LEARNING Cellular Physiology

2 Dr. Murtadha Al-Shareifi e-Library Plasma Membrane, Membrane Transport, and Resting Membrane Potential AC TIVE L EARNING Cellular Physiology Upon mastering the material in this chapter you should be able to: • Understand how proteins and lipids are assembled to form a selectively permeable barrier known as the plasma membrane. • Explain how the plasma membrane maintains an internal environment that differs significantly from the extracellular fluid. • Understand how voltage-gated channels and ligandgated channels are opened. • Explain how carrier-mediated transport systems differ from channels. • Understand the importance of adenosine triphosphate–binding cassette transporters to T he intracellular fluid of living cells, the cytosol, has a composition very different from that of the extracellular fluid (ECF). For example, the concentrations of potassium and phosphate ions are higher inside cells than outside, whereas sodium, calcium, and chloride ion concentrations are much lower inside cells than outside. These differences are necessary for the proper function of many intracellular enzymes; for instance, the synthesis of proteins by the ribosomes requires a relatively high potassium concentration. The plasma membrane of the cell creates and maintains these differences by establishing a permeability barrier around the cytosol. The ions and cell proteins needed for normal cell function are prevented from leaking out; those not needed by the cell are unable to enter the cell freely. The plasma membrane also keeps metabolic intermediates near where they will be needed for further synthesis or processing and retains metabolically expensive proteins inside the cell. The plasma membrane is necessarily selectively permeable. Cells must receive nutrients to function, and they must dispose of metabolic waste products. To function in coordination with the rest of the organism, cells receive and send information in the form of chemical signals, such as 24 • • • • • O BJ ECTIVES lipid transport and development of multidrug resistance. Explain the difference between primary and secondary active transport. Explain how epithelial cells are organized to produce directional movement of solutes and water. Explain how many cells can regulate their volume when exposed to osmotic stress. Understand why the Goldman equation gives the value of the membrane potential. Understand why the resting membrane potential in most cells is close to the Nernst potential for K+. hormones and neurotransmitters. The plasma membrane has mechanisms that allow specific molecules to cross the barrier around the cell. A selective barrier surrounds not only the cell but also every intracellular organelle that requires an internal milieu different from that of the cytosol. The cell nucleus, mitochondria, endoplasmic reticulum, Golgi apparatus, and lysosomes are delimited by membranes similar in composition to the plasma membrane. This chapter describes the specific types of membrane transport mechanisms for ions and other solutes, their relative contributions to the resting membrane electrical potential, and how their activities are coordinated to achieve directional transport from one side of a cell layer to the other. ● PLASMA MEMBRANE STRUCTURE The first theory of membrane structure proposed that cells are surrounded by a double layer of lipid molecules, a lipid bilayer. This theory was based on the known tendency of lipid molecules to form lipid bilayers with low permeability to water-soluble molecules. However, the lipid bilayer theory did not explain the selective movement of certain Dr. Murtadha Al-Shareifi e-Library ● 25 Chapter 2 / Plasma Membrane, Membrane Transport, and Resting Membrane Potential Extracellular fluid Glycoprotein Integral proteins Sphingolipid Hormone receptor Glycolipid TK TK Phospholipid Cholesterol Channel Peripheral protein Lipid raft Cytoplasm water-soluble compounds, such as glucose and amino acids, across the plasma membrane. In 1972, Singer and Nicolson proposed the fluid mosaic model of the plasma membrane, which described the organization and interaction of proteins with the lipid bilayer (Fig. 2.1). With minor modifications, this model is still accepted as the correct picture of the structure of the plasma membrane. Plasma membrane consists of different types of membrane lipids with different functions. Lipids found in cell membranes can be classified into two broad groups: those that contain fatty acids as part of the lipid molecule and those that do not. Phospholipids are an example of the first group, and cholesterol is the most important example of the second group. Phospholipids The fatty acids present in phospholipids are molecules with a long hydrocarbon chain and a carboxyl terminal group. The hydrocarbon chain can be saturated (no double bonds between the carbon atoms) or unsaturated (one or more double bonds present). The long hydrocarbon chain tends to avoid contact with water and is described as hydrophobic. The carboxyl group at the other end is compatible with water and is termed hydrophilic. Fatty acids are said to be amphipathic because both hydrophobic and hydrophilic regions are present in the same molecule. Phospholipids are the most abundant complex lipids found in cell membranes. They are amphipathic molecules formed by two fatty acids (normally, one saturated and one unsaturated) and one phosphoric acid group substituted on the backbone of a glycerol or sphingosine molecule. This arrangement produces a hydrophobic area formed by the two fatty acids and a polar hydrophilic head. When phospholipids are arranged in a bilayer, the polar heads are on the outside and the hydrophobic fatty acids on the inside (see Fig. 2.1). It is difficult for water-soluble molecules and ions to pass directly through the hydrophobic interior of the lipid bilayer. ● Figure 2.1 The fluid mosaic model of the plasma membrane. Lipids are arranged in a bilayer. Cholesterol provides rigidity to the bilayer. Integral proteins are embedded in the bilayer and often span it. Some membranespanning proteins form pores and channels; others are receptors. Peripheral proteins do not penetrate the bilayer. Lipid rafts form stable microdomains composed of sphingolipids and cholesterol. The phospholipids, with a backbone of sphingosine (a long amino alcohol), are usually called sphingolipids and are present in all plasma membranes in small amounts. They are especially abundant in brain and nerve cells. Ceramide is a lipid second messenger that is generated from the sphingolipid sphingomyelin (see Chapter 1, “Homeostasis and Cellular Signaling,” for a discussion of lipid secondmessenger signaling). Glycolipids are lipid molecules that contain sugars and sugar derivatives (instead of phosphoric acid) in the polar head. They are located mainly in the outer half of the lipid bilayer, with the sugar molecules facing the ECF. Proteins can associate with the plasma membrane by linkage to the extracellular sugar moiety of glycolipids. Cholesterol Cholesterol is an important component of mammalian plasma membranes. The proportion of cholesterol in plasma membranes varies from 10% to 50% of total lipids. Cholesterol has a rigid structure that stabilizes the cell membrane and reduces the natural mobility of the complex lipids in the plane of the membrane. Increasing amounts of cholesterol make it more difficult for lipids and proteins to move in the membrane. Some cell functions, such as the response of immune system cells to the presence of an antigen, depend on the ability of membrane proteins to move in the plane of the membrane to bind the antigen. A decrease in membrane fluidity resulting from an increase in cholesterol will impair these functions. Lipid microdomains Aggregates of sphingolipids and cholesterol can form stable microdomains termed lipid rafts that diffuse laterally in the phospholipid bilayer. The protein caveolin is present in a subset of lipid rafts (termed caveolae), causing the raft to form a cavelike structure. It is believed that one function of both noncaveolar and caveolar lipid rafts is to facilitate interactions between specific proteins by selectively including (or excluding) these proteins from the raft microdomain. ● Dr. Murtadha Al-Shareifi e-Library 26 Part I / Cellular Physiology Clinical Focus / 2.1 Hereditary Spherocytosis Red blood cells (erythrocytes) contain hemoglobin, which binds oxygen for transport from lungs to other organs and tissues. A normal erythrocyte has a marked biconcave structure, which permits the cell to deform for easier passage through narrow capillaries, especially in the spleen. The unusual shape is maintained by the cytoskeleton, formed by both integral and peripheral plasma membrane proteins. A key player is the long filamentous protein known as spectrin, a peripheral protein that forms a meshwork on the cytoplasmic surface. Linkages between spectrin and several integral proteins serve to anchor the plasma membrane to the cytoskeleton. Hereditary spherocytosis (HS) is a genetic disease that affects proteins in the erythrocyte membrane, and the result is a defective cytoskeleton. The incidence of HS is 1 in 5,000. The most common defect is deficiency of spectrin, and the result is that regions of the membrane break off because they are no longer anchored to the cytoskeleton. The remaining membrane reseals spontaneously, but after several “sheddings,” the cell eventually becomes small and spherical. The presence of these microspherocytes is the hallmark of HS disease, which can range from asymptomatic to severe hemolytic anemia. Hemolysis (cell bursting) is present because the spherocytes are fragile to osmotic stress. Any entry of water will increase intracellular volume, and the cell membrane will break and release the hemoglobin. In contrast, normal biconcave eryth- For example, lipid rafts can mediate the assembly of membrane receptors and intracellular signaling proteins as well as the sorting of plasma membrane proteins for internalization. Proteins are integrally and peripherally associated with the plasma membrane. Proteins are the second major component of the plasma membrane, present in about equal proportion by weight with the lipids. Two different types of proteins are associated with the plasma membrane. Integral proteins (or intrinsic proteins) are embedded in the lipid bilayer; many span it completely, being accessible from the inside and outside of the membrane. The polypeptide chain of these proteins may cross the lipid bilayer once or may make multiple passes across it. The membrane-spanning segments usually contain amino acids with nonpolar side chains and are arranged in an ordered a-helical conformation. Peripheral proteins (or extrinsic proteins) do not penetrate the lipid bilayer. They are in contact with the outer side of only one of the lipid layers— either the layer facing the cytoplasm or the layer facing the ECF (see Fig. 2.1). Many membrane proteins have carbohydrate molecules, in the form of specific sugars, attached to the parts of the proteins that are exposed to the ECF. These molecules are known as glycoproteins. Some of the integral membrane proteins can move in the plane of the membrane, like small boats floating in the “sea” formed by the lipid bilayer. Other membrane proteins are anchored to the cytoskeleton