUVs are heated from the gel state and approach the T m , domains of l d start to form in the gel state. Solutes can then pass more readily through the newly formed l d domains than the gel domains resulting in an increase in permeability. At T m there is a maximum amount of coexisting gel and l d state domains that exhibit extremely porous domain boundaries. It is through these boundaries that most permeability occurs. As the temperature is further increased, the LUVs pass into the l d state and the interface boundaries disappear, reducing permeability to that observed for the single-component l d state. Thus, maximum permeability is observed at the T m .

1.1. Fick's First Law

The tendency for solutes to move from a region of higher concentration to one of lower concentration was first defined in 1855 by the physiologist Adolf Fick ( Fig. 19.2 ). His work is summarized in what is now the very well-known Fick's Laws of Diffusion [3] . The laws apply to both free solution and diffusion across membranes. Fick developed his laws by measuring concentrations and fluxes of salt diffusing between two reservoirs through connecting tubes of water.

Figure 19.2.

Adolf Fick, 1829–1901.

Fick's First Law describes diffusion as:

Where D  = diffusion coefficient (bigger molecules have lower D s); A  = cross-sectional area over which diffusion occurs; dc / dx is the solute concentration gradient (diffusion occurs from a region of higher concentration to one of lower concentration).

The relationship between a solute's molecular weight and its diffusion coefficient is shown in Table 19.1 . Large solutes have low diffusion coefficients and therefore diffuse more slowly than small solutes. The diffusion rate for a particular solute under physiological conditions is a constant and cannot be increased. This defines the theoretical limit for an enzymatic reaction rate and also limits the size of a cell. If a solute starts at the center of a bacterial cell, it takes about 10 −3  s to diffuse to the plasma membrane. For this reason, typical cells are microscopic (see Chapter 1). At about 3.3 pounds and the size of a cantaloupe, the largest cell on Earth today is the ostrich egg. However a fossilized dinosaur egg in the American Museum of Natural History in New York is about the size of basketball. Since an egg's only function is to store nutrients for a developing embryo, its size is many orders of magnitude larger than a normal cell.

Table 19.1.

Relationship Between a Solute's Molecular Weight and Its Diffusion Coefficient, D

1.2. Osmosis

Osmosis is a special type of diffusion, namely the diffusion of water across a semipermeable membrane. Water readily crosses a membrane down its potential gradient from high to low potential ( Fig. 19.3 ) [4] . Osmotic pressure is the force required to prevent water movement across the semipermeable membrane. Net water movement continues until its potential reaches zero. An early application of the basic principles of osmosis came from the pioneering work on hemolysis of red blood cells by William Hewson in the 1770s (see Chapter 2). It has also been discussed that MLVs (multilamellar vesicles, liposomes) behave as almost perfect osmometers, swelling in hypotonic solutions and shrinking in hypertonic solutions (see Chapter 3) [5] , [6] . Liposome swelling and shrinking can be easily followed by changes in absorbance due to light scattering using a simple spectrophotometer. Therefore, osmosis has been investigated for many years using common and inexpensive methodologies and a lot is known about the process.

Figure 19.3.

Osmosis and osmotic pressure. Water is placed in a U-shaped tube where each of the tube arms is separated by a semipermeable membrane with pores of a size that water can easily pass through but a solute cannot. Upon addition of the solute to the tube's right arm, water diffuses from left to right (high water potential to low). The column of water in the tube's right arm (the one containing the solute) rises until the extra weight of the column equals the osmotic pressure caused by the solute. A pump could then be used to counter the osmotic pressure whereupon the solution columns in the right and left arms of the tube are made the same. The pump pressure required to equalize the height of the two columns is the osmotic pressure [4] . Note a small amount of the solute leaks from right to left since no filter is perfect.

Membranes are rarely, if ever, perfectly semipermeable. Deviation from ideality is defined by a reflection coefficient ( σ ). For an ideal semipermeable membrane where a solute is totally impermeable, σ  = 1. If a solute is totally permeable (its permeability is equal to water), σ  = 0. Biological membranes are excellent semipermeable barriers with σ  = 0.75 to 1.0.

2. Simple Passive Diffusion

Movement of solutes across membranes can be divided into two basic types: passive diffusion and active transport [7] . Passive diffusion requires no additional energy source other than what is found in the solute's electrochemical (concentration) gradient and results in the solute reaching equilibrium across the membrane. Passive diffusion can be either simple passive diffusion where the solute crosses the membrane anywhere by simply dissolving into and diffusing through the lipid bilayer, or facilitated passive diffusion where the solute crosses the membrane at specific locations where diffusion is assisted by solute-specific facilitators or carriers. Active transport requires additional energy, often in the form of ATP, and results in a nonequilibrium, net accumulation (uptake) of the solute on one side of the membrane. The basic types of membrane transport, simple passive diffusion, facilitated diffusion (by channels and carriers) and active transport are summarized in Fig. 19.4 [8] . There are countless different examples of each type of membrane transport process [7] . Only a few representative examples will be discussed here.

Figure 19.4.

Basic types of membrane transport, simple passive diffusion, facilitated diffusion (by channels and carriers), and active transport [8] .

Even simple passive diffusion requires energy to cross a bilayer membrane. In order to cross a membrane, the solute must first lose its waters of hydration, diffuse across the membrane, and then regain its waters on the opposite side. The limiting step involves the energy required to lose the waters of hydration. Table 19.2 shows the relationship between the waters of hydration (proportional to the number of —OH groups on a homologous series of solutes) and the activation energy for transmembrane diffusion. As the number of waters of hydration increases from glycol < glycerol < erythritol, the activation energy for diffusion also increases. The activation energy compares very well with the energy of hydration.

Table 19.2.

Relationship Between the Waters of Hydration (Number of —OH Groups on a Homologous Series of Solutes) and the Activation Energy for Transmembrane Diffusion

However, water diffusion does not fit this model. Water permeability is just too high. Several possibilities have been suggested to account for the abnormally high membrane permeability of water:

Water is very small and so it just dissolves in bilayers better than larger solutes.

Due to its size, water can readily enter very small statistical pores (∼4.2 Å in diameter). Statistical pores result from the simultaneous lateral movement of adjacent membrane phospholipids in opposite directions. Statistical pores have only a fleeting existence and cannot be isolated or imaged.

Passage down water chains.

Water can be carried down kinks in acyl chains that result from acyl chain melting (see lipid melting in Chapter 9).

Water may rapidly cross membranes through nonlamellar regions (eg, micelles, cubic or H II phase—see Chapter 10).

High water permeability will occur at regions of packing defect (eg, surface of integral membrane proteins, boundary between membrane domains).

Through pores or channels used to conduct ions.

Through specific water channels known as aquaporins (see below, Chapter 19, Section 3.5 ).

The only molecules that can cross a membrane by simple passive diffusion are water, small noncharged solutes, and gasses. Charged or large solutes are virtually excluded from membranes and so require more than just simple passive diffusion to cross a membrane.

3. Facilitated Diffusion

Facilitated diffusion (also known as carrier-mediated diffusion) is, like simple passive diffusion, dependent on the inherent energy in a solute gradient. No additional energy is required to transport the solute and the final solute distribution reaches equilibrium across the membrane. Facilitated diffusion, unlike simple passive diffusion, requires a highly specific transmembrane integral protein or carrier to assist in the solute's membrane passage. Facilitators come in two basic types: carriers and gated channels. Facilitated diffusion exhibits Michaelis-Menton saturation kinetics ( Fig. 19.5 , Part A, right), indicating the carrier has an enzyme-like active site. Like enzymes, facilitated diffusion carriers exhibit saturation kinetics and recognize their solute with exquisite precision, easily distinguishing chemically similar isomers like d -glucose from l -glucose. Fig. 19.5 (Part A) compares simple passive diffusion to facilitated diffusion. The figure is not to scale, however, as facilitated diffusion is orders of magnitude faster than simple passive diffusion.

