Molecular Biology of the Cell - Volume 1 - Alberts B., Bray D., Lewis J., Raff M., Roberts K., Watson J. 1994

Molecular Organization of Cells
Plasma Membrane
Transport of Small Molecules Across the Membrane

Because the interior of Cell/29.html">The Lipid Bilayer is hydrophobic, it acts as a virtually impermeable barrier to most polar molecules. This barrier prevents the leakage of Water-soluble cellular contents. However, because of this barrier, Cells have had to develop specialized pathways for transporting water-soluble molecules across their membranes. Cells must import essential nutrients and excrete harmful Metabolic waste products. In addition, cells need to regulate intracellular ion concentrations, which requires The transport of specific ions into or out of The Cell. The transport of small water-soluble molecules across the lipid bilayer is mediated by specialized transmembrane Proteins, each responsible for transporting a specific molecule or group of closely related molecules. Cells also have mechanisms for transporting macromolecules, such as proteins, and even large particles across their Plasma Membranes. However, these mechanisms differ significantly from those involved in small-molecule transport and will therefore be discussed in another section (see Section 6.5).

In this section, we will see that the selective permeability of The Plasma Membrane, combined with Active Transport across it, creates significant differences in the ionic COMPOSITION OF THE Cytosol and the extracellular fluid (Table 6-3). This allows cell membranes to store potential energy in the form of ion concentration gradients. Transmembrane ion gradients are used to drive various transport processes, to transmit electrical signals, and to synthesize ATP in Mitochondria, METABOLISM/14.html">Chloroplasts, and Bacteria. Before discussing The properties of transport proteins and the ion gradients generated by some of these proteins, we must first examine the permeability of a protein-free synthetic lipid bilayer.

Class="center">Table 6-3. Comparison of Ion Concentrations Inside and Outside a Typical Animal Cell

¹⁾ Because cells must contain equal numbers of positive and negative charges (to be electrically neutral), the substantial deficit of intracellular anions reflects the fact that most cellular constituents are negatively charged molecules (HCO^-₃, PO^3-, proteins, Nucleic Acids, metabolites carrying phosphate or carboxyl groups, etc.). The concentrations of Ca²⁺ and Mg²⁺ are given for free ions. There is about 20 mM Mg²⁺ and 1-2 mM Ca²⁺ in cells, but these are mostly bound to proteins and other substances, and in the case of Ca²⁺, are stored inside various Organelles.

6.4.1. Protein-Free Lipid bilayers are Impermeable to Ions but Freely Permeable to Water [19]

In principle, given enough time, virtually any molecule will diffuse across a protein-free lipid bilayer down its concentration gradient. However, the rates at which different molecules diffuse across such a bilayer vary enormously, depending primarily on the size of the molecule and its relative solubility in oil. In general, the smaller the molecule and the more soluble it is in oil (that is, the more hydrophobic, or nonpolar, it is), the more rapidly it will diffuse across the bilayer. Small nonpolar molecules, such as O₂, readily dissolve in lipid bilayers and therefore diffuse rapidly across them. Uncharged polar molecules also diffuse rapidly if they are small enough. For example, CO₂ (44 Da), ethanol (46 Da), and urea (60 Da) cross the bilayer rapidly, glycerol (92 Da) does so more slowly, and glucose (180 Da) is hardly able to cross at all (Fig. 6-41). Crucially, water (18 Da) diffuses very rapidly across a lipid bilayer, even though water molecules are relatively insoluble in oil. This is because water molecules are small and uncharged.

Fig. 6-41. Relative permeability of a synthetic lipid bilayer to different classes of molecules. The smaller the molecule and, more importantly, the fewer Hydrogen Bonds it forms, the more rapidly it diffuses across the membrane.

Fig. 6-42. Permeability coefficients (cm/s) of a synthetic lipid bilayer for various molecules. The rate of flow of solute molecules across the bilayer is directly proportional to the difference in concentration of the substance on the Two Sides of the membrane. Multiplying the concentration difference (mol/cm³) by the permeability coefficient (cm/s) yields the solute flux in moles per second through a square centimeter of membrane. For example, a Tryptophan concentration difference of 10^-4 mol/cm³ (10^-4/ 10^-3 L = 0.1 M) will drive a flux of 10^-4 mol/cm³ x 10^-7 cm/s = 10^-11 mol/s through 1 cm² of membrane, or 6 x 10⁴ molecules/s through 1 µm² of membrane.

In contrast, lipid bilayers are highly impermeable to all charged molecules (ions), regardless of their size: the charge and high degree of Hydration of these molecules prevent them from entering the hydrocarbon phase of the bilayer. This is why synthetic bilayers are 10⁹ times more permeable to water than to even small ions such as Na⁺ or K⁺ (Fig. 6-42).

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6.4.2. Membrane Transport Proteins Can Act as Carriers or Channels [19]

Like artificial lipid bilayers, cell membranes allow water and nonpolar molecules to pass through by simple physical diffusion. However, cell membranes are also permeable to various polar molecules, such as sugars, Amino Acids, NUCLEOTIDES, and many other metabolites, which cross synthetic bilayers extremely slowly. The transport of these solutes across cell membranes is mediated by specific proteins called membrane transport proteins. These are found in all types of Introduction/36.html">Biological Membranes and can vary greatly. Each specific protein is designed for a particular class of molecules (such as inorganic ions, sugars, or amino acids), and often only for a specific member of these classes. The Specificity of transport proteins was first demonstrated when Mutations in a single Gene were found to abolish the ability of bacteria to transport specific sugars across the plasma membrane. Similar mutations are now known in humans suffering from various inherited diseases that impair the transport of specific substances in the Kidneys or intestine. For example, individuals with the inherited disease cystinuria lack The ability to transport Certain amino acids (including cystine, a disulfide-linked dimer of Cysteine) from the urine or intestine into the Blood. As a result, cystine accumulates in the urine, leading to The formation of cystine "stones" in the kidneys.