inside the cell or to proteins of the extracellular matrix. rocytes can swell without bursting to accommodate osmotic water entry. Hence the lifespan of a spherocyte is considerably shorter than the 90- to 120-day lifespan of an erythrocyte. The shorter life is often compensated by accelerated production of new red blood cells (erythropoiesis) in an otherwise healthy individual. However, the anemia may be severe during an illness involving fever because erythropoiesis is slowed by fever. The anemia is usually the reason that HS patients complain of tiredness or loss of stamina and shortness of breath during exercise. Splenomegaly (enlarged spleen) is present in 75% of HS patients. The spherocytes cannot deform their shape, and they are trapped and destroyed (by hemolysis) in the spleen, which may be the reason it increases in size. Removal of spherocytes by the spleen is a major cause of the anemia and the shortened lifespan. Surgery to remove the spleen is often recommended if the anemia is severe and will improve spherocyte survival significantly. This enables most patients with HS to maintain a normal hemoglobin level. Treatment of patients with HS has produced an appreciation of the role of the spleen in maintaining the integrity of the red blood cell population. Laboratory investigation of HS has helped the understanding of the structure and function of the membrane cytoskeleton, which is present in almost all types of cells, and the point mutations that produce defective spectrin have been identified. The proteins in the plasma membrane play a variety of roles. Many peripheral membrane proteins are enzymes, and many membrane-spanning integral proteins are carriers or channels for the movement of water-soluble molecules and ions into and out of the cell. Another important role of membrane proteins is structural; for example, certain membrane proteins in the erythrocyte help maintain the biconcave shape of the cell. Finally, some membrane proteins serve as highly specific receptors on the outside of the cell membrane to which extracellular molecules, such as hormones, can bind. If the receptor is a membrane-spanning protein, it provides a mechanism for converting an extracellular signal into an intracellular response. ● SOLUTE TRANSPORT MECHANISMS All cells must import oxygen, sugars, amino acids, and small ions and export carbon dioxide, metabolic wastes, and secretions. At the same time, specialized cells require mechanisms to transport molecules such as enzymes, hormones, and neurotransmitters. The movement of large molecules is carried out by endocytosis and exocytosis, that is, the transfer of substances into or out of the cell, respectively, by vesicle formation and vesicle fusion with the plasma membrane. Cells also have mechanisms for the rapid movement of ions and solute molecules across the plasma membrane. These mechanisms are of two general types: passive transport, which requires no direct expenditure of metabolic energy, Dr. Murtadha Al-Shareifi e-Library ● 27 Chapter 2 / Plasma Membrane, Membrane Transport, and Resting Membrane Potential and active transport, which uses metabolic energy to move solutes across the plasma membrane. Import of extracellular materials occurs through phagocytosis and endocytosis. Phagocytosis is the ingestion of large particles or microorganisms, usually occurring only in specialized cells such as macrophages (Fig. 2.2). An important function of macrophages is to remove invading bacteria from the body. The phagocytic vesicle (1–2 mm in diameter) is almost as large as the phagocytic cell itself. Phagocytosis requires a specific stimulus. It occurs only after the extracellular particle has bound to the extracellular surface. The particle is then enveloped by expansion of the cell membrane around it. Endocytosis is a general term for the process in which a region of the plasma membrane is pinched off to form an endocytic vesicle inside the cell. During vesicle formation, some fluid, dissolved solutes, and particulate material from the extracellular medium are trapped inside the vesicle and internalized by the cell. Endocytosis produces much smaller endocytic vesicles (0.1–0.2 mm in diameter) than phagocytosis. It occurs in almost all cells and is termed a constitutive process, because it occurs continually and specific stimuli are not required. In further contrast to phagocytosis, endocytosis originates with the formation of depressions in the cell membrane. The depressions pinch off within a few minutes after forming and give rise to endocytic vesicles inside the cell. Two types of endocytosis can be distinguished (see Fig. 2.2). Fluid-phase endocytosis is the nonspecific uptake of the ECF and all its dissolved solutes. The material is trapped inside the endocytic vesicle as it is pinched off inside the cell. The amount of extracellular material internalized by this process is directly proportional to its concentration in the extracellular solution. Receptor-mediated endocytosis is a more efficient process, which uses receptors on the cell surface to bind specific molecules. These receptors accumulate at specific depressions known as coated pits, so named because the cytosolic surface of the membrane at this site is covered with a coat of several proteins. The coated pits pinch off continually to form endocytic vesicles, providing the cell with a mechanism for rapid internalization of a large amount of a specific molecule without the need to endocytose large volumes of ECF. The receptors also aid the cellular uptake of molecules present at low concentrations outside the cell. Receptor-mediated endocytosis is the mechanism by which cells take up a variety of important molecules, including hormones, growth factors, and serum transport proteins such as transferrin (an iron carrier). Foreign substances, such as diphtheria toxin and certain viruses, also enter cells by this pathway. Export of macromolecules occurs through exocytosis. Many cells synthesize important macromolecules that are destined for exocytosis or export from the cell. These molecules are synthesized in the endoplasmic reticulum, modified in the Golgi apparatus, and packed inside transport vesicles. The vesicles move to the cell surface, fuse with the cell membrane, and release their contents outside the cell (see Fig. 2.2). There are two exocytic pathways—constitutive and regulated. Some proteins are secreted continuously by the cells Endocytosis Phagocytosis Extracellular fluid Exocytosis Fluid-phase endocytosis Receptor-mediated endocytosis Ligand Receptor Plasma membrane Coated pit Cytoplasm ● Figure 2.2 The transport of macromolecules across the plasma membrane by the formation of vesicles. Particulate matter in the extracellular fluid (ECF) is engulfed and internalized by phagocytosis. During fluid-phase endocytosis, ECF and dissolved macromolecules enter the cell in endocytic vesicles that pinch off at depressions in the plasma membrane. Receptor-mediated endocytosis uses membrane receptors at coated pits to bind and internalize specific solutes (ligands). Exocytosis is the release of macromolecules destined for export from the cell. These are packed inside secretory vesicles that fuse with the plasma membrane and release their contents outside the cell. ● Dr. Murtadha Al-Shareifi e-Library 28 Part I / Cellular Physiology that make them. Secretion of mucus by goblet cells in the small intestine is a specific example. In this case, exocytosis follows the constitutive pathway, which is present in all cells. In other cells, macromolecules are stored inside the cell in secretory vesicles. These vesicles fuse with the cell membrane and release their contents only when a specific extracellular stimulus arrives at the cell membrane. This pathway, known as the regulated pathway, is responsible for the rapid “ondemand” secretion of many specific hormones, neurotransmitters, and digestive enzymes. Diffusion High Membrane Low Uncharged solutes cross the plasma membrane by passive diffusion. Any solute will tend to uniformly occupy the entire space available to it. This movement, known as diffusion, is a result of the spontaneous Brownian (random) movement that all molecules experience. A drop of ink placed in a glass of water will diffuse and slowly color all the water. The net result of diffusion is the movement of substances from regions of high concentration to regions of low concentration. Diffusion is an effective way for substances to move short distances. The speed with which the diffusion of a solute in water occurs depends on the difference of concentration, the size of the molecules, and the possible interactions of the diffusible substance with water. These different factors appear in the Fick law, which describes the diffusion of any solute in water. In its simplest formulation, the Fick law (also known as Fick's law) can be written as J = DA(C1 - C2)/DX (1) where J is the flow of solute from region 1 to region 2 in the solution; D is the diffusion coefficient of the solute, which is determined by factors such as solute molecular size and interactions of the solute with water; A is the cross-sectional area through which the flow of solute is measured; C is the concentration of the solute at regions 1 and 2; and DX is the distance between regions 1 and 2. Sometimes, J is expressed in units of amount of substance per unit area per unit time, for example, mol/cm2/h, and is also referred to as the solute flux. The principal force driving the passive diffusion of an uncharged solute across the plasma membrane is the difference of concentration between the inside and the outside of the cell (Fig. 2.3). In the case of an electrically charged solute, such as an ion, diffusion is also driven by the membrane potential, which is the electrical gradient across the membrane. Movement of charged solutes and the membrane potential will be discussed in greater detail later in this chapter. Diffusion across a membrane has no preferential direction; it can occur from the outside of the cell toward the inside or from the inside of the cell toward the outside. For any substance, it is possible to measure the permeability coefficient (P), which gives the speed of the diffusion across a unit area of plasma membrane for a defined driving force. The Fick law for the diffusion of an uncharged solute across a membrane can be written as J = PA(C1 - C2) (2) which is similar to equation 1. P includes the membrane thickness, the diffusion coefficient of the solute within the ● Figure 2.3 The diffusion of gases and lipid-soluble molecules through the lipid bilayer. In this example, the diffusion of a solute across a plasma membrane is driven by the difference in concentration on the two sides of the membrane. The solute molecules move randomly by Brownian movement. Initially, random movement from left to right across the membrane is more frequent than movement in the opposite direction because there are more molecules on the left side. This results in a net movement of solute from left to right across the membrane until the concentration of solute is the same on both sides. At this point, equilibrium (no net movement) is reached because solute movement from left to right is balanced by equal movement from right to left. membrane, and the solubility of the solute in the membrane. Dissolved gases, such as oxygen and carbon