Figure 19.5.

(A) Simple passive diffusion (top, left) and facilitated passive diffusion (top, right) both result in a final equilibrium distribution of a solute across the membrane. For a noncharged solute, the final distribution of the solute would find equal amounts of S on both sides of the membrane. Facilitated diffusion employs a specific transporter and exhibits Michaelis–Menten saturation kinetics. (A, center right) Active transport (bottom) utilizes energy, often in the form of ATP, to drive solute uptake against its gradient resulting in a net accumulation of the solute.

3.1. Glucose Transporter

A well-studied example of a facilitated diffusion carrier is the glucose transporter, or GLUT [9] . From the activation energies for transmembrane simple passive diffusion of glycol, glycerol and erythritol presented in Table 19.2 , it can be estimated that the activation energy for glucose should be well over 100 kJ/mol, but instead it is only 16 kJ/mol. This large discrepancy is attributed to the presence of a glucose-facilitated diffusion carrier. Fig. 19.6 demonstrates the mode of action of one of these transporters, GLUT-1, from the erythrocyte [10] . GLUTs occur in nearly all cells and are particularly abundant in cells lining the small intestine. GLUTs are but one example in a superfamily of transport facilitators. GLUTs are integral membrane proteins whose membrane-spanning region is composed of 12 α-helices. GLUTs function through a typical membrane transport mechanism [10] . Glucose binds to the membrane outer surface site causing a conformational change associated with transport across the membrane. At the inner side of the membrane, glucose is released into the internal aqueous solution ( Fig. 19.6 ).

Figure 19.6.

Glucose-facilitated diffusion transporter GLUT-1 [10] .

3.2. Potassium Channels

In virtually all organisms there exists a wide variety of ion channels, the most widely distributed being potassium channels [11] . There are four basic classes of potassium channels, all of which provide essential membrane-associated functions including setting and shaping action potentials and hormone secretion:

Calcium-activated potassium channel

Inwardly rectifying potassium channel

Tandem pore domain potassium channel

Voltage-gated potassium channel

Potassium channels are composed of four protein subunits that can be the same (homotetramer) or closely related (heterotetramer). All potassium channel subunits have a distinctive pore-loop structure that sits at the top of the channel and is responsible for potassium selectivity [12] . This is often referred to as a selectivity or filter loop. The selectivity filter strips the waters of hydration from the potassium ion, allowing it into the channel. Farther down the structure is a 10-Å-diameter, transmembrane, water-filled central channel that conducts potassium across the membrane. Elucidating the three-dimensional structure of this important integral membrane protein by X-ray crystallography ( Fig. 19.7 ) [12] was a seminal accomplishment in the field of membrane biophysics. For this work from 1998, Rod MacKinnon ( Fig. 19.8 ) of Rockefeller University was awarded the 2003 Nobel Prize in Chemistry. Until the potassium channel work, just obtaining the structure of non–water-soluble proteins was next to impossible. MacKinnon's work elucidated not only the structure of the potassium channel but also its molecular mechanism. It has served as a blueprint for determining the structure of other membrane proteins and has greatly stimulated interest in the field.

Figure 19.7.

Three-dimensional structure of the potassium channel [12] . The channel itself is the clear opening in the center of the structure and a single K + is shown in the center of the channel.

Figure 19.8.

Rod MacKinnon, 1956–.

3.3. Sodium Channel

In some ways, Na + channels [13] parallel the action of K + channels. They are both facilitated diffusion carriers that conduct the cation down the ion's electrochemical gradient. In excitable cells such as neurons, myocytes, and some glia, Na + channels are responsible for the rising phase of action potentials (see Chapter 18). Therefore agents that block Na + channels also block nerve conduction and so are deadly neurotoxins. There are two basic types of Na + channels: voltage-gated and ligand-gated. The opening of a Na + channel has a selectivity filter that attracts Na + . From there the Na + ions flow into a constricted part of the channel that is about 3–5 Å wide. This is just large enough to allow the passage of a single Na + with one attached water. Since the larger K + cannot squeeze through, the channel is selective for Na + . Of particular interest are two extremely potent biological toxins, tetrodotoxin (TTX) and saxitoxin (STX) ( Fig. 19.9 , [14] ), that, in seafood, have killed and injured many humans. Both toxins shut down Na + channels by binding from the extracellular side.

Figure 19.9.

Structures of the extremely potent neurotoxins, tetrodotoxin (TTX) and saxitoxin (STX). Both neurotoxins function by blocking the Na + channel.

TTX is encountered primarily in puffer fish but also in porcupine fish, ocean sunfish, and triggerfish. TTX ( Fig. 19.9 , left) is a potent neurotoxin that blocks Na + channels while having no effect on K + channels. Puffer fish is the second most poisonous vertebrate in the world, trailing only the Golden Poison Frog that is endemic to the rain forests on the Pacific Coast of Colombia. In some parts of the world puffer fish are considered to be a delicacy but must be prepared by chefs who really know their business, as a slight error can be fatal. Puffer poisoning usually results from consumption of incorrectly prepared puffer soup, and TTX has no known antidote!

Saxitoxin (STX, Fig. 19.9 , right) is a Na + channel–blocking neurotoxin produced by some marine dinoflagellates that can accumulate in shellfish during toxic algal blooms known as Red Tide. Saxitoxin is one of the most potent natural toxins, and it has been estimated that a single contaminated mussel has enough STX to kill 50 humans! STX's toxicity has not escaped the keen eye of the United States military, which has weaponized the toxin and given it the designation TZ.

3.4. Solute Equilibrium

The driving force for transmembrane solute movement by simple or passive diffusion is determined by the free energy change, Δ G .

Where Δ G is the free energy change; [ s o ′ ] is the solute concentration on the right side of a membrane; [ s o ] is the solute concentration on the left side of a membrane; R is the gas constant; T is the temperature in K; Z is the charge of the solute; F is the Faraday; ΔΨ is the transmembrane electrical potential.

Solute movement will continue until Δ G  = 0. If Δ G is negative, solute movement is left to right (it is favorable as drawn). If Δ G is positive, solute movement is right to left (it is unfavorable in the left-to-right direction) or energy must be added for the solute to go from left to right. The equation has two parts; a transmembrane chemical gradient ( [ s o ′ ] / [ s o ] ) and a transmembrane electrical gradient (ΔΨ). The net movement of a solute is therefore determined by a combination of the solute's chemical gradient and an electrical gradient inherent to the cell. If the solute has no charge, Z  = 0 (as is the case for glucose) and the right hand part of the equation ( Z FΔΨ) drops out. Therefore, the final equilibrium distribution of glucose across the membrane will have the internal glucose concentration equal to the external glucose concentration and is independent of ΔΨ, the electrical potential. At equilibrium for a noncharged solute, Δ G = R T ln [ s o ′ ] / [ s o ] and Δ G can only be = zero if [ s o ′ ] = [ s o ] .

The situation for a charged solute like K + is more complicated. The net Δ G is determined by both the chemical gradient ( [ s o ′ ] / [ s o ] ) and electrical gradient (ΔΨ). The ΔΨ results from the sum of all charged solutes on both sides of the membrane, not just K + . Therefore even if the K + concentration is higher inside the cell than outside (the chemical gradient is unfavorable for K + uptake), the ΔΨ may be in the correct direction (negative interior) and of sufficient magnitude to drive K + uptake against its chemical gradient.