All membrane transport proteins that have been studied in sufficient detail to determine their membrane topology have been found to be transmembrane proteins whose polypeptide chain crosses the lipid bilayer multiple times. These proteins enable the transport of specific substances across the membrane without direct contact with the hydrophobic interior of the lipid bilayer by forming continuous pathways through it.

There are two Major Classes of membrane transport proteins: carrier proteins and channel proteins. Carrier proteins (also called carriers or transporters) bind the specific solute to be transported and undergo conformational changes that transfer the solute across the membrane. In contrast, channel proteins form water-filled pores that extend across the lipid bilayer. When these pores are open, they allow specific solutes (usually inorganic ions of appropriate size and charge) to pass through them and, consequently, across the membrane (Fig. 6-43).

Fig. 6-43. Simplified schematic representation of the two classes of membrane transport proteins. A. A carrier protein can alternate between two Conformations, so that the binding site for a specific solute is sequentially exposed on one side of the bilayer and then on the other. B. A channel protein forms a water-filled pore in the lipid bilayer through which specific ions can diffuse.

6.4.3. Active Transport Is Mediated by Carrier Proteins Coupled to an Energy Source [21]

All channel proteins and many carrier proteins allow solutes to cross the membrane only passively ('downhill'). This process is called passive transport (or Facilitated Diffusion). If the solute molecule is uncharged, the direction of passive transport is determined solely by its concentration difference on the two sides of the membrane (the concentration gradient). However, if the solute is charged, both its concentration gradient and the electrical potential difference across the membrane (the Membrane Potential) influence its transport. Together, the concentration gradient and the electrical gradient form the electrochemical gradient. In fact, almost all plasma membranes have an electrical potential gradient across them, with the inside of the membrane usually being negatively charged relative to the outside (see Section 6.4.15). This potential favors The entry of positively charged ions into the cell but opposes the entry of negatively charged ions.

Cells also require transport proteins that actively pump specific solutes against their electrochemical gradients ('uphill'). This process, known as active transport, is always mediated by carrier proteins. As discussed below, in active transport, the pumping activity of the carriers is directional because it is tightly coupled to a source of metabolic energy, such as ATP Hydrolysis or an ion gradient. Thus, transport mediated by carrier Proteins can be either active or passive, whereas transport through channels is always passive (Fig. 6-44).

Fig. 6-44. Schematic representation of passive transport down an electrochemical gradient and active transport against it. Simple diffusion and passive transport mediated by transport proteins (facilitated diffusion) occur spontaneously. Active transport requires the expenditure of metabolic energy. Only nonpolar and small uncharged polar molecules can cross the lipid bilayer by simple diffusion. The transport of other polar molecules is mediated at significant rates by carrier proteins or channel proteins.

Fig. 6-45. Kinetics of simple diffusion and carrier protein-mediated diffusion. In the former case, the rate is always proportional to the concentration of the transported substance, whereas in the latter, the rate reaches a maximum value, V_max, when the carrier proteins are saturated. The concentration at which the rate is half-maximal is defined as the binding constant, K_m, of the transported molecules for the carrier (analogous to K_m in an enzyme-substrate system).

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6.4.4. Carrier proteins function as membrane-bound Enzymes [19]

The process by which carrier proteins specifically bind and transport solute molecules across the lipid bilayer resembles an enzymatic reaction, with transport proteins acting as specialized membrane-bound enzymes. All types of carrier proteins possess binding sites for the transported molecule (substrate). When the protein is saturated (i.e., when all binding sites are occupied), the rate of transport is maximal. This rate, designated V_max, is characteristic of the given carrier protein. In addition, each carrier protein has a characteristic binding constant, K_m, which is equal to the concentration of the solute at which the transport rate is half-maximal (Fig. 6-45). The binding of a solute can be specifically blocked by either Competitive Inhibitors (which compete for the same binding site) or noncompetitive inhibitors (which bind elsewhere and specifically alter The Structure of the carrier). However, the analogy with an enzyme-substrate reaction is incomplete because transported solutes are not usually covalently modified by carrier proteins.

Some transport proteins simply carry a single solute from one side of the membrane to the other. This simple transfer is called uniport. Other proteins function as cotransport systems, in which The transfer of one solute depends on the simultaneous or sequential transfer of another solute, either in the same direction (symport) or in the opposite direction (antiport) (Fig. 6-46). For example, most animal cells take up glucose from the extracellular fluid, where its concentration is relatively high, by passive transport mediated by specific glucose carriers operating as uniporters. In contrast, intestinal and Kidney cells take up glucose from the lumen of the intestine and kidney tubules, where the concentration of this sugar is low. In this case, glucose is symported with Na⁺ ions, whose extracellular concentration is very high. As discussed earlier, the anion transporter (band 3 protein) in human red Blood Cells operates via an antiport mechanism, exchanging Cl^- for HCO₃^-.

The Molecular Mechanism of carrier protein function is still unknown. They are proposed to transport solutes across the bilayer by undergoing reversible conformational changes that allow them to alternately expose solute-binding sites first on one side and then on the other. A schematic model of how this process might occur is shown in Fig. 6-47. We now know that carriers are transmembrane proteins with polypeptide chains that span the bilayer multiple times. It is highly unlikely that such proteins flip-flop continuously from one monolayer to the other or move back and forth across the lipid bilayer, as was previously proposed.

Fig. 6-46. Schematic diagram of carrier proteins functioning via uniport, symport, and antiport mechanisms.

Fig. 6-47. A hypothetical model showing how Conformational Changes in a carrier protein could mediate the facilitated diffusion of solute A. The carrier protein can exist in two conformational states: in the "pong" state, the binding sites for A are exposed on the exterior of the bilayer; in the "ping" state, the same sites are exposed on the other side. The transition between the two states occurs randomly and is completely reversible. Therefore, when the concentration of A is higher on the outside of the bilayer, more molecules of A will bind to the carrier protein in the "pong" state, resulting in the net transport of solute A down its concentration gradient.