dioxide, have high permeability coefficients and diffuse across the plasma membrane rapidly. As a result, gas exchange in the lungs is very effective. Because diffusion across the plasma membrane usually implies that the diffusing solute enters the lipid bilayer to cross it, the solute’s solubility in a lipid solvent (e.g., olive oil or chloroform) compared with its solubility in water is important in determining its permeability coefficient. A substance’s solubility in oil compared with its solubility in water is its partition coefficient. Lipophilic (lipidsoluble) substances, such as gases, steroid hormones, and anesthetic drugs, which mix well with the lipids in the plasma membrane, have high partition coefficients and, as a result, high permeability coefficients; they tend to cross the plasma membrane easily. Hydrophilic (water-soluble) substances, such as ions and sugars, do not interact well with the lipid component of the membrane, have low partition coefficients and low permeability coefficients, and diffuse across the membrane more slowly. Solutes such as oxygen readily diffuse across the lipid part of the plasma membrane by simple diffusion. Thus, the relationship between the rate of movement and the difference in concentration between the two sides of the membrane is linear (Fig. 2.4). The larger the difference in concentration (C1 − C2), the greater the amount of substance crossing the membrane per unit time. Dr. Murtadha Al-Shareifi e-Library ● 29 Chapter 2 / Plasma Membrane, Membrane Transport, and Resting Membrane Potential Carrier-mediated transport 10 Rate of solute entry (mmol/min) Rate of solute entry (mmol/min) Simple diffusion 5 1 2 3 Solute concentration (mmol/L) outside cell ● Figure 2.4 A graph of solute transport across a plasma membrane by simple diffusion. The rate of solute entry increases linearly with extracellular concentration of the solute. Assuming no change in intracellular concentration, increasing the extracellular concentration increases the gradient that drives solute entry. Integral membrane proteins facilitate diffusion of solutes across the plasma membrane. For many solutes of physiologic importance, such as ions, sugars, and amino acids, the rate of transport across the plasma membrane is much faster than expected for simple diffusion through a lipid bilayer. Furthermore, the relationship between transport rate and concentration difference of these hydrophilic substances follows a curve that reaches a plateau (Fig. 2.5). Membrane transport with these characteristics is often called facilitated diffusion or carriermediated diffusion, because an integral membrane protein facilitates (or assists) the movement of a solute through the membrane. Integral membrane proteins can form pores, channels, or carriers, each of which facilitates the transport of specific molecules across the membrane. There are a limited number of pores, channels, and carriers in any cell membrane; thus, increasing the concentration of the solute initially uses the existing “spare” pores, channels, or carriers to transport the solute at a higher rate than by simple diffusion. As the concentration of the solute increases further and more solute molecules associate with the pore, channel, or carrier, the transport system eventually reaches saturation, when all the pores, channels, and carriers are involved in translocating molecules of solute. At this point, additional increases in solute concentration do not increase the rate of solute transport (see Fig. 2.5). The types of integral membrane protein transport mechanisms considered here can transport a solute along its concentration gradient only, as in simple diffusion. Net movement stops when the concentration of the solute has the same value on both sides of the membrane. At this point, with reference to equation 2, C1 = C2 and the value of J is 0. The transport systems function until the solute concentrations 10 Vmax 5 1 2 Solute concentration (mmol/L) outside cell 3 ● Figure 2.5 A graph of solute transport across a plasma membrane by facilitated diffusion. The rate of transport is much faster than that of simple diffusion (see Fig. 2.4) and increases linearly as the extracellular solute concentration increases. The increase in transport is limited, however, by the availability of channels and carriers. Once all are occupied by solute, further increases in extracellular concentration have no effect on the rate of transport. A maximum rate of transport (Vmax) is achieved that cannot be exceeded. have equilibrated. However, equilibrium is attained much faster than with simple diffusion. Membrane pores A pore provides a conduit through the lipid bilayer that is always open to both sides of the membrane. Aquaporins in the plasma membranes of specific kidney and gastrointestinal tract cells permit the rapid movement of water. Within the nuclear pore complex, which regulates movement of molecules into and out of the nucleus, is an aqueous pore that only allows the passive movement of molecules smaller than 45 kDa and excludes molecules larger than 62 kDa. The mitochondrial permeability transition pore and mitochondrial voltage-dependent anion channel (VDAC), which cross the inner and outer mitochondrial membranes, promote mitochondrial failure when formed, resulting in the generation of reactive oxygen species and cell death. Gated channels Small ions, such as Na+, K+, Cl−, and Ca2+, cross the plasma membrane faster than would be expected based on their partition coefficients in the lipid bilayer. The electrical charge of an ion makes it difficult for the ion to move across the lipid bilayer. The rapid movement of ions across the membrane, however, is an aspect of many cell functions. The excitation of nerves, the contraction of muscle, the beating of the heart, and many other physiologic events are possible because of the ability of small ions to enter or leave the cell rapidly. This movement occurs through selective ion channels. Ion channels are composed of several polypeptide subunits that span the plasma membrane and contain a gate ● Dr. Murtadha Al-Shareifi e-Library 30 Part I / Cellular Physiology Number of channels open Lipid bilayer 0 Selectivity filter 1 2 3 pA 3 100 msec Figure 2.7 A patch clamp recording from a frog muscle fiber. Ions flow through the channel when it opens, generating a current. The current in this experiment is about 3 pA and is detected as a downward deflection in the recording. When more than one channel open, the current and the downward deflection increase in direct proportion to the number of open channels. This record shows that up to three channels are open at any instant. ● Integral protein Gate Aqueous pore ● Figure 2.6 An ion channel. Ion channels are formed between the polypeptide subunits of integral proteins that span the plasma membrane, providing an aqueous pore through which ions can cross the membrane. Different types of gating mechanisms are used to open and close channels. Ion channels are often selective for a specific ion. that determines if the channel is open or closed. Specific stimuli cause a conformational change in the protein subunits to open the gate, creating an aqueous channel through which the ions can move (Fig. 2.6). In this way, ions do not have to enter the lipid bilayer to cross the membrane; they are always in an aqueous medium. When the channels are open, the ions diffuse rapidly from one side of the membrane to the other down the concentration gradient. Specific interactions between the ions and the sides of the channel produce an extremely rapid rate of ion movement; in fact, ion channels permit a much faster rate of solute transport (about 108 ions/s) than the carrier-mediated systems discussed below. Ion channels have a selectivity filter, which regulates the transport of certain classes of ions such as anions or cations or specific ions such as Na+, K+, Ca2+, and Cl− (see Fig 2.6). The amino acid composition of the channel protein does not appear to confer the ion selectivity of the channel. The patch clamp technique has revealed a great deal of information about the characteristic behavior of channels for different ions. The small electrical current caused by ion movement when a channel is open can be detected with this technique, which is so sensitive that the opening and closing of a single ion channel can be observed (Fig. 2.7). In general, ion channels exist either fully open or completely closed, and they open and close very rapidly. The frequency with which a channel opens is variable, and the time the channel remains open (usually a few milliseconds) is also variable. The overall rate of ion transport across a membrane can be controlled by changing the frequency of a channel opening or by changing the time a channel remains open. Most ion channels usually open in response to a specific stimulus. Ion channels can be classified according to their gating mechanisms, the signals that make them open or close. There are voltage-gated channels and ligand-gated channels. Some ion channels are more like membrane pores in that they are always open; these ion transport proteins are referred to as nongated channels (see Chapter 3, “Action Potential, Synaptic Transmission, and Maintenance of Nerve Function”). Voltage-gated ion channels open when the membrane potential changes beyond a certain threshold value. Channels of this type are involved in conducting the excitation signal along nerve axons and include sodium and potassium channels (see Chapter 3, “Action Potential, Synaptic Transmission, and Maintenance of Nerve Function”). Voltage-gated ion channels are found in many cell types. It is thought that some charged amino acids located in a membrane-spanning a-helical segment of the channel protein are sensitive to the transmembrane potential. Changes in the membrane potential cause these amino acids to move and induce a conformational change of the protein that opens the way for the ions. Ligand-gated ion channels cannot open unless they first bind to a specific agonist. The opening of the gate is produced by a conformational change in the protein induced by the ligand binding. The ligand can be a neurotransmitter arriving from the extracellular medium. It can also be an intracellular second messenger, produced in response to some cell activity or hormone that reaches the ion channel from the inside of the cell. The nicotinic acetylcholine receptor channel found in the postsynaptic neuromuscular junction (see Chapter 3, “Action Potential, Synaptic Transmission, and Maintenance of Nerve Function” and Chapter 9, “Blood Components”) is a ligand-gated ion channel that is opened by an extracellular ligand (acetylcholine). Examples of ion channels gated by intracellular messengers also abound in nature. This type of gating mechanism allows the channel to open or close in response to events that occur at other locations in the cell. For example, a sodium channel gated by intracellular cyclic guanosine monophosphate (cGMP) is involved in the process of vision (see Chapter 4, “Sensory Physiology”). This channel is located in the rod cells of the retina and opens in the presence of cGMP. The generalized structure of one subunit of an ion channel gated by cyclic nucleotides is shown in Figure 2.8. There are six membrane-spanning Dr. Murtadha Al-Shareifi e-Library ● 31 Chapter 2 / Plasma