3.5. Aquaporins

Aquaporins are also known as water channels and are considered to be “the plumbing system for cells” [15] , [16] . For decades it was assumed that water simply leaked through biological membranes by numerous processes described above (Chapter 19, Section 2 ). However, these methods of water permeability could not come close to explaining the rapid movement of water across some cells. Although it had been predicted that water pores must exist in very leaky cells, it was not until 1992 that Peter Agre ( Fig. 19.10 ) at Johns Hopkins University identified a specific transmembrane water pore that was later called aquaporin-1. For this accomplishment Agre shared the 2003 Nobel Prize in Chemistry with Rod MacKinnon for his work on the potassium channel. Aquaporins are usually specific for water permeability and exclude the passage of other solutes. A type of aquaporin known as aqua-glyceroporins can also conduct some very small uncharged solutes such as glycerol, CO 2 , ammonia, and urea across the membrane. However, all aquaporins are impermeable to charged solutes. Water molecules traverse the aquaporin channel in single file ( Fig. 19.11 ) [17] .

Figure 19.10.

Peter Agre, 1949–.

Figure 19.11.

Aquaporin. Water molecules pass through the aquaporin channel in single file.

4. Active Transport

A characteristic of all living membranes is the formation and maintenance of transmembrane gradients of all solutes including salts, biochemicals, macromolecules, and even water. In living cells, large gradients of Na + and K + are particularly important. Typical cell concentrations are:

Living cells will also have a ΔΨ from −30 to −200 mV (negative interior) resulting from the uneven distribution of all ionic solutes including Na + and K + . The chemical and electrical gradients are maintained far from equilibrium by a multitude of active transport systems. Active transport requires a form of energy (often ATP) to drive the movement of solutes against their electrochemical gradient, resulting in a nonequilibrium distribution of the solute across the membrane. A number of nonexclusive and overlapping terms are commonly used to describe the different types of active transport. Some of these are depicted in Fig. 19.12 [18] .

Figure 19.12.

Basic types of active transport [18] .

4.1. Primary Active Transport

Primary active transport is also called direct active transport or uniport. It involves using energy (usually ATP) to directly pump a solute across a membrane against its electrochemical gradient.

The most studied example of primary active transport is the plasma membrane Na + ,K + -ATPase discussed below (Chapter 19, Section 4.2 ). Other familiar examples of primary active transport are the redox H + -gradient generating system of mitochondria (see Chapter 18), the light-driven H + -gradient generating system of photosynthetic thylakoid membranes, and the ATP-driven acid (H + ) pump found in the epithelial lining of the stomach. There are four basic types of ATP-utilizing primary active transport systems ( Table 19.3 ).

Table 19.3.

Four Types of ATP-Using Primary Active Transport Systems

4.2. Na + ,K + -ATPase

Arguably the most important active transport protein is the plasma membrane-bound Na + ,K + -ATPase. This single enzyme accounts for one-third of human energy expenditure and is often referred to as the “pacemaker for metabolism.” As a result the Na + ,K + -ATPase has been extensively studied for more than 50 years. The enzyme was discovered in 1957 by Jens Skou ( Fig. 19.13 ) who, 40 years later, was awarded the 1997 Nobel Prize in Chemistry.

Figure 19.13.

Jens Skou, 1918–.

As is often the case in biochemistry, a serendipitous discovery of a natural product from the jungles of Africa has been instrumental in unraveling the enzyme's mechanism of action. The compound is ouabain ( Fig. 19.14 ), a cardiac glycoside first discovered in a poison added to the tip of Somali tribesmen's hunting arrows. In fact the name ouabain comes from the Somali word waabaayo that means “arrow poison.” The sources of ouabain are ripe seeds and bark of certain African plants and ouabain is potent enough to kill a hippopotamus with a single arrow. For decades after its discovery, ouabain was routinely used to treat atrial fibrillation and congestive heart failure in humans. More recently, ouabain has been replaced by digoxin, a structurally related, but more lipophilic cardiac glycoside.

Figure 19.14.

Structure of ouabain.

There are several important observations about Na + ,K + -ATPase that had to be factored in before a mechanism of action could be proposed. These include:

Na + ,K + -ATPase is an example of active antiport and primary active transport.

Na + ,K + -ATPase is inhibited by ouabain, a cardiac glycoside.

Ouabain binds to the outer surface of Na + ,K + -ATPase and blocks K + transport into the cell.

Na + binds better from the inside.

K + binds better from the outside.

ATP phosphorylates an aspartic acid on the enzyme from the inside.

Phosphorylation is related to Na + transport.

Dephosphorylation is related to K + transport.

Dephosphorylation is inhibited by ouabain.

Three Na + ions are pumped out of the cell as two K + ions are pumped in, driven by hydrolysis of one ATP.

Na + ,K + -ATPase is electrogenic.

Mechanism of Na + ,K + -ATPase [19] is based on toggling back and forth between two conformational states of the enzyme, ENZ-1 and ENZ-2 ( Fig. 19.15 ). Three Na + s bind from the inside to Na + ,K + -ATPase in one conformation (ENZ-1). This becomes phosphorylated by ATP causing a conformation change producing ENZ-2∼P. ENZ-2∼P does not bind Na + , but does bind two K + ions. Therefore, three Na + ions are released to the outside and two K + ions are bound from the outside, generating ENZ-2∼P (2K + ). Upon hydrolysis of ∼P, Na + ,K + -ATPase (ENZ II) reverts back to the original ENZ-1 conformation that releases two K + ions and binds three Na + ions from the inside. Ouabain blocks the dephosphorylation step.

Figure 19.15.

Mechanism of the Na + ,K + -ATPase.

4.3. Secondary Active Transport

Secondary active transport (also known as cotransport) systems are composed of two separate functions. The energy-dependent movement of an ion (eg, H + , Na + , or K + ) generates an electrochemical gradient of the ion across the membrane. This ion gradient is coupled to the movement of a solute in either the same direction (symport) or in the opposite direction (antiport, see Fig. 19.12 , [18] ). Movement of the pumped ion down its electrochemical gradient is by facilitated diffusion. The purpose of both types of co-transport is to use the energy in an electrochemical gradient to drive the movement of another solute against its gradient. An example of symport is the SGLT1 (sodium-glucose transport protein-1) in the intestinal epithelium [20] . SGLT1 uses the energy in a downhill transmembrane movement of Na + to transport glucose across the apical membrane against an uphill glucose gradient so that the sugar can be transported into the bloodstream.

4.4. Bacterial Lactose Transport

The secondary active symport system for lactose uptake in Escherichia coli is shown in Fig. 19.16 [21] . Lactose uptake is driven through a channel by a H + gradient generated by the bacterial electron transport system [22] . The free energy equation for transport described above ( Δ G = R T ln [ s o ′ ] / [ s o ] + Z F Δ Ψ ) can be rearranges for cases employing H + gradients (see Chapter 18) to:

Where Δ μ H + is the proton motive force; ΔΨ is the transmembrane electrical potential; R is the gas constant; T is the temperature in °K; n is the solute charge (+1 for protons); F is the Faraday; ΔpH is the transmembrane pH gradient.

Figure 19.16.

Lactose transport system in Escherichia coli [21] . Uptake of lactose is coupled to the movement of an H + down its electrochemical gradient. This is an example of active transport, co-transport, and active.

It is the force on an H + (called the proton motive force) that drives lactose uptake. Note that the ability to take up lactose is a combination of the electrical gradient and the pH gradient. Although lactose uptake is directly coupled to H + transmembrane movement, it is possible to take up lactose even if the pH gradient is zero (ie, if the ΔΨ is sufficiently large).