As we shall see, a relatively minor Modification of the model shown in Fig. 6-47 allows the carrier protein to be coupled to a source of energy, such as ATP hydrolysis (see Fig. 6-49). A remarkable example of a carrier protein that uses the energy of ATP hydrolysis to pump ions is the (Na⁺ + K⁺) pump, which plays a crucial role in generating the membrane potential across the plasma membranes of animal cells.

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6.4.5. The plasma membrane (Na⁺ + K⁺) pump is an ATPase [22]

The intracellular concentration of K⁺ is typically 10 to 20 times higher than the extracellular concentration. For Na⁺ ions, the situation is exactly the opposite (see Table 6-3). This difference in ion concentrations is maintained by the (Na⁺ + K⁺) pump, which is found in the plasma membranes of virtually all animal cells. This pump operates as an antiport, actively pumping Na⁺ out of cells and K⁺ into cells against their steep electrochemical gradients. As discussed below, the Na⁺ gradient generated by the pump regulates cell volume through osmotic effects. It is also used to drive the transport of sugars and amino acids into the cell. Nearly one-third of all the energy required by an animal cell is expended on running this pump. In electrically active Nerve Cells, the propagation of an Action Potential leads to repeated entry of small amounts of Na⁺ and loss of small amounts of K⁺ (see below). Restoring these gradients consumes about two-thirds of the cell's total energy budget.

A major step forward in understanding the molecular mechanism of the sodium-potassium pump was made in 1957, when it was discovered that the optimal activity of the enzyme that hydrolyzes ATP to ADP and phosphate requires Na⁺ and K⁺. Furthermore, ouabain, a well-known inhibitor of the (Na⁺ + K⁺) pump, was shown to inhibit the ATPase as well. Thus, a link was established between the (Na⁺ + K⁺)-ATPase and the sodium-potassium pump. However, the definitive proof that ATP hydrolysis directly provides the energy for the pump came from studies on sealed erythrocyte ghosts. In these experiments, the ion concentrations on either side of the membrane could be varied to observe how these changes affected Ion transport and ATP hydrolysis. It was found that: 1) the transport of sodium and potassium ions and ATP hydrolysis are tightly coupled, so that neither process can occur without the other; 2) ion transport and ATP hydrolysis occur only when Na⁺ and ATP are present inside the ghosts, and K⁺ is outside; 3) ouabain inhibits the ATPase only when applied to the outside of the ghosts, where it competes for the K⁺-binding site; 4) for every molecule of ATP hydrolyzed (one ATPase molecule can hydrolyze 100 ATP molecules per second), three sodium ions are pumped out and two potassium ions are pumped in (Fig. 6-48).

Fig. 6-48. The (Na⁺ + K⁺)-ATPase actively pumps Na⁺ out and K⁺ into the cell against their electrochemical gradients. For every molecule of ATP hydrolyzed inside the cell, three Na⁺ ions are pumped out of the cell and two K⁺ ions are pumped in. Ouabain, a specific pump inhibitor, and K⁺ ions compete for a common site on the extracellular side of the ATPase.

These experiments provided unequivocal proof that ATP provides the energy for pumping sodium and potassium ions across the plasma membrane; however, it remained unclear exactly how ATP hydrolysis is coupled to ion transport. Subsequent studies showed that, in the presence of Na⁺, the terminal phosphate group of ATP is transferred to an aspartic acid residue in the ATPase molecule. The ATPase-bound phosphate group is then hydrolyzed in the presence of K⁺, and it is this final step that is inhibited by ouabain. Na⁺-dependent phosphorylation is coupled to a conformational change in the ATPase, which leads to the extrusion of sodium from the cell. Conversely, the subsequent K⁺-dependent dephosphorylation drives the transport of potassium ions into the cell and returns the ATPase to its original state (Fig. 6-49).

The (Na⁺ + K⁺) pump in erythrocyte ghosts can be made to run in reverse to synthesize ATP. If the concentration gradients of sodium and potassium ions are experimentally increased to such an extent that the energy of their electrochemical gradients exceeds the chemical energy of ATP hydrolysis, the ions will flow down their electrochemical gradients, and ATP will be synthesized from inorganic phosphate and ADP by the sodium-potassium ATPase. Thus, the phosphorylated form of the ATPase (state 2 in Fig. 6-49) can relax either by transferring its phosphate to ADP (from state 2 to state 1) or by changing its conformation (from state 2 to state 3). Whether the overall free-energy change is used to synthesize ATP or to pump Na⁺ out of the erythrocyte ghosts depends on the relative concentrations of ATP, ADP, and phosphate, and on the electrochemical gradients of sodium and potassium ions.

Fig. 6-49. A model of (Na⁺ + K⁺)-ATPase function. The binding of Na⁺ (1) and subsequent phosphorylation (2) of the ATPase from the cytoplasmic side induce a conformational change in the protein, which translocates Na⁺ across the membrane and releases it into the extracellular space (3). Then, the binding of K⁺ on the outer surface (4) and subsequent dephosphorylation (5) return the protein to its original conformation, translocating K⁺ across the membrane and releasing it into the Cytoplasm (6). These conformational changes are analogous to the 'ping-pong' transitions shown in Fig. 6-47, except that here the conformational transitions are driven by Na⁺-dependent phosphorylation and K⁺-dependent dephosphorylation of the protein, enabling it to perform useful work. For simplicity, only one binding site for Na⁺ and one for K⁺ are shown. In the actual pump, there are likely three Na⁺-binding sites and two K⁺-binding sites.