Membrane, Membrane Transport, and Resting Membrane Potential (Fig. 2.9). During carrier-mediated transport, binding of the solute to one side of the carrier induces a conformational change in the protein, which closes one gate and opens the second gate, allowing the solute to pass through the membrane. As with pores and channels, carriers function until the solute concentrations have equilibrated. Carrier-mediated transport systems have several characteristics: Out 1 2 3 6 5 4 In Binding site H2N COOH A • They eventually reach saturation at high substrate concentration (see Fig 2.5). • They have structural specificity, meaning each carrier system recognizes and binds specific chemical structures (a carrier for d-glucose will not bind or transport l-glucose). IV I III II B ● Figure 2.8 Structure of a cyclic nucleotide-gated ion channel. (A) The secondary structure of a single subunit has six membrane-spanning regions and a binding site for cyclic nucleotides on the cytosolic side of the membrane. (B) Four identical subunits (I–IV) assemble together to form a functional channel that provides a hydrophilic pathway across the plasma membrane. regions, and a cyclic nucleotide-binding site is exposed to the cytosol. The functional protein is a tetramer of four identical subunits. Other cell membranes have potassium channels that open when the intracellular concentration of calcium ions increases. Several known channels respond to inositol 1,4,5-trisphosphate, the activated part of G proteins, or adenosine triphosphate (ATP). The epithelial chloride channel that is mutated in cystic fibrosis is normally gated by ATP. Carrier-mediated transport moves a range of ions and organic solutes passively across membranes. In contrast to pores and ion channels, integral membrane proteins that form carriers provide a conduit through the membrane that is never open to both sides of the membrane at the same time. This is due to the presence of two gates A • They allow the transport of polar (hydrophilic) molecules at rates much higher than that expected from the partition coefficient of these molecules. B • They show competitive inhibition by molecules with similar chemical structure. For example, carrier-mediated transport of d-glucose occurs at a slower rate when molecules of d-galactose are also present. This is because galactose, structurally similar to glucose, competes with glucose for the available glucose carrier proteins. A specific example of carrier-mediated transport is the movement of glucose from the blood to the interior of cells. Most mammalian cells use blood glucose as a major source of cellular energy, and glucose is transported into cells down its concentration gradient. The transport process in many cells, such as erythrocytes and the cells of fat, liver, and muscle tissues, involves a plasma membrane protein called GLUT1 (glucose transporter-1). The erythrocyte GLUT1 has an affinity for d-glucose that is about 2,000fold greater than the affinity for l-glucose. It is an integral membrane protein that contains 12 membrane-spanning a-helical segments. Carrier-mediated transport, like simple diffusion, does not have a directional preference. It functions equally well bringing its specific solutes into or out of the cell, depending on the concentration gradient. Net movement by carriermediated transport ceases once the concentrations inside and outside the cell become equal. ● Figure 2.9 The role of a carrier protein in facilitated diffusion of solute molecules across a plasma membrane. In this example, solute transport into the cell is driven by the high solute concentration outside compared with inside. (A) Binding of extracellular solute to the carrier, a membrane-spanning integral protein, may trigger a change in protein conformation that exposes the bound solute to the interior of the cell. (B) Bound solute readily dissociates from the carrier because of the low intracellular concentration of solute. The release of solute may allow the carrier to revert to its original conformation (A) to begin the cycle again. ● Dr. Murtadha Al-Shareifi e-Library 32 Part I / Cellular Physiology The anion exchange protein (AE1), the predominant integral protein in the mammalian erythrocyte membrane, provides a good example of the reversibility of transporter action. AE1 is folded into at least 12 transmembrane a helices and normally permits the one-for-one exchange of Cl− and HCO3− ions across the plasma membrane. The direction of ion movement is dependent only on the concentration gradients of the transported ions. AE1 has an important role in transporting CO2 from the tissues to the lungs. The erythrocytes in systemic capillaries pick up CO2 from tissues and convert it to HCO3−, which exits the cells via AE1. When the erythrocytes enter pulmonary capillaries, the AE1 allows plasma HCO3− to enter erythrocytes, where it is converted back to CO2 for expiration by the lungs (see Chapter 21, “Control of Ventilation”). Active transport systems move solutes against gradients. All the passive transport mechanisms tend to bring the cell into equilibrium with the ECF. Cells must oppose these equilibrating systems and preserve intracellular concentrations of solutes, in particular ions that are compatible with life. Primary active transport Integral membrane proteins that directly use metabolic energy to transport ions against a gradient of concentration or electrical potential are known as ion pumps. The direct use of metabolic energy to carry out transport defines a primary active transport mechanism. The source of metabolic energy is ATP synthesized by mitochondria, and the different ion pumps hydrolyze ATP to adenosine diphosphate (ADP) and use the energy stored in the third phosphate bond to carry out transport. Because of this ability to hydrolyze ATP, ion pumps also are called ATPases. The most abundant ion pump in higher organisms is the sodium–potassium pump or Na+/K+-ATPase. It is found in the plasma membrane of practically every eukaryotic cell and is responsible for maintaining the low sodium and high potassium concentrations in the cytoplasm by transporting sodium out of the cell and potassium ions in. The sodium–potassium pump is an integral membrane protein consisting of two subunits. The a subunit has 10 transmembrane segments and is the catalytic subunit that mediates active transport. The smaller b subunit has one transmembrane segment and is essential for the proper assembly and membrane targeting of the pump. The Na+/K+-ATPase is known as a P-type ATPase because the protein is phosphorylated during the transport cycle (Fig. 2.10). The pump counterbalances the tendency of sodium ions to enter the cell passively and the tendency of potassium ions to leave passively. It maintains a high intracellular potassium concentration, which is necessary for protein synthesis. It also plays a role in the resting membrane potential by maintaining ion gradients. The sodium–potassium pump can be inhibited either by metabolic poisons that stop the synthesis and supply of ATP or by specific pump blockers, α subunit β subunit K+ K+ Out Lipid bilayer ATP In 1 ● Figure 2.10 Function of the sodium–potassium pump. The pump is composed of two large a subunits that hydrolyze adenosine triphosphate (ATP) and transport the ions. The two smaller b subunits are molecular chaperones that facilitate the correct integration of the a subunits into the membrane. In step 1, three intracellular Na+ bind to the a subunit, and ATP is hydrolyzed. Phosphorylation (Pi) of the a subunit results in a conformational change, exposing the Na+ to the extracellular space (step 2). In step 3, the Na+ diffuses away and two K+ bind, resulting in dephosphorylation of the a subunit. Dephosphorylation returns the a subunit to an intracellular conformation. The K+ diffuses away, and ATP is rebound to start the cycle over again (step 6). ADP, adenosine diphosphate. ADP Na+ Na+ Na+ 5 Na + Na + Na + K+ Pi K+ 2 4 Na+ Na+ Na+ K+ K+ 3 K+ Pi Pi Dr. Murtadha Al-Shareifi e-Library ● 33 Chapter 2 / Plasma Membrane, Membrane Transport, and Resting Membrane Potential From Bench to Bedside / 2.1 Multidrug Resistance and Cancer Some studies indicate that up to 40% of human cancers develop resistance to multiple anticancer agents, and it is a major problem when treating patients with malignant disease. A number of cellular mechanisms are known to lead to drug resistance, but it is now recognized that the most common mechanism is the efflux of chemotherapeutic drugs from the tumor cell by adenosine triphosphate–binding cassette (ABC) family drug transporters. The reduced accumulation of drugs in tumor cells was originally thought to result from overexpression of a 170-kDa protein termed P-glycoprotein (now known as MDR1 or ABCB1) in the plasma membrane, resulting in expulsion of cytotoxic drugs, thereby reducing the intracellular concentration to below the threshold for cell killing. Since the discovery of P-glycoprotein, a number of additional drug resistance transporters have been identified. These include the 190-kDa multidrug resistance (MDR)-associated proteins (MRP1), or ABCC1, and MRP2, or ABCC2, and the breast cancer–resistance protein (BCRP), or ABCG2. Unlike the classical mammalian transport systems, which can be very selective in their substrate preferences, the multidrug transporters are highly promiscuous and can recognize and transport a wide range of substrates. P-glycoprotein and BCRP preferentially exclude large positively charged molecules, whereas the MRP family can exclude both uncharged molecules and water-soluble anionic compounds. Thus, the most logical approach to reversing MDR is to find compounds that can block ABC transporter activity. First-generation modulators of MDR were the calciumchannel blocker, verapamil, and the immunosuppressant, cyclosporine A. Although these compounds were effective in such as digoxin, a cardiac glycoside used to treat a variety of cardiac conditions. As the Na+/K+-ATPase specifically moves sodium and potassium ions against their concentration or electrical potential, a number of other pumps move specific substrates across membranes utilizing the energy released by ATP hydrolysis. • Calcium pumps are P-type ATPases located in the plasma membrane and the membrane of intracellular organelles. Plasma membrane Ca2+ ATPases pump calcium out of the cell. Calcium pumps in the membrane of the endoplasmic reticulum and in the sarcoplasmic reticulum membrane within muscle cells (termed SERCAs for sarcoplasmic and endoplasmic reticulum calcium ATPases) pump calcium into the lumen of these organelles. The organelles store calcium and, as a result, help maintain a low cytosolic concentration of this ion (see Chapter 1, “Homeostasis and Cellular Signaling”). • The H+/K+-ATPase is another example of a P-type ATPase present in the luminal membrane of the parietal cells in the oxyntic (acid-secreting) glands of the stomach. By pumping protons into the lumen of the stomach in exchange for potassium ions, this pump maintains the low pH in the stomach that is necessary for proper digestion. cell culture and animal models, side effects due to the high dose of drug required to reverse chemotherapeutic drug resistance halted clinical trials. A number of derivatives (structurally similar molecules) for verapamil and cyclosporine A were subsequently