4.5. Vectorial Metabolism, Group Translocation

Over 50 years ago, Peter Mitchell (see Chapter 18, Fig. 18.26) recognized the importance of what he termed “vectorial metabolism” [23] , [24] . Water-soluble enzymes convert substrate to product without any directionality. Mitchell proposed that many enzymes are integral membrane proteins that have a specific transmembrane orientation. When these enzymes convert substrate to product they do so in one direction only. This enzymatic conversion is therefore unidirectional, or “vectorial.” Mitchell expanded this basic concept into his now famous “chemiosmotic hypothesis” for ATP synthesis in oxidative phosphorylation (Chapter 18) [25] , [26] . For this revolutionary idea Mitchell was awarded the 1997 Nobel Prize in Chemistry.

Vectorial metabolism has been used to describe the mechanism for several membrane transport systems. For example, it has been reported in some cases the uptake of glucose into a cell may be faster if the external source of glucose is sucrose rather than free glucose. Through a vectorial transmembrane reaction, membrane-bound sucrase may convert external sucrose into internal glucose plus fructose more rapidly than the direct transport of free glucose through its transport system.

Mitchell defined one type of vectorial transport as group translocation, the best example being the PTS (phosphotransferase system) discovered by Saul Roseman in 1964.

PTS is a multicomponent active transport system that uses the energy of intracellular phosphoenol pyruvate (PEP) to take up extracellular sugars in bacteria. Transported sugars include glucose, mannose, fructose, and cellobiose. Components of the system include both plasma membrane and cytosolic enzymes. PEP is a high-energy phosphorylated compound (Δ G of hydrolysis is −61.9 kJ/mol) that drives the system. The high-energy phosphoryl group is transferred through an enzyme bucket brigade from PEP to glucose producing glucose-6-phosphate in several steps (PEP → EI → HPr →EIIA → EIIB → EIIC → glucose-6-phosphate). The sequence is depicted in more detail in Fig. 19.17 [27] . HPr stands for heat-stable protein that carries the high-energy ∼P from EI (enzyme-I) to EIIA. EIIA is specific for glucose and transfers ∼P to EIIB that sits next to the membrane where it takes glucose from the transmembrane EIIC and phosphorylates it producing glucose-6-phosphate. Although it is glucose that is being transported across the membrane, it never actually appears inside the cell as free glucose but rather as glucose-6-phosphate. Free glucose could leak back out of the cell via a glucose transporter, but glucose-6-phosphate is trapped inside the cell where it can rapidly be metabolized through glycolysis. Group translocation is defined by a transported solute appearing in a different form immediately after crossing the membrane.

Figure 19.17.

The bacterial PTS system for glucose transport [27] .

5. Ionophores

The term ionophore means “ion bearer.” Ionophores are small, lipid-soluble molecules, usually of microbial origin, whose function is to conduct ions across membranes [28] , [29] . They are facilitated diffusion carriers that transport ions down their electrochemical gradient. Ionophores can be divided into two basic classes: channel formers and mobile carriers ( Fig. 19.18 ) [30] . Channel formers are long lasting, stationary structures that allow many ions at a time to rapidly flow across a membrane. Mobile carriers bind to an ion on one side of a membrane, dissolve in and cross the membrane bilayer and release the ion on the other side. They can only carry one ion at a time. Four representative ionophores will be discussed: the K + ionophore valinomycin, the proton ionophore 2,4-dinitrophenol, synthetic crown ethers, and the channel-forming ionophore nystatin ( Fig. 19.19 ).

Figure 19.18.

Two basic types of ionophores: channel formers (left) and mobile carriers (right) [30] .

Figure 19.19.

Representative examples of ionophores: the K + ionophore valinomycin, the proton ionophore 2,4-dinitrophenol, the synthetic crown ether 18-crown-6, and the channel forming ionophore nystatin.

5.1. Valinomycin

Superficially valinomycin resembles a cyclic peptide ( Fig. 19.19 ). However, upon closer examination the ionophore is actually a 12-unit (dodeca) depsi peptide where amino acid peptide bonds are alternated with amino alcohol ester bonds. Therefore the linkages that hold the molecule together alternate between nitrogen esters (peptide bonds) and oxygen esters. The units that comprise valinomycin are d - and l -valine (hence the name “valinomycin”), hydroxyvaleric acid and l -lactic acid. The circular structure is a macrocyclic molecule with the 12 carbonyl oxygens facing the inside of the ring where they chelate a single K + . The outside surface of valinomycin is coated with nine hydrophobic side chains of d - and l -valine and l -hydroxyvaleric acid. The polar interior of valinomycin precisely fits one K + . The binding constant for K + -valinomycin is 10 6 while Na + -valinomycin is only 10. This emphasizes the high selectivity valinomycin has for K + over Na + . Valinomycin, therefore, has an oily surface that readily dissolves in a membrane lipid bilayer, carrying K + across the membrane down its electrochemical gradient.

Valinomycin was first recognized as a potassium ionophore by Bernard Pressman in the early 1960's [31] , [32] . He reported that valinomycin, a known antibiotic, stimulated K + uptake and H + efflux from mitochondria. Many studies showed that valinomycin dissipates essential transmembrane electrochemical gradients causing tremendous metabolic upheaval in many organisms including microorganisms. It is for this reason that valinomycin was recognized as an antibiotic long before it was identified as an ionophore. Currently several ionophores are added to animal feed as antibiotics and growth enhancing additives [33] . Recently valinomycin has been reported to be the most potent agent against SARS-CoV (severe acute respiratory-syndrome coronavirus), a severe form of pneumonia first identified in 2003 [34] .

5.2. 2,4-Dinitrophenol

2,4-Dinitrophenol (DNP, Fig. 19.19 ) is considered to be the classic uncoupler of oxidative phosphorylation (see Chapter 18). It is a synthetic lipid-soluble proton ionophore that dissipates proton gradients across bioenergetic membranes (mitochondrial inner, thylakoid, bacterial plasma). An uncoupler is therefore an H + -facilitated diffusion carrier. Elucidating the role of DNP in uncoupling oxidative phosphorylation was an essential component in support of Peter Mitchell's chemiosmotic hypothesis [25] . Electron movement from NADH or FADH 2 to O 2 via the mitochondrial electron transport system generates a considerable amount of electrical energy that is partially captured as a transmembrane pH gradient (see Chapter 18). The movement of H + s back across the membrane, driven by the electrochemical gradient, is through a channel in the F 1 ATPase (an F-type primary active transport system discussed above, (Chapter 19, Section 4.1 )) that is coupled to ATP synthesis. DNP short-circuits the H + gradient before it can pass through the F 1 ATPase, thus uncoupling electron transport, the energy source for the H + gradient, from ATP synthesis. Therefore, in the presence of DNP, electron transport continues, even at an accelerated rate, but ATP production is diminished. The energy that should have been converted to chemical energy in the form of ATP is then released as excess heat.

This combination of properties led to the medical application of DNP to treat obesity from 1933 to 1938 [35] . Upon addition of DNP:

The patient became weak due to low ATP levels.

Breathing increased due to increased electron transport to rescue ATP production.

Metabolic rate increased.

Body temperature increased due to inability to trap electrical energy as chemical energy in the form of ATP, releasing heat.

Body weight decreased due to increased respiration burning more stored fat.