Once the (Na⁺ + K⁺)-ATPase was purified, it was found to consist of two subunits: a large transmembrane subunit (about 1000 amino acid residues long) that spans the bilayer multiple times and possesses catalytic activity, and an associated smaller glycoprotein. The larger subunit has binding sites for Na⁺ and ATP on its cytoplasmic side, and for K⁺ and ouabain on its extracellular side. Furthermore, it is reversibly phosphorylated and dephosphorylated. The function of the glycoprotein is unknown. A functional sodium-potassium pump can be reconstituted from the purified complex: the ATPase is solubilized in detergent, purified, and mixed with appropriate Phospholipids. Upon removal of the detergent, membrane vesicles are formed that, in the presence of ATP, pump Na⁺ and K⁺ in opposite directions (see Fig. 6-21).

6.4.6. The (Na⁺ + K⁺)-ATPase is required to maintain osmotic balance and stabilize cell volume [23]

Because the (Na⁺ + K⁺)-ATPase pumps out three positively charged ions for every two it pumps into the cell, it is electrogenic. This means that a net current flows across the membrane, generating an electrical potential that is negative on the inside of the cell relative to the outside. However, this direct effect of the pump contributes no more than 10% to the membrane potential. The remaining 90% of the potential is generated, as we shall see, indirectly by the pump's action and is due to the difference in K⁺ concentration on either side of the membrane. The high concentration of potassium inside the cell is required to balance the large net negative charge of the cell's fixed anions—the multitude of negatively charged organic molecules that reside inside the cell and cannot cross the plasma membrane.

Figure 6-50. Response of human red blood cells to changes in osmotic conditions in the extracellular fluid. Water is absorbed into or leaves the cell down its concentration gradient because the plasma membrane is highly permeable to water molecules. This process is called osmosis. When cells are placed in a hypotonic solution (i.e., a solution with a low salt concentration and, consequently, a high water concentration), water molecules move into the cells, causing them to swell and burst (lyse). Conversely, when cells are placed in a hypertonic solution, they shrink (see also Scheme 2-1).

(Na⁺ + K⁺)-ATPase plays a direct role in regulating cell volume. It controls the concentration of solutes inside the cell, and therefore the osmotic forces that cause the cell to swell or shrink (Figure 6-50). As explained in Scheme 6-1, the solutes inside the cell (including fixed anions and the accompanying cations required to balance their charge) maintain a large osmotic gradient that draws water into the cell. In animal cells, this effect is counteracted by the high concentrations of inorganic ions (mostly Na⁺ and Cl^-) in the extracellular fluid. The sodium-potassium ATPase maintains osmotic equilibrium by pumping out Na⁺ ions that leak into the cell down their electrochemical gradient; Cl^- is kept out of the cell by the membrane potential.

The crucial role of the (Na⁺ + K⁺)-ATPase in regulating cell volume is demonstrated by the fact that when animal cells are treated with ouabain, which inhibits the sodium-potassium ATPase, they swell and burst. Cells can solve osmotic problems in other ways as well. In many bacteria and plant cells, the plasma membrane is surrounded by a semi-rigid wall that prevents the cell from bursting. In amoebae, excess water entering by osmosis is collected in contractile vacuoles, which periodically discharge their contents to the exterior (Scheme 6-1). However, in most animal cells, the primary role in preventing bursting due to osmotic pressure belongs to the (Na⁺ + K⁺)-ATPase.

6.4.7. Some Ca²⁺ pumps are also membrane-bound ATPases [24]

The concentration of Ca²⁺ ions in the cytosol of Eukaryotic cells is maintained at a much lower level (~10^-7 M) compared to its concentration outside the cell (~10^-3 M). Even a small influx of Ca²⁺ from the outside significantly increases the concentration of free Ca²⁺ in the cytosol, and the flow of Calcium Ions rushing down their electrochemical gradient in response to extracellular signals is one of the ways these signals are transmitted across the plasma membrane (see Section 12.3.7). The Ca²⁺ gradient is partially maintained by Ca²⁺ pumps in the plasma membrane that actively transport calcium out of the cell. One such pump is known to be an ATPase, while another operates as an antiport driven by the Na⁺ electrochemical gradient (see below).

Scheme 6-1. Intracellular water balance: the problem and its solution.

The best-understood Ca²⁺ pump is the membrane-bound ATPase of the sarcoplasmic reticulum in Muscle cells. The sarcoplasmic reticulum forms a network of fine tubules in the cytoplasm of muscle cells and serves as an intracellular store of calcium ions. When an action potential depolarizes the muscle cell membrane, Ca²⁺ is released from the sarcoplasmic reticulum into the cytosol, stimulating the muscle to contract (see Section 11.1.4). The calcium pump is responsible for pumping Ca²⁺ from the cytosol back into the sarcoplasmic reticulum. Like the sodium-potassium pump, the Ca²⁺ pump is an ATPase that is phosphorylated and dephosphorylated in each operational cycle, pumping two calcium ions into the sarcoplasmic reticulum for every molecule of ATP hydrolyzed. Because the Ca²⁺-ATPase is the predominant protein in the sarcoplasmic reticulum membrane (accounting for about 90% of the total membrane protein), it is relatively easy to purify. This protein consists of a single long polypeptide chain (about 1000 amino acid residues) that spans the membrane several times; when reconstituted into phospholipid vesicles, the calcium ATPase mediates Ca²⁺ transport coupled to ATP hydrolysis. DNA Cloning and sequencing experiments indicate Homology between the Ca²⁺-ATPase and the large catalytic subunit of the (Na⁺ + K⁺)-ATPase, suggesting an evolutionary relationship between these two ion pumps.

In non-muscle cells, organelles equivalent to the sarcoplasmic reticulum also contain a Ca²⁺-ATPase that pumps Ca²⁺ out of the cytosol. In response to specific extracellular signals, this sequestered Ca²⁺ is released back into the cytosol.