tested. Unfortunately, these compounds interfered with drug metabolism and elimination, resulting in overexposure to cytotoxic chemotherapeutic agents. Using structure–activity relationships and combinatorial chemistry, a third generation of MDR modulators are being studied that are effective at nanomolar concentrations and do not affect the pharmacokinetics of chemotherapeutic agents. The search for modulators of MDR is also currently focused on naturally occurring compounds, with the rationale that such products would be less toxic. Naturally occurring modulators of MDR include curcumin, coumarin, flavonoids, chokeberry, and mulberry leaves. Curcumin, in particular, has been shown to inhibit all three major ABC transporters, MDR1, MRP, and BCRP. Low oral bioavailability and rapid metabolism have prompted investigation into improved methods of delivery such as encapsulation in liposomes. Finally, the use of transcriptional and translational inhibitors to specifically block synthesis of MDR1, MRP, and BCRP is under investigation. However, the recent discovery of single-nucleotide polymorphisms among the ABC drug transporters may complicate this approach. Polymorphisms in MDR1 have been shown to alter expression and function of the protein. Such changes in transporter function could contribute to variation between different individuals and ethnic groups in both chemotherapeutic drug response and the effect of inhibitors on transporter activity. It is also found in the colon and in the collecting ducts of the kidney. Its role in the kidney is to secrete H+ ions into the urine, when blood pH falls, and to reabsorb K+ ions (see Chapter 25, “Neurogastroenterology and Motility”). • Proton pumps or H+-ATPases are found in the membranes of the lysosomes and the Golgi apparatus. They pump protons from the cytosol into these organelles, keeping the inside of the organelles more acidic (at a lower pH) than the rest of the cell. These pumps, classified as V-type ATPases because they were first discovered in intracellular vacuolar structures, have now been detected in plasma membranes. For example, the proton pump in the plasma membrane of specialized bone and kidney cells is characterized as a V-type ATPase. The secretion of protons by osteoclasts helps to solubilize the bone mineral and creates an acidic environment for bone breakdown by enzymes. The proton pump in the kidney is present in the same cells as the H+/K+-ATPase and helps to secrete H+ ions into the urine when blood pH falls. • ATP-binding cassette (ABC) transporters are a super family of transporters composed of two transmembrane domains and two cytosolic nucleotide-binding domains. The transmembrane domains recognize specific solutes and transport them across the membrane using a number ● Dr. Murtadha Al-Shareifi e-Library 34 Part I / Cellular Physiology of different mechanisms, including conformational change. The nucleotide-binding domain, or ABC domain, has a highly conserved sequence. ABC transporters are involved in a number of cellular processes, including nutrient uptake, cholesterol and lipid trafficking, resistance to cytotoxic drugs and antibiotics, cellular immune response, and stem cell biology. • ABCA1, a member of the ABC subfamily A, has an important role in effluxing cholesterol, phospholipids, and other metabolites out of cells. ABCA1 transfers lipids and cholesterol to lipid-poor high-density lipoproteins (HDLs). ABCA1 is a unique ABC transporter because it is also a receptor, binding the lipid-poor HDL to facilitate the loading of the cholesterol that the transporter is moving out of the cell. • ABC subfamily C transporters play a crucial role in the development of multidrug resistance (MDR). There are a number of different transporters encoded by multiple MDR genes. The MDR1 transporter is widely distributed in the liver, brain, lung, kidney, pancreas, and small intestine and transports a wide range of antibiotics, antivirals, and chemotherapeutic drugs out of the cell. MDR-associated protein transporters are a related class of ABCC transporters that also interfere with antibiotic and chemotherapy. The cystic fibrosis transmembrane conductance regulator (ABCC7) is another member of this family. • Organic anion transporting polypeptides (OATPs) are members of the solute carrier family and are highly expressed in the liver, kidney, and brain. OATPs transport anionic and cationic chemicals, steroid, and peptide backbones generally into cells. Thyroxine, bile acids, and bilirubin are important solutes transported by OATPs. These transporters also import agents such 3-hydroxy3-methylglutaryl-CoA reductase inhibitors (statins), angiotensin-converting enzyme inhibitors, angiotensin receptor II antagonists, and cardiac glycosides into cells. • F-type ATPases are located in the inner mitochondrial membrane. This type of proton pump normally functions in reverse. Instead of using the energy stored in ATP molecules to pump protons, its principal function is to synthesize ATP by using the energy stored in a gradient of protons that is crossing the inner mitochondrial membrane down its concentration gradient. The proton gradient is generated by the respiratory chain. Secondary active transport The net effect of ion pumps is maintenance of the various environments needed for the proper functioning of organelles, cells, and organs. Metabolic energy is expended by the pumps to create and maintain the differences in ion concentrations. Besides the importance of local ion concentrations for cell function, differences in concentrations represent stored energy. An ion releases potential energy when it moves down an electrochemical gradient, just as a body releases energy when falling to a lower level. This energy can be used to perform work. Cells have developed Out In Out Na+ Na+ Na+ Na+ Na+ Na+ Na+ In Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ Na+ 1. Na+ binds 2. Solute ( ) binds 3. Translocation 4. Na+ released 5. Solute released ● Figure 2.11 Mechanism of secondary active transport. A solute is moved against its concentration gradient by coupling it to Na+ moving down a favorable gradient. Binding of extracellular Na+ to the carrier protein (step 1) may increase the affinity of binding sites for solute, so that solute also can bind to the carrier (step 2), even though its extracellular concentration is low. A conformational change in the carrier protein (step 3) exposes the binding sites to the cytosol, where Na+ readily dissociates because of the low intracellular Na+ concentration (step 4). The release of Na+ decreases the affinity of the carrier for solute and forces the release of the solute inside the cell (step 5), where solute concentration is already high. The free carrier then reverts to the conformation required for step 1, and the cycle begins again. several carrier mechanisms to transport one solute against its concentration gradient by using the energy stored in the favorable gradient of another solute. In mammals, most of these mechanisms use sodium as the driver solute and use the energy of the sodium gradient to carry out the “uphill” transport of another important solute (Fig. 2.11). Because the sodium gradient is maintained by the action of the Na+/ K+-ATPase, the function of these transport systems depends on the function of the Na+/K+-ATPase. Although they do not directly use metabolic energy for transport, these systems ultimately depend on the proper supply of metabolic energy to the sodium–potassium pump. They are called secondary active transport mechanisms. Disabling the pump with metabolic inhibitors or pharmacologic blockers causes these transport systems to stop when the sodium gradient has been dissipated. Similar to passive carrier-mediated systems, secondary active transport systems are integral membrane proteins; they have specificity for the solute they transport and show saturation kinetics and competitive inhibition. They differ, however, in two respects. First, they cannot function in the absence of the driver ion, the ion that moves along its electrochemical gradient and supplies energy. Second, they transport the solute against its own concentration or electrochemical gradient. Functionally, the different secondary active transport systems can be classified into two groups: symport (cotransport) systems, in which the solute being transported moves in the same direction as the sodium ion; and antiport (exchange) systems, in which the sodium ion and the solute move in opposite directions (Fig. 2.12). Dr. Murtadha Al-Shareifi e-Library ● 35 Chapter 2 / Plasma Membrane, Membrane Transport, and Resting Membrane Potential Examples of symport mechanisms are the sodiumcoupled sugar transport system and the several sodiumcoupled amino acid transport systems found in the small intestine and the renal tubule. The symport systems allow efficient absorption of nutrients even when the nutrients are present at low concentrations. The sodium-dependent glucose transporter-1 (SGLT1) in the human intestine contains 664 amino acids in a single polypeptide chain with 14 membrane-spanning segments (Fig. 2.13). One complete cycle or turnover of a single SGLT1 protein, illustrated in Figure 2.11, can occur 1,000 times/s at 37°C. In reality, this cycle probably involves a coordinated trapping–release cycle and/or tilt of membrane-spanning segments rather than the simplistic view presented in Figure 2.11. Another example of a symport system is the family of sodium-coupled phosphate transporters (termed NaPi, types I and II) in the intestine and renal proximal tubule. These transporters have six to eight membrane-spanning segments and contain 460 to 690 amino acids. Sodium-coupled chloride transporters in the kidney are targets for inhibition by specific diuretics. The Na+–Cl− cotransporter in the distal tubule, known as NCC, is inhibited by thiazide diuretics, and the Na+–K+–2Cl− cotransporter in the ascending limb of the loop of Henle, referred to as NKCC, is inhibited by bumetanide. The most important examples of antiporters are the Na+/ + H exchange and Na+/Ca2+ exchange systems, found mainly in the plasma membrane of many cells. The first uses the sodium gradient to remove protons from the cell, controlling Hexose Malabsorption in the Intestine Malabsorption of hexoses in the intestine can be the indirect result of a number of circumstances, such as an increase in intestinal motility or defects in digestion because of pancreatic insufficiency. Although less common, malabsorption may be a direct result of a specific defect in hexose transport. Regardless of the cause, the symptoms are common and include diarrhea, abdominal pain, and gas. The challenge is to identify the cause so proper treatment can be applied. Some infants develop a copious watery diarrhea when fed milk that contains glucose or galactose or the disaccharides lactose and sucrose. The latter are degraded to glucose, galactose, and fructose by enzymes in the intestine. The dehydration can begin during the first day of life and can lead to rapid death if not corrected. Fortunately, the symptoms disappear when a carbohydrate-free formula fortified with fructose is used instead of milk. This condition is a rare inherited disease known as glucose–galactose malabsorption (GGM), and about 200 severe cases have been reported worldwide. At least 10% of the general