DNP was indeed a successful weight loss drug. Two of the early proponents of DNP use as a diet drug, Cutting and Tainter at Stanford University, estimated that more than 100,000 people in the United States had tested the drug during its first year in use [35] . DNP, however, did have one disturbing side effect—death! Fatality was not caused by a lack of ATP, but rather by a dangerous increase in body temperature (hyperthermia). In humans, 20–50 mg/kg of DNP can be lethal. Although general use of DNP in the United States was discontinued in 1938, it is still employed in other countries and by bodybuilders to eliminate fat before competitions.

5.3. Crown Ethers

Crown ethers are a family of synthetic ionophores that are generally similar in function to the natural product valinomycin [36] . The first crown ether was synthesized by Charles Pederson ( Fig. 19.20 ) while working at DuPont in 1967. For this work Pedersen was co-awarded the 1987 Nobel Prize in Chemistry. Crown ethers are cyclic compounds composed of several ether groups. The most common crown ethers are oligomers of ethylene oxide with repeating units of (—CH 2 CH 2 O—) n where n  = 4 (tetramer), n  = 5 (pentamer), or n  = 6 (hexamer). Crown ethers are given structural names, X -crown- Y , where X is the total number of atoms in the ring and Y is the number of these atoms that are oxygen. Crown refers to the crown-like shape the molecule takes. Crown ether oxygens form complexes with specific cations that depend on the number of atoms in the ring. For example, 18-crown-6 ( Fig. 19.19 ) has high affinity for K + , 15-crown-5 for Na + , and 12-crown-4 for Li + . Like valinomycin, the exterior of the ring is hydrophobic, allowing crown ethers to dissolve in the membrane lipid bilayer while carrying the sequestered cation down its electrochemical gradient. It is now possible to tailor make crown ethers of different sizes that can encase a variety of catalysts for phase transfer into the bilayer hydrophobic interior where they can be used to catalyze reactions inside the membrane.

Figure 19.20.

Charles Pedersen, 1904–1989.

5.4. Nystatin

Nystatin ( Fig. 19.19 ) is a channel-forming ionophore that creates a hydrophobic pore across a membrane [37] , [38] . Channel-forming ionophores allow for the rapid facilitated diffusion of various ions that depend on the dimensions of the pore. Nystatin, like other channel-forming ionophores (eg, amphotericin B and natamycin), is a commonly used antifungal agent. Finding medications that can selectively attack fungi in the presence of normal animal cells presents a difficult challenge since both cell types are eukaryotic. Bacteria, being prokaryotes, are sufficiently different to present a variety of anti-bacterial approaches not amenable to fungi. However, fungi do have an Achilles heel. Fungal plasma membranes have as their dominant sterol ergosterol, not the animal sterol cholesterol (see Chapter 5). Nystatin binds preferentially to ergosterol, thus targeting fungi in the presence of animal cells. When present at sufficient levels, nystatin complexes with ergosterol and forms transmembrane channels that lead to K + leakage and death of the fungus. Nystatin is a polyene antifungal ionophore that is effective against many molds and yeast including Candida . A major use of nystatin is as a prophylaxis for AIDS patients who are at risk for fungal infections.

6. Gap Junctions

Gap junctions are a common structural feature of many animal plasma membranes [39] , [40] . In plants similar structures are known as plasmodesmata. Gap junctions were introduced earlier in Chapter 11 (see Fig. 11.6). Gap junctions represent a primitive type of intercellular communication that allows transmembrane passage of small solutes like ions, sugars, amino acids, and nucleotides while preventing migration of organelles and large polymers like proteins and nucleic acids. Gap junctions connect the cytoplasms of two adjacent cells through nonselective channels. Connections through adjacent cells are at locations where the gap between cells is only 2–3 nm. This small gap is where the term “gap junction” originated. Gap junctions are normally clustered from a few to over a 1000 in select regions of a cell plasma membrane.

Early experiments involved injecting fluorescent dyes, initially fluorescein (molecular weight 300), into a cell and observing the dye movement into adjacent cells with a fluorescence microscope [41] , [42] . Currently Lucifer Yellow has become the fluorescent dye of choice for gap junction studies, replacing fluorescein. At first, the dye only appeared in the initially labeled cell. With time, however, the dye was observed to spread to adjacent cells through what appeared to be points on the plasma membrane. These points were later recognized as gap junctions. By varying the size of the fluorescent dye, it was shown that there was an upper size limit for dye diffusion. Solutes had to have a molecular weight of less than ∼1200 to cross from one cell to another [41] .

Although gap junctions were obviously channels that connected the cytoplasms of adjacent cells, it was years before their structure, shown in Fig. 19.21 , was determined [43] , [44] . Each channel in a gap junction is made up of 12 proteins called connexins. Six hexagonally arranged connexins are associated with each of the adjacent cell plasma membranes that the gap junction spans. Each set of six connexins is called a connexon and forms half of the gap junction channel. Therefore, one gap junction channel is composed of 2 aligned connexons and 12 connexins. Each connexin has a diameter of about 7 nm and the hollow center formed between the 6 connexins (the channel) is about 3 nm in diameter. Gap junctions allow adjacent cells to be in constant electrical and chemical communication with one another. Of particular importance is the rapid transmission of small second messengers, such as inositol triphosphate (IP 3 ) and Ca 2+ .

Figure 19.21.

Gap junction [43] . Six connexins form a connexon and one connexon from each cell unite to form a gap junction.

It appears that all cells in the liver are interconnected through gap junctions. This presents a possible dilemma. If even a single cell is damaged, deleterious effects may be rapidly spread throughout the entire liver. Preventing this is one important function of Ca 2+ . Extracellular Ca 2+ is ∼10 −3  mol/L while intracellular levels are maintained at ∼10 −6  mol/L. If a cell is damaged, Ca 2+ rushes in, dramatically increasing intracellular Ca 2+ . Gap junction channels close if intracellular Ca 2+ reaches 10 −3  mol/L, thus preventing the spread of damage.

Gap junctions are particularly important in cardiac muscle as the electrical signals for contraction are passed efficiently through these channels [45] . As would be expected, malfunctions of gap junctions lead to a number of human disorders including demyelinating neurodegenerative diseases, skin disorders, cataracts, and even some types of deafness.

7. Other Ways to Cross the Membrane

There are several other ways that solutes, including large macromolecules, can cross membranes. These methods include receptor-mediated endocytosis (RME, discussed in Chapter 17), phagocytosis, pinocytosis, exocytosis, and membrane blebbing. These methods involve large sections of a membrane containing many lipids and proteins.

Two similar transport processes that have been known for a long time are pinocytosis and phagocytosis [46] . Both involve nonspecific uptake (endocytosis) of many things from water and ions through to large macromolecules and, for phagocytosis, even whole cells. Pinocytosis is Greek for “cell drinking” and involves the plasma membrane invaginating a volume of extracellular fluid and anything it contains including water, salts, biochemicals and even soluble macromolecules. Phagocytosis is Greek for “cell eating” and involves the plasma membrane invaginating large insoluble solids.

7.1. Pinocytosis

Pinocytosis is a form of endocytosis involving fluids containing many solutes. In humans, this process occurs in cells lining the small intestine and is used primarily for absorption of fat droplets. In endocytosis the cell plasma membrane extends and folds around desired extracellular material, forming a pouch that pinches off creating an internalized vesicle ( Fig. 19.22 , [19] , [20] , [21] , [22] , [23] , [24] , [25] , [26] , [27] , [28] , [29] , [30] , [31] , [32] , [33] , [34] , [35] , [36] , [37] , [38] , [39] , [40] , [41] , [42] , [43] , [44] , [45] , [46] , [47] ). The invaginated pinocytosis vesicles are much smaller than those generated by phagocytosis. The vesicles eventually fuse with the lysosome whereupon the vesicle contents are digested. Pinocytosis involves a considerable investment of cellular energy in the form of ATP and so is many 1000 times less efficient than RME (see Chapter 17). Also, in sharp contrast to RME, pinocytosis is nonspecific for the substances it accumulates. Pinocytosis is not a recent discovery as it was first observed decades before the other transport systems discussed in Chapter 19. Its discovery is attributed to Warren Lewis in 1929.