6.4.8. Membrane-bound enzymes that synthesize ATP are transport ATPases operating in reverse [25]

In the plasma membranes of bacteria, the inner membranes of mitochondria, and the thylakoid membranes of chloroplasts, enzymes are found that are very similar to the two transport ATPases discussed above. Here, however, they normally work in reverse. Instead of hydrolyzing ATP to drive ion transport, they catalyze the synthesis of ATP from ADP and phosphate, driven by a proton gradient across these membranes. The H⁺ gradient is generated during electron transport in Oxidative Phosphorylation (in aerobic bacteria and mitochondria) or Photosynthesis (in chloroplasts), or by a light-activated proton pump (Bacteriorhodopsin in Halobacterium). These enzymes, which normally synthesize ATP, are called ATP synthases. Like transport ATPases, they can work in either direction depending on conditions: they can either hydrolyze ATP to pump H⁺ across the membrane into the inner space, or synthesize ATP when H⁺ ions flow back through the enzyme. ATP synthases are responsible for producing almost all the ATP in most cells and are discussed in more detail in Chapter 9.

6.4.9. Active transport can be driven by ion gradients [26]

Many active transport systems are driven by the energy stored in ion gradients rather than by direct ATP hydrolysis. These operate as coupled transporters: some function as symports, others as antiports. In animal cells, the co-transported ion is usually Na⁺, whose electrochemical gradient provides the energy to actively transport a second solute. The Na⁺ that enters the cell during this process is pumped out by the (Na⁺ + K⁺)-ATPase, which, by maintaining the Na⁺ gradient, indirectly drives the transport. For example, intestinal and kidney epithelial cells contain a variety of symport systems driven by the transmembrane Na⁺ gradient. Each system is specific for transporting a small group of related sugars or amino acids into the cell. In these systems, the solute and sodium ions bind to different sites on the carrier protein; Na⁺ tends to enter the cell down its electrochemical gradient and, in doing so, "drags" the sugar or amino acid molecule into the cell with it. The greater the Na⁺ gradient, the faster the rate of solute uptake. Conversely, if the extracellular Na⁺ concentration is significantly reduced, solute transport stops. A hypothetical (and highly simplified) diagram of how such a symport system works is shown in Figure 6-51.

Figure 6-51. Principles of using a Na⁺ gradient to drive a glucose pump. The pump oscillates randomly between two states, "ping" and "pong", as in Figure 6-47. Na⁺ binds equally well to the protein in either conformation. The binding of Na⁺ induces an allosteric transition in the protein, greatly increasing its affinity for glucose. Because the Na⁺ concentration is higher outside the cell than in the cytosol, glucose binding to the pump is much more likely in the "pong" conformation. Therefore, the co-transport of Na⁺ and glucose into the cell (the "pong" → "ping" transition) occurs much more frequently than the reverse, resulting in net transport. By maintaining the Na⁺ gradient, the (Na⁺ + K⁺)-ATPase indirectly provides energy for this transport system. Transporters operating on this principle are said to mediate secondary active transport, whereas the ATPase mediates primary active transport.

In bacteria and plants, most active transport systems driven by ion gradients utilize H⁺ rather than Na⁺ as the co-transported ion. In particular, The active transport of most sugars and amino acids into bacterial cells is driven by an H⁺ gradient across the plasma membrane. The best-studied example of this type is the lactose carrier (permease). This transmembrane protein, consisting of a single polypeptide chain (about 400 amino acid residues), apparently spans the lipid bilayer at least nine times. It mediates H⁺-dependent symport: one proton is co-transported into the cell with each lactose molecule.

6.4.10. Antiports in the plasma membrane regulate intracellular pH [27]

Almost all vertebrate cells have a (Na⁺ + H⁺) exchanger in their plasma membrane. It plays a key role in maintaining the intracellular pH (pH_i), which is normally about 7.1 to 7.2. This exchanger couples the efflux of H⁺ ions to the influx of Na⁺ ions, thereby removing excess H⁺ ions generated by cellular metabolic reactions. The activity of the (Na⁺ + H⁺) exchanger is regulated by pH: for example, when the pH_i in chick muscle cells is above 7.7, the exchanger becomes inactive; as pH_i falls, the activity of the exchanger increases, reaching half its maximum activity at pH_i 7.4. This regulation is due to the binding of H⁺ to a regulatory site on the cytoplasmic side of the exchanger. The Importance of the (Na⁺ + H⁺) exchanger in maintaining pH_i was demonstrated by experiments with mutant fibroblasts lacking this exchanger: they died very rapidly when placed in acidic conditions, whereas normal fibroblasts survived. In many nucleated cells, a (Cl^- + HCO₃^-) exchanger, similar to the band 3 protein of THE RED BLOOD cell membrane (see Section 6.2.6), also plays an important role in maintaining pH_i. Like the (Na⁺ + H⁺) exchanger, the (Cl^- + HCO₃^-) exchanger is regulated by pH_i, but in the opposite direction. Its activity increases as pH_i rises (i.e., when the cytosol becomes too alkaline), increasing the rate of HCO₃^- efflux in exchange for Cl^-, thereby lowering pH_i.

There is evidence suggesting that the (Na⁺ + H⁺) exchanger may be involved not only in maintaining pH_i but also in transducing extracellular signals into intracellular ones. For example, most protein growth factors activate this type of antiport system during The stimulation of cell proliferation, increasing pH_i from 7.1 or 7.2 to about 7.3. In at least some cases, they do this indirectly by activating a specific protein kinase (protein kinase C—see Section 12.3.10), which in turn phosphorylates the exchanger. This increases the affinity of the H⁺-binding regulatory site, so that the exchanger remains active even at higher pH values. Mutant cells lacking the (Na⁺ + H⁺) exchanger, or cells treated with the drug amiloride, which inhibits it, fail to respond to growth factors. These findings suggest that the activation of the exchanger and the subsequent rise in pH_i play an important role in initiating cell proliferation. Similarly, the rise in pH_i that occurs upon Fertilization of sea urchin eggs, driven by the activation of the (Na⁺ + H⁺) exchanger, appears to stimulate DNA and Protein Synthesis. It remains unclear which intracellular proteins are responsible for the rise in pH_i in response to this activation.