population has glucose or lactose intolerance, however, and it is possible that these people may have milder forms of the disease. A specific defect in absorption of glucose and galactose can be demonstrated by tolerance tests in which oral administration of these monosaccharides produces little or no increase in plasma glucose or galactose. The primary defect lies in the Na+–glucose cotransporter protein Out In Symport Na+ S Na+ S Na+ S Na+ S Antiport ● Figure 2.12 Secondary active transport systems. In a symport system (top), the transported solute (S) is moved in the same direction as the Na+ ion. In an antiport system (bottom), the solute is moved in the opposite direction to Na+. Boldfaced and lightfaced fonts indicate high and low concentrations, respectively, of Na+ ions and solute. Clinical Focus / 2.2 (SGLT, Fig. 2.13), located in the apical plasma membrane of intestinal epithelial cells (Fig. 2.14). Glucose and galactose have very similar structures, and both are substrates for transport by SGLT. Fructose transport is not affected by a defect in SGLT because a specific fructose transporter named GLUT5 is present in the apical membrane. Human SGLT was cloned in 1989, and almost 30 different mutations have been identified in GGM patients. Many of the mutations produce premature cessation of SGLT protein synthesis or disrupt the trafficking of mature SGLT to the apical plasma membrane. In a few cases, the SGLT reaches the apical membrane but is no longer capable of glucose transport. The result in all cases is that functional SGLT proteins are not present in the apical membrane so glucose and galactose remain in the lumen of the intestine. As these solutes accumulate in the lumen, the osmolality of the fluids increases and retards absorption of water, leading to diarrhea and severe water loss from the body. Identification of specific changes in defective SGLT proteins in patients has provided clues about the specific amino acids that are essential for the normal function of SGLT. At the same time, advances in molecular biology have allowed a better understanding of the genetic defect at the cellular level and how this leads to the clinical symptoms. GGM is an example of how information from a disease can further understanding of physiology and vice versa. ● Dr. Murtadha Al-Shareifi e-Library 36 Part I / Cellular Physiology ● Figure 2.13 A model of the secondary structure of the Na+glucose cotransporter protein (SGLT1) in the human intestine. The polypeptide chain of 664 amino acids passes back and forth across the membrane 14 times. Each membranespanning segment consists of 21 amino acids arranged in an a-helical conformation. Both the NH2 and the COOH ends are located on the extracellular side of the plasma membrane. In the functional protein, the membrane-spanning segments are clustered together to provide a hydrophilic pathway across the plasma membrane. The N-terminal portion of the protein, including helices 1 to 9, is required to couple Na+ binding to glucose transport. The five helices (10–14) at the C terminus may form the transport pathway for glucose. NH2 COOH Out 1 2 3 4 5 6 7 8 9 10 12 11 13 14 In the intracellular pH and counterbalancing the production of protons in metabolic reactions. It is an electroneutral system because there is no net movement of charge. One Na+ enters the cell for each H+ that leaves. The second antiporter removes calcium from the cell and, together with the different calcium pumps, helps maintain a low cytosolic calcium concentration. It is an electrogenic system because there is a net movement of charge. Three Na+ enter the cell and one Ca2+ ion leaves during each cycle. The structures of the symport and antiport protein transporters that have been characterized (see Fig. 2.13) share a common property with ion channels (see Fig. 2.9) and equilibrating carriers, namely the presence of multiple membrane-spanning segments within the polypeptide chain. This supports the concept that, regardless of the mechanism, the membrane-spanning regions of a transport protein form a hydrophilic pathway for rapid transport of ions and solutes across the hydrophobic interior of the membrane lipid bilayer. An example is the absorption of glucose in the small intestine. Glucose enters the intestinal epithelial cells by active transport using the electrogenic Na+–glucose cotransporter system (SGLT) in the apical membrane. This increases the Transcellular transport GLUT2 Epithelial cells occur in layers or sheets that allow the directional movement of solutes not only across the plasma membrane but also from one side of the cell layer to the other. Such regulated movement is achieved because the plasma membranes of epithelial cells have two distinct regions with different morphologies and different transport systems. These regions are the apical membrane, facing the lumen, and the basolateral membrane, facing the blood supply (Fig. 2.14). The specialized or polarized organization of the cells is maintained by the presence of tight junctions at the areas of contact between adjacent cells. Tight junctions prevent proteins on the apical membrane from migrating to the basolateral membrane and those on the basolateral membrane from migrating to the apical membrane. Thus, the entry and exit steps for solutes can be localized to opposite sides of the cell. This is the key to transcellular transport across epithelial cells. Apical (luminal) side Na+ Glucose Amino Na+ acid Tight junctions Lumen SGLT Cell layer Na+ Na Glucose + K+ + K Amino acid Intercellular spaces Blood Basolateral side ● Figure 2.14 The localization of transport systems to different regions of the plasma membrane in epithelial cells of the small intestine. A polarized cell is produced, in which entry and exit of solutes, such as glucose, amino acids, and Na+, occur at opposite sides of the cell. Active entry of glucose and amino acids is restricted to the apical membrane, and exit requires equilibrating carriers located only in the basolateral membrane. For example, glucose enters on sodium-dependent glucose transporter (SGLT) and exits on glucose transporter-2 (GLUT2). Na+ that enters via the apical symporters is pumped out by the Na+/K+-ATPase on the basolateral membrane. The result is a net movement of solutes from the luminal side of the cell to the basolateral side, ensuring efficient absorption of glucose, amino acids, and Na+ from the intestinal lumen. Dr. Murtadha Al-Shareifi e-Library ● 37 Chapter 2 / Plasma Membrane, Membrane Transport, and Resting Membrane Potential intracellular glucose concentration above the blood glucose concentration, and the glucose molecules move passively out of the cell and into the blood via an equilibrating carrier mechanism (GLUT2) in the basolateral membrane (see Fig. 2.14). The intestinal GLUT2, like the erythrocyte GLUT1, is a sodium-independent transporter that moves glucose down its concentration gradient. Unlike GLUT1, the GLUT2 transporter can accept other sugars, such as galactose and fructose, that are also absorbed in the intestine. The Na+/K+-ATPase that is located in the basolateral membrane pumps out the sodium ions that enter the cell with the glucose molecules on SGLT. The polarized organization of the epithelial cells and the integrated functions of the plasma membrane transporters form the basis by which cells accomplish transcellular movement of both glucose and sodium ions. ● WATER MOVEMENT ACROSS THE PLASMA MEMBRANE Water can move rapidly in and out of cells, but the partition coefficient of water into lipids is low; therefore, the permeability of the membrane lipid bilayer for water is also low. Specific membrane proteins that function as water channels explain the rapid movement of water across the plasma membrane. These water channels are small (molecular weight of about 30 kDa), integral membrane proteins known as aquaporins. Ten different forms have been discovered so far in mammals. At least six forms are expressed in cells in the kidney and seven forms in the gastrointestinal tract, tissues in which water movement across plasma membranes is particularly rapid. In the kidney, aquaporin-2 (AQP2) is abundant in the collecting duct and is the target of the hormone arginine vasopressin, also known as antidiuretic hormone. This hormone increases water transport in the collecting duct by stimulating the insertion of AQP2 proteins into the apical plasma membrane. Several studies have shown that AQP2 has a critical role in inherited and acquired disorders of water reabsorption by the kidney. For example, diabetes insipidus is a condition in which the kidney loses its ability to reabsorb water properly, resulting in excessive loss of water and excretion of a large volume of very dilute urine (polyuria). Although inherited forms of diabetes insipidus are relatively rare, it can develop in patients receiving chronic lithium therapy for psychiatric disorders, giving rise to the term lithium-induced polyuria. Both of these conditions are associated with a decrease in the number of AQP2 proteins in the collecting ducts of the kidney. Water movement across the plasma membrane is driven by differences in osmotic pressure. The spontaneous movement of water across a membrane driven by a gradient of water concentration is the process known as osmosis. The water moves from an area of high concentration of water to an area of low concentration. Because concentration is defined by the number of particles per unit of volume, a solution with a high concentration of solutes has a low concentration of water, and vice versa. Osmosis can, therefore, be viewed as the movement of water from a solution of high water concentration (low concentration of solute) toward a solution with a lower concentration of water (high solute concentration). Osmosis is a passive transport mechanism that tends to equalize the total solute concentrations of the solutions on both sides of every membrane. If a cell that is in osmotic equilibrium is transferred to a more dilute solution, water will enter the cell, the cell volume will increase, and the solute concentration of the cytoplasm will be reduced. If the cell is transferred to a more concentrated solution, water will leave the cell, the cell volume will decrease, and the solute concentration of the cytoplasm will increase. As discussed below, many cells have regulatory mechanisms that keep cell volume within a certain range. Other cells such as mammalian erythrocytes do not have volume regulatory mechanisms, and large volume changes occur when the solute concentration of the ECF is changed. The driving force for the movement of water across the plasma membrane is the difference in water concentration between the two sides of the membrane. For historical reasons, this driving force is not called the chemical gradient of water but the difference in osmotic pressure. The osmotic pressure of a solution is defined as the pressure necessary to stop the net movement of water across a selectively permeable membrane that separates the solution from pure water. When a membrane separates two solutions of different osmotic pressure, water will move from the solution with low osmotic pressure (high water and low solute concentrations) to the solution of high osmotic pressure (low water and high solute concentrations). In this context, the term selectively permeable