Figure 19.22.

Pinocytosis, a type of endocytosis. An invagination of the plasma membrane encapsulates many water-soluble solutes ranging in size from salts to macromolecules.

7.2. Phagocytosis

Phagocytosis is a type of endocytosis that involves uptake of large solid particles, often >0.5 mm [47] . The particles are aggregates of macromolecules, parts of other cells, and even whole microorganisms and, in contrast to pinocytosis (shown in Fig. 19.22 ), phagocytosis has surface proteins that specifically recognize and bind to the solid particles. Fig. 19.23 [48] depicts events in phagacytosis. Phagocytosis is a routine process that ameba and ciliated protozoa use to obtain food. In humans, phagocytosis is restricted to specialized cells called phagocytes that include white blood cell neutrophils and macrophages. As with pinocytosis, phagocytosis generates intracellular vesicles called phagosomes that have sequestered solid particles they transport to the lysosome for digestion. Phagocytosis is a major mechanism used by the immune system to remove pathogens and cell debris. In fact, very early studies of the immune system led Elie Metchnikoff to discover phagocytosis in 1882. For this work Metchnikoff shared the 1908 Nobel Prize in Medicine with Paul Ehrlich.

Figure 19.23.

Phagocytosis, a type of endocytosis that involves uptake of large solid particles.

7.3. Exocytosis

Exocytosis is the process by which cells excrete waste and other large molecules from the cytoplasm to the cell exterior [49] and therefore is the opposite of endocytosis. Exocytosis generates vesicles referred to as secretory or transport vesicles (Chapter 17). In exocytosis, intracellular (secretory) vesicles fuse with the plasma membrane and release their aqueous sequestered contents to the outside at the same time that the vesicular membrane hydrophobic components (mostly lipids and proteins) are added to the plasma membrane ( Fig. 19.24 , [50] ). Steady state composition of the plasma membrane results from a balance between endocytosis and exocytosis. The resultant process of plasma membrane recycling is amazingly fast. For example, pancreatic secretory cells recycles an amount of membrane equal to the whole surface of the cell in ∼90 min. Even faster are macrophages that can recycle contents of their plasma membrane in only 30 min.

Figure 19.24.

Exocytosis. Intracellular secretory vesicles fuse with the plasma membrane releasing their water-soluble contents to the outside and adding membrane material to the plasma membrane [50] .

Before approaching the plasma membrane for fusion, exocytosis vesicles had a prior life that is considered in Chapter 17. The vesicles must first dock with the plasma membrane, a process that keeps the two membranes separated by <5–10 nm. During docking, complex molecular rearrangements occur to prepare the membranes for fusion. The process of vesicle fusion and release of aqueous compartment components is driven by SNARE proteins (see Chapters 10 and 17Chapter 10Chapter 17) [51] , [52] .

7.4. Blebbing

Blebbing of the plasma membrane is a morphological feature of cells undergoing late stage apoptosis (programmed cell death, see Chapter 24) [53] . A bleb is an irregular bulge in the plasma membrane of a cell caused by localized decoupling of the cytoskeleton from the plasma membrane. The bulge eventually blebs off from the parent plasma membrane taking part of the cytoplasm with it. It is clear in Fig. 19.25 [54] that the plasma membrane of an apoptotic cell is highly disintegrated and has lost the integrity required to maintain essential transmembrane gradients. Blebbing is also involved in some normal cell processes, including cell locomotion and cell division.

Figure 19.25.

Membrane blebbing during apoptosis [54] .

Carefully controlled solute movement into and out of cells is an essential feature of life. There are many ways solutes are transported across the thin (∼40 Å) membrane hydrophobic barrier. Transport is divided into passive diffusion and active transport. A biological membrane is semipermeable, being permeable to some molecules, most notably water (osmosis), while being very impermeable to most solutes that require some form of transporter. Passive diffusion (simple and facilitated) only requires the energy inherent in the solute's electrochemical gradient and results in its equilibrium across the membrane. In contrast, active transport requires additional energy (ie, ATP), and results in a nonequilibrium, net accumulation of the solute. Passive transport can involve simple diffusion or facilitated carriers including ionophores and channels. Active transport comes in many, often complex forms. Examples of active transport include primary active transport (uniport), secondary active transport (co-transport, antiport), and group translocation. Besides the multitude of transport systems, transport can be accomplished by gap junctions, receptor mediated endocytosis, phagocytosis, pinocytosis, exocytosis, and apoptotic membrane blebbing.

Chapter 20 will discuss bioactive lipids, highly specialized lipids that are functional at very low levels. Discussed bioactive lipids include ceramides, diacylglycerol, eicosanoids, steroid hormones, and phosphatidic acid.

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Investigating Transport Across Membranes (A-level Biology)

Investigating transport across membranes, investigating diffusion.

We can investigate how diffusion occurs in biological cells by using cubes of agar jelly. The basic concept of this experiment is outlined below:

Table of Contents

• The agar jelly contains a pH indicator. We can make up agar jelly with an alkaline solution (e.g. sodium hydroxide) and add a few drops of phenolphthalein to it before the jelly sets. Phenolphthalein is a pH indicator which turns pink in the presence of alkaline solutions, thus, the jelly will have a bright pink colour.
• The agar jelly is placed in an acidic solution. Once the jelly has set, we can cut it up into cubes and place it in an acidic solution, such as dilute hydrochloric acid.
• The agar jelly is neutralised by the diffusion of the acid. The acidic solution will slowly diffuse into the agar jelly and neutralise the alkaline solution. As it does, the jelly will lose its pink colour and become colourless, as phenolphthalein turns colourless in non-alkaline environments.

We can alter different parts of this experiment to model how different factors affect the rate of diffusion.

Investigating the effects of surface area on diffusion

• Cut the agar jelly into different sized cubes to investigate the effects of surface area . Cut the jelly into cubes of different sizes and work out each cube’s surface area to volume ratio . For example, a cube with 2cm edges will have a surface area to volume ratio of 3:1.
• Place the cubes in the same volume and concentration of acid. Put the cubes into containers which hold the same volume and concentration of hydrochloric acid. Then measure the time it takes for the different cubes to go colourless.
• The cube with the largest surface area: volume ratio will go colourless the quickest. The cube with the largest surface area: volume ratio has the greatest amount of space available for the hydrochloric acid to diffuse into the jelly so it will be neutralised the fastest.

Investigating the effects of concentration on diffusion

• Place the agar jelly cubes in different concentrations of acid. Cut the agar jelly into equal sized cubes and put them in different containers, each with a different concentration of hydrochloric acid. Measure the time it takes for the different cubes to go colourless.
• The cube placed in the highest concentration of acid will go colourless the quickest. The cube placed in the container with the highest concentration will have the greatest concentration of acid being diffused into the jelly per minute. As such, it will go colourless the quickest.

Investigating the effects of temperature on diffusion

• Place the agar jelly cubes in different temperatures. Cut the agar jelly into equal sized cubes and put them in different containers, each with the same concentration of hydrochloric acid. Put the containers in water baths heated to different temperatures. Be careful not to heat the water baths over 65° as the agar jelly will melt.
• The cube placed in the highest temperature of acid will go colourless the quickest. As high temperatures speed up the rate of diffusion, the cube in the hottest container will be neutralised the quickest.