6.4.11. Transcellular transport of solutes depends on the asymmetrical distribution of carrier proteins in epithelial cells [28]

In the plasma membrane of some epithelial cells involved in nutrient Absorption in the gut, carrier proteins are distributed asymmetrically, thereby facilitating the transcellular transport of absorbed solutes across the cell. As shown in Figure 6-52, proteins localized in the plasma membrane on the apical (absorptive) surface of the epithelial cell mediate Na⁺-dependent symport, importing nutrients into the cell. At the same time, Na⁺-independent proteins in the basal and lateral membranes allow nutrients to leave the cell down their concentration gradients. The Na⁺ gradient across the plasma membrane of these cells is maintained by the (Na⁺ + K⁺)-ATPase located in the basolateral region. Apparently, similar mechanisms are used by intestinal and renal epithelial cells to pump water molecules from one extracellular space to another.

Figure 6-52. The asymmetrical distribution of transport proteins in the plasma membrane of an intestinal epithelial cell results in the transcellular transport of glucose from the gut lumen, through the cell, and into the extracellular fluid (from where it enters the blood). Glucose enters the cell across the apical membrane via Na⁺-dependent glucose symport and leaves the cell (down its concentration gradient) by facilitated diffusion mediated by a different glucose carrier protein localized in the basal and lateral domains. The Na⁺ gradient that drives the glucose symport is maintained by the (Na⁺ + K⁺)-ATPase located in the plasma membrane of the basolateral domain; this enzyme keeps the intracellular Na⁺ concentration low. Adjacent cells are joined by impermeable junctions (called tight junctions) that serve two Functions in the transport process shown here. These junctions prevent solutes from leaking through the spaces between epithelial cells and help maintain a glucose gradient across the epithelial sheet. Tight junctions also act as barriers within the plasma membrane, preventing the diffusion of Membrane Proteins. This keeps the different carrier proteins restricted to their respective membrane domains (see Figure 6-36).

Figure 6-53. Active transport of sugars into bacterial cells by group translocation. A specialized "phosphotransferase protein system" in the bacterial membrane phosphorylates the sugar as it is transported across the membrane. Phosphoenolpyruvate, rather than ATP, serves as the phosphate donor.

In many epithelial cells, the surface area of the plasma membrane is greatly increased by the presence of thousands of microvilli, which project from the apical surface as thin, finger-like projections (Fig. 6-52). These microvilli can increase the total absorptive surface area up to 25-fold, thereby significantly enhancing the cell's transport capacity. The apical surface of an intestinal epithelial cell is also the site where immobilized hydrolytic enzymes involved in the final stages of food Digestion are localized. The increase in epithelial surface area provided by microvilli greatly facilitates the DIGESTION AND ABSORPTION of food.

6.4.12. Active transport in bacteria can occur via group translocation [29]

As we now know, active transport in cells can be driven by light (for example, in bacteriorhodopsin), ATP hydrolysis, or ion gradients. A fourth pathway used by some bacteria is to "trap" a molecule that has entered the cell via passive transport by chemically modifying it, which prevents the molecule from leaving by the same route. For example, after being transported across the plasma membrane of certain bacteria, sugars are phosphorylated. As a result of this modification, they become charged, cannot escape, and therefore accumulate inside the cell. Moreover, because the transported sugars are phosphorylated, the concentration of their unphosphorylated analogs inside the cell remains very low, so that the sugar concentration gradient continues to "push" these molecules into the cell. Because phosphate groups are transferred to solute molecules after their transport, this type of active transport is called group translocation. In most well-studied Examples, the phosphorylation mechanism is quite complex and tightly regulated. It involves at least four distinct membrane proteins, and phosphoenolpyruvate, rather than ATP, is used as the high-energy phosphate donor (Fig. 6-53).

6.4.13. Double-membrane bacteria possess transport systems that depend on water-soluble substrate-binding proteins [30]

As mentioned above, the plasma membranes of all bacteria contain carrier proteins that use a H⁺ ion gradient to pump various nutrients into the cell. However, many bacteria, including E. coli, also have an outer membrane surrounding them, through which solutes with a Molecular Weight of up to 600 daltons can pass relatively freely via various channel-forming proteins (collectively known as porins) (Fig. 6-54). In these bacteria, a two-component transport system is used to transport certain sugars, amino acids, and small Peptides across the inner (plasma) membrane, utilizing water-soluble proteins located in the periplasmic space between the two membranes. These periplasmic substrate-binding proteins bind specific molecules that need to be transported across the membrane. This binding induces a conformational change in the proteins, allowing them to bind to another component of the transport system—a transmembrane carrier protein located in the inner membrane (Fig. 6-55). Apparently, the substrate-binding proteins hand over their bound solute to the specific carrier protein, which then uses the energy of ATP hydrolysis to transport it across the inner membrane. These same periplasmic substrate-binding proteins also serve as receptors in chemotaxis—an adaptive process that allows bacteria to swim toward higher concentrations of a specific nutrient.

We now turn our attention back to channel-forming proteins.

Fig. 6-54. Schematic diagram of a small section of the double membrane of E. coli. The inner membrane is the cell's plasma membrane. Between the inner and outer lipid bilayer membranes lies a highly porous, resilient peptidoglycan layer composed of proteins and Polysaccharides that make up the Bacterial Cell wall. It is attached to lipoprotein molecules in the outer membrane and fills the periplasmic space, which contains various soluble proteins. The dark filaments On the surface of the outer membrane represent the polysaccharide chains of specific lipopolysaccharide molecules that form the outer monolayer of the outer membrane. For simplicity, only a few chains are shown. Bacteria with a double membrane are called Gram-negative because they do not stain dark blue with Gram stain. Bacteria with a single membrane (but thick cell walls), such as staphylococci or streptococci, retain the Gram stain and are called Gram-positive. Their single membrane is analogous to the inner membrane of Gram-negative bacteria.