means that the membrane is permeable to water but not solutes. In reality, most biologic membranes contain membrane transport proteins that permit solute movement. The osmotic pressure of a solution depends on the number of particles dissolved in it, the total concentration of all solutes, regardless of the type of solutes present. Many solutes, such as salts, acids, and bases, dissociate in water, so the number of particles is greater than the molar concentration. For example, NaCl dissociates in water to give Na+ and Cl−, so one molecule of NaCl will produce two osmotically active particles. In the case of CaCl2, there are three particles per molecule. The equation giving the osmotic pressure of a solution is p = nRTC (3) where p is the osmotic pressure of the solution, n is the number of particles produced by the dissociation of one molecule of solute (2 for NaCl, 3 for CaCl2), R is the universal gas constant (0.0821 L×atm/mol×K), T is the absolute temperature, and C is the concentration of the solute in mol/L. Osmotic pressure can be expressed in atmospheres (atm). Solutions with the same osmotic pressure are called isosmotic. A solution is hyperosmotic with respect to another solution if it has a higher osmotic pressure and hyposmotic if it has a lower osmotic pressure. ● Dr. Murtadha Al-Shareifi e-Library 38 Part I / Cellular Physiology Equation 3, called the van’t Hoff equation, is valid only when applied to very dilute solutions, in which the particles of solutes are so far away from each other that no interactions occur between them. Generally, this is not the case at physiologic concentrations. Interactions between dissolved particles, mainly between ions, cause the solution to behave as if the concentration of particles is less than the theoretical value (nC). A correction coefficient, called the osmotic coefficient (j) of the solute, needs to be introduced in the equation. Therefore, the osmotic pressure of a solution can be written more accurately as p = nRTFC (4) The osmotic coefficient varies with the specific solute and its concentration. It has values between 0 and 1. For example, the osmotic coefficient of NaCl is 1.00 in an infinitely dilute solution but changes to 0.93 at the physiologic concentration of 0.15 mol/L. At any given T, because R is constant, equation 4 shows that the osmotic pressure of a solution is directly proportional to the term nfC. This term is known as the osmolality or osmotic concentration of a solution and is expressed in Osm/kg H2O. Most physiologic solutions such as blood plasma contain many different solutes, and each contributes to the total osmolality of the solution. The osmolality of a solution containing a complex mixture of solutes is usually measured by freezing point depression. The freezing point of an aqueous solution of solutes is lower than that of pure water and depends on the total number of solute particles. Compared with pure water, which freezes at 0°C, a solution with an osmolality of 1 Osm/kg H2O will freeze at −1.86°C. The ease with which osmolality can be measured has led to the wide use of this parameter for comparing the osmotic pressure of different solutions. The osmotic pressures of physiologic solutions are not trivial. Consider blood plasma, for example, which usually has an osmolality of 0.28 Osm/kg H2O, determined by freezing point depression. Equation 4 shows that the osmotic pressure of plasma at 37°C is 7.1 atm, about seven times greater than the atmospheric pressure. Many cells can regulate their volume. Cell volume changes can occur in response to changes in the osmolality of ECF in both normal and pathophysiologic situations. Accumulation of solutes also can produce volume changes by increasing the intracellular osmolality. Many cells can correct these volume changes. Volume regulation is particularly important in the brain where cell swelling can have serious consequences because expansion is strictly limited by the rigid skull. Tonicity A solution’s osmolality is determined by the total concentration of all the solutes present. In contrast, the solution’s tonicity is determined by the concentrations of only those solutes that do not enter (“penetrate”) the cell. Tonicity determines cell volume, as illustrated in the following examples. Na+ behaves as a nonpenetrating solute because it is pumped out of cells by the Na+/K+-ATPase at the same rate that it enters. A solution of NaCl at 0.2 Osm/kg H2O is hypoosmotic compared with cell cytosol at 0.3 Osm/kg H2O. The NaCl solution is also hypotonic because cells will accumulate water and swell when placed in this solution. A solution containing a mixture of NaCl (0.3 Osm/kg H2O) and urea (0.1 Osm/kg H2O) has a total osmolality of 0.4 Osm/kg H2O and will be hyperosmotic compared with cell cytosol. The solution is isotonic, however, because it produces no permanent change in cell volume. The reason is that cells shrink initially as a result of loss of water, but urea is a penetrating solute that rapidly enters the cells. Urea entry increases the intracellular osmolality, so water also enters and increases the volume. Entry of water ceases when the urea concentration is the same inside and outside the cells. At this point, the total osmolality both inside and outside the cells will be 0.4 Osm/kg H2O and the cell volume will be restored to normal. By extension, it can be seen that normal blood plasma is an isotonic solution because Na+ is the predominant plasma solute and is nonpenetrating. This stabilizes cell volume while other plasma solutes (glucose, amino acids, phosphate, urea, etc.) enter and leave the cells as needed. Volume regulation mechanisms When cell volume increases because of extracellular hypotonicity, the response of many cells is rapid activation of transport mechanisms that tend to decrease the cell volume (Fig. 2.15A). Different cells use different regulatory volume decrease (RVD) mechanisms to move solutes out of the cell and decrease the number of particles in the cytosol, causing water to leave the cell. Because cells have high intracellular concentrations of potassium, many RVD mechanisms involve an increased efflux of K+, either by stimulating the opening of potassium channels or by activating symport mechanisms for KCl. Other cells activate the efflux of some amino acids, such as taurine or proline. The net result is a decrease in intracellular solute content and a reduction of cell volume close to its original value (see Fig. 2.15A). When placed in a hypertonic solution, cells rapidly lose water and their volume decreases. In many cells, a decreased volume triggers regulatory volume increase (RVI) mechanisms, which increase the number of intracellular particles, bringing water back into the cells. Because Na+ is the main extracellular ion, many RVI mechanisms involve an influx of sodium into the cell. Na+–Cl− symport, Na+–K+–2Cl− symport, and Na+/H+ antiport are some of the mechanisms activated to increase the intracellular concentration of Na+ and increase the cell volume toward its original value (Fig. 2.15B). Mechanisms based on an increased Na+ influx are effective for only a short time because, eventually, the sodium pump will increase its activity and reduce intracellular Na+ to its normal value. Cells that regularly encounter hypertonic ECFs have developed additional mechanisms for maintaining normal volume. These cells can synthesize specific organic solutes, enabling them to increase intracellular osmolality for a long time and avoiding altering the concentrations of ions they must maintain within a narrow range of values. The organic solutes are usually small molecules that do not interfere with normal cell function when they accumulate inside Dr. Murtadha Al-Shareifi e-Library ● 39 Chapter 2 / Plasma Membrane, Membrane Transport, and Resting Membrane Potential A Relative volume 2.0 Rapid swelling phase the gastrointestinal tract. The main ingredients of rehydration solutions are glucose, NaCl, and water. The glucose and Na+ ions are reabsorbed by SGLT1 and other transporters in the epithelial cells lining the lumen of the small intestine (see Fig. 2.14). Deposition of these solutes on the basolateral side of the epithelial cells increases the osmolality in that region compared with the intestinal lumen and drives the osmotic absorption of water. Absorption of glucose, and the obligatory increases in absorption of NaCl and water, helps to compensate for excessive diarrheal losses of salt and water. Regulatory volume decrease 1.0 ● 0 10 20 The different passive and active transport systems are coordinated in a living cell to maintain intracellular ions and other solutes at concentrations compatible with life. Consequently, the cell does not equilibrate with the ECF but rather exists in a steady state with the extracellular solution. For example, intracellular Na+ concentration (10 mmol/L in a muscle cell) is much lower than extracellular Na+ concentration (140 mmol/L), so Na+ enters the cell by passive transport through nongated (always open) Na+ channels. The rate of Na+ entry is matched, however, by the rate of active transport of Na+ out of the cell via the sodium–potassium pump (Fig. 2.16). The net result is that intracellular Na+ is maintained constant and at a low level, even though Na+ continually enters and leaves Time (min) in hypotonic solution B Relative volume 2.0 Regulatory volume increase 1.0 Rapid shrinkage 0 10 RESTING MEMBRANE POTENTIAL 20 Time (min) in hypertonic solution + Passive exit K via nongated channel Figure 2.15 The effect of tonicity changes on cell volume. Cell volume changes when a cell is placed in either a hypotonic or a hypertonic solution. (A) In a hypotonic solution, the reversal of the initial increase in cell volume is known as a regulatory volume decrease. Transport systems for solute exit are activated, and water follows movement of solute out of the cell. (B) In a hypertonic solution, the reversal of the initial decrease in cell volume is a regulatory volume increase. Transport systems for solute entry are activated, and water follows solute into the cell. Active transport by + + Na /K -ATPase ● the cell. For example, cells of the medulla of the mammalian kidney can increase the level of the enzyme aldose reductase when subjected to elevated extracellular osmolality. This enzyme converts glucose to an osmotically active solute, sorbitol. Brain cells can synthesize and store inositol. Synthesis of sorbitol and synthesis of inositol represent different answers to the problem of increasing the total intracellular osmolality, allowing normal cell volume to be maintained in the presence of hypertonic ECF. Oral rehydration therapy is driven by solute transport. Oral administration of rehydration solutions has dramatically reduced the mortality resulting from cholera and other diseases that involve excessive losses of water and solutes from + ATP K 2K+ Na + 3Na+ ADP Passive entry via nongated channel Na + ● Figure 2.16 The concept of a steady state. Na+ enters a cell through nongated Na+ channels, moving passively down the electrochemical gradient. The rate of Na+ entry is matched by the rate of active transport of Na+ out of the cell via the Na+/K+-ATPase. The intracellular concentration of Na+ remains low and constant. Similarly, the rate of passive K+ exit through nongated K+ channels is matched by the rate