Investigating Osmosis

Osmosis is the movement of water molecules from an area of high water potential to an of low water potential by osmosis. Water potential is determined by the concentration of solutes in the solutions on either side of the cell membrane.

Investigations using plant tissue

This experiment involves placing plant tissue, e.g. potato cylinders, in varying concentrations of sucrose solutions to determine the water potential of the plant tissues.

• Prepare the different concentrations of sucrose solutions . Using distilled water and 1M sucrose solution, prepare a series of dilutions such that you now have 0.0, 0.2, 0.4, 0.6, 0.8 and 1.0M sucrose. Place 5cm 3 of each dilution into separate beakers.
• Prepare equal sized pieces of potato chips. Using a cork borer, cut out 18 pieces of potato chips, all of equal sizes.
• Weigh the mass of the potato chips. Dry the potato chips gently with a paper towel. Divide them into groups of three and weigh each group.
• Place each group of potato chips in each solution . The potato chips should be left in the solutions for a minimum of 20 minutes.   All groups should be left in the solution for the same amount of time.

• Weigh the mass of the potato chips again. Once your desired amount of time has passed, remove the chips from the solutions, and dry them gently using a paper towel. Reweigh each group again.
• Calculate % change in mass. Using the mass of the potato chips before and after being placed in the solution, calculate the % change in its mass.
• Plot the % change in mass on a calibration curve. The calibration curve helps us determine the water potential in the potato sample. Plot the % change in mass against concentration of sucrose solution.   The point at which the curve crosses the x axis is when the sucrose solution is isotonic with the potato samples i.e. the water potential of the sucrose solution is the same as the water potential of the potatoes. At this point, there is no movement of water in or out of the potato. Overall:
• The potato samples in the dilute solutions will have a net increase in mass – the water potential is greater in the potato than in the sucrose solution, so water moves into the potato samples via osmosis.
• The potato samples in the concentrated solutions will have a net decrease in mass – the water potential is lower in the potato than in the sucrose solution, so water moves out of the potato samples via osmosis.

Investigations using Visking tubing

Visking tubing is an artificial membrane that is selectively permeable as it has many microscopic pores. This allows smaller molecules such as water and glucose to pass through it, while larger molecules such as starch and sucrose are unable to cross the membrane.

• Prepare three equal-sized pieces of Visking tubings. Run the tubing under tap water to soften it and knot each tubing on one end to create a bag.
• Place a rubber bung at the open end of the Visking tubing. Find rubber bungs with an opening in the centre that will fit the open end of the Visking tubing. Then seal the tubing using the bung and fix it in place using a rubber band.
• Prepare sucrose solutions with concentrations of 0.5M and 1.0M. You may wish to add a food dye to the 0.5M solution so that it is easier to see later on.
• Pipette in the 0.5M sucrose solution. Using a pipette or a syringe, fill each tubing through the opening of the rubber bung with the 0.5M sucrose solution. Make sure it is filled completely to the brim with no air bubbles.
• Insert capillary tubes into each of the tubings . Insert a capillary tube through the rubber bung’s opening. Mark the level at which the sucrose solution has risen to in the capillary tube.
• Place each Visking tubing into containers of different solutions. Prepare three beakers, each containing distilled water, 0.5M sucrose, and 1.0M sucrose. Place each Visking tubing into each of the beakers and leave them in for the same amount of time.
• Measure the change in liquid level. Mark the new liquid level on the capillary tube before removing the Visking tubing from its beaker. Measure the change in the liquid level. Overall:
• The liquid level of the Visking tubing placed in distilled water will have risen as the sucrose solution in the tubing is hypertonic to the water i.e. the sucrose is more concentrated. Thus, there is net movement of water into the Visking tubing via osmosis.
• The liquid level of the Visking tubing placed in 0.5M sucrose will remain the same as the solution inside the tubing and outside the tubing are isotonic i.e. the solutions are the same concentration.
• The liquid level of the Visking tubing placed in 1.0M sucrose will have decreased as the solution inside the tubing is hypotonic to the solution outside the tubing i.e. the solution inside the tubing is less concentrated.

Transport across membranes is the movement of substances such as ions, molecules, and fluids from one side of a biological membrane to the other. This process is crucial for maintaining cellular homeostasis and allowing cells to exchange materials with their environment.

Investigating transport across membranes is important because it helps us understand the mechanisms by which cells regulate the flow of substances in and out of the cell. This is essential for understanding cellular processes such as metabolic reactions, waste removal, and communication between cells.

There are several methods used to investigate transport across membranes, including: Diffusion experiments to study the movement of substances through the lipid bilayer Osmosis experiments to study the movement of water across a semi-permeable membrane Active transport experiments to study the movement of substances against a concentration gradient with the use of energy Electrochemical experiments to study the movement of ions across the membrane

Factors that can affect transport across membranes include the size of the substance being transported, the charge of the substance, the concentration gradient, and the presence of specific transport proteins.

Transport across membranes can be measured in a variety of ways, including measuring changes in substance concentration, changes in electrical potential, and changes in fluid movement.

The limitations of investigating transport across membranes include the difficulty of obtaining pure and intact biological membranes, the potential for damage to the membrane during experimentation, and the limitations of experimental techniques.

In A-Level Biology, knowledge of transport across membranes can be applied to understand cellular processes such as the movement of nutrients and waste, the regulation of cell volume, and the communication between cells. This knowledge is also important for understanding diseases and disorders related to the malfunction of transport processes, such as cystic fibrosis and diabetes.