6.4.14. Protein channels form Pores in the plasma membrane [31]

Unlike carrier proteins, protein channels (or channel-forming proteins) form water-filled pores in membranes. The channel-forming Proteins of the outer membranes of bacteria (as well as Mitochondria and chloroplasts) form large, relatively non-specific pores, whereas in the plasma membranes of animal and plant cells, these pores are small and highly specific. Almost all protein channels serve for the specific transport of ions and are discussed here under the term Ion Channels. Ion channels allow the passage of approximately 10⁶ ions per second, which is more than 100 times faster than the transport rate of any known carrier protein. Ion channels are never coupled to an energy source; the transport they mediate is always passive ("downhill"), allowing specific ions, mainly Na⁺, K⁺, Ca²⁺, or Cl^-, to diffuse down their electrochemical gradients across the lipid bilayer.

Fig. 6-55. Transport system dependent on periplasmic substrate-binding proteins in double-membrane bacteria. Solutes diffuse through channel-forming proteins (porins) in the outer membrane and bind to periplasmic substrate-binding proteins. This binding induces conformational Changes in the proteins, enabling them to bind to plasma membrane carrier proteins, which then capture the substrate and actively transport it across the bilayer. This step is mediated by ATP hydrolysis. Peptidoglycans are omitted for simplicity. Their porous structure allows substrate-binding proteins and water-soluble substances to move by simple diffusion.

Protein channels in the plasma membrane exhibit ion selectivity, meaning they allow only specific types of ions to diffuse through them. The pores must be narrow enough for ions to come into close contact with their walls, ensuring that only those of appropriate size and charge can pass. Along this pathway, ions likely have to shed most or even all of their associated water molecules. These two factors limit the rate of diffusion through the channel and make it a selective filter that permits the passage of only specific ions. Consequently, as the ion concentration increases, the flux of ions through the channel increases proportionally, but only up to a certain limit.

Another feature that distinguishes ion channels from simple water-filled pores is that they are not open all the time. As shown in Fig. 6-56, channels have "gates" that open briefly and then close. In most cases, the gates open in response to specific membrane perturbations. Currently, the most well-known of these are Changes in membrane potential (voltage-gated channels), mechanical stimulation (mechanically gated channels—see Section 19.6.3), or the binding of signaling molecules (Ligand-gated channels). Signaling ligands can be either extracellular messengers called Neurotransmitters (transmitter-gated channels) or intracellular messengers, such as ions (ion-gated channels—see Section 21.1.1), nucleotides (nucleotide-gated channels—see Section 3.3.5), or GTP-binding regulatory proteins (G-protein-gated channels—see Section 12.3.12).

Fig. 6-56. Schematic diagram of a gated ion channel in closed and open conformations. The transmembrane protein, shown in cross-section, forms a water-filled continuous pore in the lipid bilayer when the gate is open. The walls of the pore apparently consist of hydrophilic amino acid residues, while hydrophobic residues interact with the lipid bilayer. The ion selectivity of the channel is determined by its narrowest region. The transient opening of the gate is triggered by a specific membrane perturbation, which varies for different channels (discussed in the text). The exact locations of the gate and the ion selectivity filter remain unknown for most channels.

To date, about 50 types of ion channels are known, and new types continue to be discovered. Ion channels are responsible for the electrical excitability of nerve and muscle cells; they mediate most forms of electrical signaling in The Nervous system. A single nerve cell typically contains more than five types of ion channels. However, such channels are not unique to electrically excitable cells. They are present in all animal cells and are found in some PLANT CELLS AND microorganisms. For example, these channels are responsible for leaf closure in response to Touch in Mimosa, or for the reversal of swimming direction in the unicellular Paramecium.

Apparently, the most common ion channels are those permeable mainly to K+. They are found in the plasma membranes of almost all animal cells. Because their opening likely does not require specific membrane perturbations, they are sometimes called potassium leak channels. These channels play a key role in establishing the membrane potential—the electrical voltage difference observed across all types of membranes.

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6.4.15. The membrane potential depends on K⁺ leak channels and the K⁺ gradient across the membrane [32]

The membrane potential depends on the distribution of ions on both sides of the membrane. As mentioned above, the (Na⁺ + K⁺)-ATPase helps establish osmotic equilibrium in the cell by maintaining a low intracellular Na⁺ concentration. Because the sodium concentration inside the cell is low, an excess of other cations is required to balance the charge of fixed cellular anions—the negatively charged organic molecules confined within the cell. This role is primarily played by potassium ions, thanks to K⁺ leak channels, which allow these ions to cross the membrane freely and be drawn into the cell by the negative charge of the fixed anions (even in the absence of any activity by the (Na⁺ + K⁺)-ATPase). Thus, an equilibrium is established in which the electrical force pulling potassium ions into the cell is balanced by the tendency of K⁺ to flow out of the cell down its concentration gradient. The membrane potential is an expression of this electrical energy, and its magnitude can be calculated from the steepness of the K⁺ concentration gradient required to balance the electrical forces. To illustrate this with an example, suppose that there is initially no electrical gradient across the plasma membrane (i.e., the membrane potential is zero), but the K⁺ concentration inside the cell is higher (to balance the charge of fixed anions) than outside. In this case, potassium ions will tend to leave the cell through the K⁺ leak channels down their concentration gradient. As these ions leave the cell, a net negative charge is left behind, creating an electrical field—in other words, a membrane potential that tends to pull K⁺ ions back into the cell. The efflux of potassium ions will stop as soon as the generated membrane potential reaches a value at which the electrical driving force acting on the potassium ions exactly balances The Effect of the K⁺ concentration gradient, i.e., when the electrochemical gradient for potassium ions becomes zero. In a similar manner, an equilibrium is simultaneously established for Cl^- ions, but because they are negatively charged, they are held outside the cell. The equilibrium conditions under which there is no net current flow across the membrane define the resting membrane potential of the cell. There is a simple but very important formula that quantitatively expresses these equilibrium conditions: the Nernst equation. As shown in Scheme 6-2, it allows the resting membrane potential to be calculated if The ratio of internal to external ion concentrations is known.