of active transport of K+ into the cell via the pump. The intracellular K+ concentration remains high and constant. During each cycle of the ATPase, two K+ are exchanged for three Na+, and one molecule of adenosine triphosphate (ATP) is hydrolyzed. Boldfaced and lightfaced fonts indicate high and low ion concentrations, respectively. ADP, adenosine diphosphate. ● Dr. Murtadha Al-Shareifi e-Library 40 Part I / Cellular Physiology the cell. The reverse is true for K+, which is maintained at a high concentration inside the cell relative to the outside. The passive exit of K+ through nongated K+ channels is matched by active entry via the pump (see Fig. 2.16). Maintenance of this steady state with ion concentrations inside the cell different from those outside the cell is the basis for the difference in electrical potential across the plasma membrane or the resting membrane potential. will be Dm = 0. Substituting this condition into equation 5, we obtain æC ö 0 = RT ln ç i ÷ + zF(Ei - Eo ) è Co ø RT æ C i ö Ei - E o = ln zF çè C o ÷ø Ei - E o = + Ion movement is driven by the electrochemical potential. (6) RT æ C o ö ln zF çè C i ÷ø If there are no differences in temperature or hydrostatic pressure between the two sides of a plasma membrane, two forces drive the movement of ions and other solutes across the membrane. One force results from the difference in the concentration of a substance between the inside and the outside of the cell and the tendency of every substance to move from areas of high concentration to areas of low concentration. The other force results from the difference in electrical potential between the two sides of the membrane and applies only to ions and other electrically charged solutes. When a difference in electrical potential exists, positive ions tend to move toward the negative side, whereas negative ions tend to move toward the positive side. The sum of these two driving forces is called the gradient (or difference) of electrochemical potential across the membrane for a specific solute. It measures the tendency of that solute to cross the membrane. The expression of this force is given by Equation 6, known as the Nernst equation, gives the value of the electrical potential difference (Ei − Eo) necessary for a specific ion to be at equilibrium. This value is known as the Nernst equilibrium potential for that particular ion and it is expressed in millivolts (mV). At the equilibrium potential, the tendency of an ion to move in one direction because of the difference in concentrations is exactly balanced by the tendency to move in the opposite direction because of the difference in electrical potential. At this point, the ion will be in equilibrium and there will be no net movement. By converting to log10 and assuming a physiologic temperature of 37°C and a value of +1 for z (for Na+ or K+), the Nernst equation can be expressed as Dm = RT ln Ci/C0 + zF(E1-E0) The resting membrane potential is the electrical potential difference across the plasma membrane of a normal living cell in its unstimulated state. It can be measured directly by the insertion of a microelectrode into the cell with a reference electrode in the ECF. The resting membrane potential is determined by those ions that can cross the membrane and are prevented from attaining equilibrium by active transport systems. Potassium, sodium, and chloride ions can cross the membranes of every living cell, and each of these ions contributes to the resting membrane potential. By contrast, the permeability of the membrane of most cells to divalent ions is so low that it can be ignored in this context. The Goldman equation gives the value of the membrane potential (in mV) when all the permeable ions are accounted for (5) where m represents the electrochemical potential (Dm is the difference in electrochemical potential between two sides of the membrane); Ci and C o are the concentrations of the solute inside and outside the cell, respectively; Ei is the electrical potential inside the cell measured with respect to the electrical potential outside the cell (Eo); R is the universal gas constant (2 cal/mol×K); T is the absolute temperature (K); z is the valence of the ion; and F is the Faraday constant (23 cal/mV×mol). By inserting these units in equation 5 and simplifying, the electrochemical potential will be expressed in cal/mol, which is the unit of energy. If the solute is not an ion and has no electrical charge, then z = 0 and the last term of the equation becomes zero. In this case, the electrochemical potential is defined only by the different concentrations of the uncharged solute, called the chemical potential. The driving force for solute transport becomes solely the difference in chemical potential. Net ion movement is zero at the equilibrium potential. Net movement of an ion into or out of a cell continues as long as the driving force exists. Net movement stops and equilibrium is reached only when the driving force of electrochemical potential across the membrane becomes zero. The condition of equilibrium for any permeable ion EI – EO = 61 LOG10 (C O /C I ) (7) Because Na+ and K+ (and other ions) are present at different concentrations inside and outside a cell, it follows from equation 7 that the equilibrium potential will be different for each ion. Resting membrane potential is determined by the passive movement of several ions. Ei - E o = + + RT æ PK [K ]o + PNa [Na ]o + PCl [Cl ]i ö ln ç ÷ F è PK [K + ]i + PNa [Na + ]i + PCl [Cl - ]o ø (8) where PK, PNa, and PCl represent the permeability of the membrane to potassium, sodium, and chloride ions, respectively, and brackets indicate the concentration of the ion inside (i) and outside (o) the cell. If a specific cell is not permeable to one of these ions, the contribution of the impermeable ion to the membrane potential will be zero. For a cell that is permeable to an ion other than the three considered in the Goldman equation, that ion will contribute to the membrane potential and must be included in equation 8. Dr. Murtadha Al-Shareifi e-Library ● 41 Chapter 2 / Plasma Membrane, Membrane Transport, and Resting Membrane Potential It can be seen from equation 8 that the contribution of any ion to the membrane potential is determined by the membrane’s permeability to that particular ion. The higher the permeability of the membrane to one ion relative to the others, the more that ion will contribute to the membrane potential. The plasma membranes of most living cells are much more permeable to potassium ions than to any other ion. Making the assumption that PNa and PCl are zero relative to PK, equation 8 can be simplified to Ei - E o = RT æ PK [K + ]o ö ln F çè PK [K + ]i ÷ø (9) RT æ [K + ]o ö Ei - E o = ln F çè [K + ]i ÷ø which is the Nernst equation for the equilibrium potential for K+ (see equation 6). This illustrates two important points: • In most cells, the resting membrane potential is close to the equilibrium potential for K+. • The resting membrane potential of most cells is dominated by K+ because the plasma membrane is more permeable to this ion compared with the others. As a typical example, the K+ concentrations outside and inside a muscle cell are 3.5 and 155 mmol/L, respectively. Substituting these values in equation 7 gives a calculated equilibrium potential for K+ of −100 mV, negative inside the cell relative to the outside. Measurement of the resting membrane potential in a muscle cell yields a value of −90 mV (negative inside). This value is close to, although not the same as, the equilibrium potential for K+. The reason the resting membrane potential in the muscle cell is less negative than the equilibrium potential for K+ is as follows. Under physiologic conditions, there is passive entry of Na+ ions. This entry of positively charged ions has a small but significant effect on the negative potential inside the cell. Assuming intracellular Na+ to be 10 mmol/L and extracellular Na+ to be 140 mmol/L, the Nernst equation gives a value of +70 mV for the Na+ equilibrium potential (positive inside the cell). This is far from the resting membrane potential of −90 mV. Na+ makes only a small contribution to the resting membrane potential because membrane permeability to Na+ is low compared with that of K+. The contribution of Cl− ions need not be considered because the resting membrane potential in the muscle cell is the same as the equilibrium potential for Cl−. Therefore, there is no net movement of chloride ions. In most cells, as shown above using a muscle cell as an example, the equilibrium potentials of K+ and Na+ are different from the resting membrane potential, which indicates that neither K+ ions nor Na+ ions are at equilibrium. Consequently, these ions continue to cross the plasma membrane via specific nongated channels, and these passive ion movements are directly responsible for the resting membrane potential. The Na+/K+-ATPase is important indirectly for maintaining the resting membrane potential because it sets up the gradients of K+ and Na+ that drive passive K+ exit and Na+ entry. During each cycle of the pump, two K+ ions are moved into the cell in exchange for three Na+, which are moved out (see Fig. 2.16). Because of the unequal exchange mechanism, the pump’s activity contributes slightly (about −5 mV) to the negative potential inside the cell. Chapter Summary • The plasma membrane consists of proteins in a phospholipid bilayer. Integral proteins are embedded in the bilayer, whereas peripheral proteins are attached to the outer surface. • Macromolecules cross the plasma membrane by endocytosis and exocytosis. • Passive movement of a solute across a membrane dissipates the gradient (driving force) and reaches an equilibrium at which point there is no net movement of solute. • Simple diffusion is the passage of lipid-soluble solutes across the plasma membrane by diffusion through the lipid bilayer. • Facilitated diffusion is the passage of water-soluble solutes and ions through a hydrophilic pathway created by a membrane-spanning integral protein. • Facilitated diffusion of small ions is mediated by specific pores and ion channel proteins. • Active transport uses a metabolic energy source to move solutes against gradients, and the process prevents a state of equilibrium. • Polarized organization of epithelial cells ensures directional movement of solutes and water across the epithelial layer. • Water crosses plasma membranes rapidly via channel proteins termed aquaporins. Water movement is a passive process driven by differences in osmotic pressure. • Cells regulate their volume by moving solutes in or out to drive osmotic entry or exit of water, respectively. • The driving force for ion transport is the sum of the electrical and chemical gradients, known as the gradient of electrochemical potential across the membrane. • The resting membrane potential is determined by the passive movements of several ions through nongated channels, which are always open. It is described most accurately by the Goldman equation, which takes into account the differences in membrane permeability of different ions. In a muscle cell, for example, the membrane permeability to Na+ is low compared with K+ and the resting membrane potential is a result primarily of passive exit of K+. Visit http://thePoint.lww.com for chapter review Q&A, case studies, animations, and more!