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CIE 1 Cell structure

Roles of atp (a-level biology), atp as an energy source (a-level biology), the synthesis and hydrolysis of atp (a-level biology), the structure of atp (a-level biology), magnification and resolution (a-level biology), calculating cell size (a-level biology), studying cells: confocal microscopes (a-level biology), studying cells: electron microscopes (a-level biology), studying cells: light microscopes (a-level biology), life cycle and replication of viruses (a-level biology), cie 10 infectious disease, bacteria, antibiotics, and other medicines (a-level biology), pathogens and infectious diseases (a-level biology), cie 11 immunity, types of immunity and vaccinations (a-level biology), structure and function of antibodies (a-level biology), the adaptive immune response (a-level biology), introduction to the immune system (a-level biology), primary defences against pathogens (a-level biology), cie 12 energy and respiration, anaerobic respiration in mammals, plants and fungi (a-level biology), anaerobic respiration (a-level biology), oxidative phosphorylation and chemiosmosis (a-level biology), oxidative phosphorylation and the electron transport chain (a-level biology), the krebs cycle (a-level biology), the link reaction (a-level biology), the stages and products of glycolysis (a-level biology), glycolysis (a-level biology), the structure of mitochondria (a-level biology), the need for cellular respiration (a-level biology), cie 13 photosynthesis, limiting factors of photosynthesis (a-level biology), cyclic and non-cyclic phosphorylation (a-level biology), the 2 stages of photosynthesis (a-level biology), photosystems and photosynthetic pigments (a-level biology), site of photosynthesis, overview of photosynthesis (a-level biology), cie 14 homeostasis, ectotherms and endotherms (a-level biology), thermoregulation (a-level biology), plant responses to changes in the environment (a-level biology), cie 15 control and co-ordination, the nervous system (a-level biology), sources of atp during contraction (a-level biology), the ultrastructure of the sarcomere during contraction (a-level biology), the role of troponin and tropomyosin (a-level biology), the structure of myofibrils (a-level biology), slow and fast twitch muscles (a-level biology), the structure of mammalian muscles (a-level biology), how muscles allow movement (a-level biology), the neuromuscular junction (a-level biology), features of synapses (a-level biology), cie 16 inherited change, calculating genetic diversity (a-level biology), how meiosis produces variation (a-level biology), cell division by meiosis (a-level biology), importance of meiosis (a-level biology), cie 17 selection and evolution, types of selection (a-level biology), mechanism of natural selection (a-level biology), types of variation (a-level biology), cie 18 biodiversity, classification and conservation, biodiversity and gene technology (a-level biology), factors affecting biodiversity (a-level biology), biodiversity calculations (a-level biology), introducing biodiversity (a-level biology), the three domain system (a-level biology), phylogeny and classification (a-level biology), classifying organisms (a-level biology), cie 19 genetic technology, cie 2 biological molecules, properties of water (a-level biology), structure of water (a-level biology), test for lipids and proteins (a-level biology), tests for carbohydrates (a-level biology), protein structures: globular and fibrous proteins (a-level biology), protein structures: tertiary and quaternary structures (a-level biology), protein structures: primary and secondary structures (a-level biology), protein formation (a-level biology), proteins and amino acids: an introduction (a-level biology), phospholipid bilayer (a-level biology), cie 3 enzymes, enzymes: inhibitors (a-level biology), enzymes: rates of reaction (a-level biology), enzymes: intracellular and extracellular forms (a-level biology), enzymes: mechanism of action (a-level biology), enzymes: key concepts (a-level biology), enzymes: introduction (a-level biology), cie 4 cell membranes and transport, transport across membranes: active transport (a-level biology), transport across membranes: osmosis (a-level biology), transport across membranes: diffusion (a-level biology), signalling across cell membranes (a-level biology), function of cell membrane (a-level biology), factors affecting cell membrane structure (a-level biology), structure of cell membranes (a-level biology), cie 5 the mitotic cell cycle, chromosome mutations (a-level biology), cell division: checkpoints and mutations (a-level biology), cell division: phases of mitosis (a-level biology), cell division: the cell cycle (a-level biology), cell division: chromosomes (a-level biology), cie 6 nucleic acids and protein synthesis, transfer rna (a-level biology), transcription (a-level biology), messenger rna (a-level biology), introducing the genetic code (a-level biology), genes and protein synthesis (a-level biology), synthesising proteins from dna (a-level biology), structure of rna (a-level biology), dna replication (a-level biology), dna structure and the double helix (a-level biology), polynucleotides (a-level biology), cie 7 transport in plants, translocation and evidence of the mass flow hypothesis (a-level biology), the phloem (a-level biology), importance of and evidence for transpiration (a-level biology), introduction to transpiration (a-level biology), the pathway and movement of water into the roots and xylem (a-level biology), the xylem (a-level biology), cie 8 transport in mammals, controlling heart rate (a-level biology), structure of the heart (a-level biology), transport of carbon dioxide (a-level biology), transport of oxygen (a-level biology), exchange in capillaries (a-level biology), structure and function of blood vessels (a-level biology), cie 9 gas exchange and smoking, lung disease (a-level biology), pulmonary ventilation rate (a-level biology), ventilation (a-level biology), structure of the lungs (a-level biology), general features of exchange surfaces (a-level biology), understanding surface area to volume ratio (a-level biology), the need for exchange surfaces (a-level biology), edexcel a 1: lifestyle, health and risk, phospholipids – introduction (a-level biology), edexcel a 2: genes and health, features of the genetic code (a-level biology), gas exchange in plants (a-level biology), gas exchange in insects (a-level biology), edexcel a 3: voice of the genome, edexcel a 4: biodiversity and natural resources, edexcel a 5: on the wild side, reducing biomass loss (a-level biology), sources of biomass loss (a-level biology), transfer of biomass (a-level biology), measuring biomass (a-level biology), net primary production (a-level biology), gross primary production (a-level biology), trophic levels (a-level biology), edexcel a 6: immunity, infection & forensics, microbial techniques (a-level biology), the innate immune response (a-level biology), edexcel a 7: run for your life, edexcel a 8: grey matter, inhibitory synapses (a-level biology), synaptic transmission (a-level biology), the structure of the synapse (a-level biology), factors affecting the speed of transmission (a-level biology), myelination (a-level biology), the refractory period (a-level biology), all or nothing principle (a-level biology), edexcel b 1: biological molecules, inorganic ions (a-level biology), edexcel b 10: ecosystems, nitrogen cycle: nitrification and denitrification (a-level biology), the phosphorus cycle (a-level biology), nitrogen cycle: fixation and ammonification (a-level biology), introduction to nutrient cycles (a-level biology), edexcel b 2: cells, viruses, reproduction, edexcel b 3: classification & biodiversity, edexcel b 4: exchange and transport, edexcel b 5: energy for biological processes, edexcel b 6: microbiology and pathogens, edexcel b 7: modern genetics, edexcel b 8: origins of genetic variation, edexcel b 9: control systems, ocr 2.1.1 cell structure, structure of prokaryotic cells (a-level biology), eukaryotic cells: comparing plant and animal cells (a-level biology), eukaryotic cells: plant cell organelles (a-level biology), eukaryotic cells: the endoplasmic reticulum (a-level biology), eukaryotic cells: the golgi apparatus and lysosomes (a-level biology), ocr 2.1.2 biological molecules, introduction to eukaryotic cells and organelles (a-level biology), ocr 2.1.3 nucleotides and nucleic acids, ocr 2.1.4 enzymes, ocr 2.1.5 biological membranes, ocr 2.1.6 cell division, diversity & organisation, ocr 3.1.1 exchange surfaces, ocr 3.1.2 transport in animals, ocr 3.1.3 transport in plants, examples of xerophytes (a-level biology), introduction to xerophytes (a-level biology), ocr 4.1.1 communicable diseases, structure of viruses (a-level biology), ocr 4.2.1 biodiversity, ocr 4.2.2 classification and evolution, ocr 5.1.1 communication and homeostasis, the resting potential (a-level biology), ocr 5.1.2 excretion, ocr 5.1.3 neuronal communication, hyperpolarisation and transmission of the action potential (a-level biology), depolarisation and repolarisation in the action potential (a-level biology), ocr 5.1.4 hormonal communication, ocr 5.1.5 plant and animal responses, ocr 5.2.1 photosynthesis, ocr 5.2.2 respiration, ocr 6.1.1 cellular control, ocr 6.1.2 patterns of inheritance, ocr 6.1.3 manipulating genomes, ocr 6.2.1 cloning and biotechnology, ocr 6.3.1 ecosystems, ocr 6.3.2 populations and sustainability, related links.

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OpenStax Biology

Supplement for general biology i.

12. Membranes & Transport

MEMBRANES & TRANSPORT

Learning Objectives

• Explain how a cell membrane regulates interactions within environments.
• Differentiate between diffusion, osmosis, and dialysis.
• Describe the solute/solvent movements into and out of a cell under hypertonic, hypotonic, and iso-tonic conditions.
• Explain and give examples of generalized endocytosis and exocytosis
• Understand the importance of selective permeability in biological systems.
• Differentiate among diffusion, facilitated diffusion and active transport.
• Describe the operation of the Na + – K + pump
• Compare and contrast cilia and flagella in terms of their structure and function.
• List and describe the various junctions, linkages, and connections that occur between cells.
• Understand the biochemistry of phospholipids and how they are organized into membranes.

Understanding Membranes

The fluid mosaic of phospholipids, proteins and cholesterol that create the selective barrier between the interior and the exterior of the cell.

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