Scheme 6-2. Derivation of the Nernst equation.

For a membrane potential to be established, only a very small number of ions need to be transferred across the membrane. Thus, the membrane potential can be thought of as a movement of charge that leaves bulk ion concentrations virtually unchanged. As a result, only a tiny redistribution of positive and negative ions occurs between the two sides of the membrane (Fig. 6-57). Moreover, this movement of charge occurs extremely rapidly, within a few milliseconds or even faster.

Let us consider what happens if the (Na⁺ + K⁺)-ATPase is inactivated. First of all, there will be a small, rapid drop in the membrane potential because the (Na⁺ + K⁺) pump is electrogenic and, when active, contributes to the membrane potential (see Section 6.4.6). However, turning off this pump does not lead to the disappearance of the main component of the Resting Potential, which is based on the potassium ion balancing mechanism (as described above). It persists as long as the Na+ concentration inside the cell remains low, i.e., for many minutes. But since the plasma membrane, although poorly, is still permeable to Na⁺ ions, Na⁺ will slowly enter the cell down its electrochemical gradient. The influx of sodium reduces the membrane potential and, thus, causes an additional efflux of K+ ions from the cell. During this time, osmotic equilibrium is disrupted (see Section 6.4.6), but if the cell does not burst, a new state of equilibrium will eventually be established between Na⁺, K⁺, and Cl^- ions. In this case, the membrane potential will be much lower than in a normal cell with an active (Na⁺ + K⁺) pump.

Exact balance of charges on both sides of the membrane; membrane potential = 0

A few positively charged ions (colored) have crossed the membrane from right to left, leaving negatively charged counterions (colored) on the other side; the membrane potential is now non-zero

Fig. 6-57. A small flow of ions carries sufficient charge to create a large change in membrane potential. The ions that determine the membrane potential are located near the membrane, held by interaction with counterions on the other side of the membrane. For a typical cell, 1 microcoulomb of charge (6 x 10¹² monovalent ions) per 1 cm² of membrane transferred from one side to the other will change the membrane potential by approximately 1 V. This means that for a spherical cell with a diameter of 10 µm, the efflux of only 1/100,000 of the K⁺ ions from the cell will change the potential by 100 mV.

The potential difference across the plasma membrane of a resting cell varies depending on the Organism or cell type from -20 mV to -200 mV. Although the K⁺ gradient always makes the largest contribution to this potential, the gradients of other ions also have a significant effect (plus the non-equilibrium effects of active ion pumps). The more permeable the membrane is to a given ion, the more the membrane potential depends on the equilibrium conditions for that ion. Consequently, almost any change in membrane permeability to ions results in A change in the membrane potential. This is a key principle linking the electrical excitability of cells to the activity of ion channels.

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6.4.16. Voltage-Gated ion channels are responsible for the electrical excitability of nerve and muscle cells [33]

The plasma membranes of electrically excitable cells (mainly nerve and muscle cells) contain many voltage-gated ion channels responsible for generating action potentials—rapid, transient, self-propagating electrical excitations of the membrane. This process begins with membrane depolarization—a shift in the membrane potential to a less negative value. A stimulus that causes momentary partial depolarization immediately opens voltage-gated Na⁺ channels, allowing a small amount of Na⁺ to enter the cell. The influx of positive charges, in turn, depolarizes the membrane further, leading to the opening of additional Na⁺ channels, which admit more sodium ions and thus cause further depolarization. This process continues until the potential of the local membrane region shifts from its resting value of about -70 mV to the Na⁺ equilibrium potential of approximately +50 mV (see Diagram 6-2). At this value, where the net Electrochemical Potential of Na⁺ is zero, the cells would reach a new state of equilibrium (or rest) in which all sodium channels are permanently open, if the open conformation of the channel were stable. However, cells are protected from such continuous electrical spasm because Na⁺ channels are controlled by an automatic inactivating mechanism. They close rapidly after opening, despite the continued depolarization of the membrane. In this inactivated state, the channels cannot reopen until a few milliseconds have passed after the membrane potential returns to its original negative value. Fig. 6-58 schematically illustrates these three distinct states of the voltage-gated Na⁺ channel—closed but not inactivated, open, and inactivated. Fig. 6-59 shows how this channel functions during the rise and fall of an action potential.

Fig. 6-58. A voltage-gated Na⁺ channel can exist in at least one of three states (conformations). Internal forces, represented here as the interaction of charges on different sides of the channel, stabilize each state and protect it from the effects of small perturbations. However, interactions with other molecules can trigger a transition from one channel state to another. The state with the lowest energy is determined by the membrane potential, as different conformations have different charge distributions. In the resting state (highly polarized membrane), the channel is closed but not inactivated. This is the most stable state with the lowest Free energy. Upon membrane depolarization, the open conformation will have lower energy, and therefore the channel will open. However, the Free energy of the inactivated state is even lower, and after some random period of time in the open state, the channel transitions into the inactivated state. Thus, the open conformation corresponds to a metastable state that exists only briefly. Black arrows show The sequence of events during membrane depolarization, and the red arrow indicates the return to the original state of lowest free energy after membrane repolarization.

Fig. 6-59. Induction of an action potential by a short electrical pulse (shown in the upper graph). The pulse partially depolarizes the membrane (middle graph). The solid line on the membrane potential graph shows the generation of an action potential during the opening and subsequent inactivation of voltage-gated Na⁺ channels. The membrane potential automatically returns to its original value of -70 mV when the Na⁺ channels close, due to the continuous efflux of K⁺ through K⁺ channels. The generation of a second action potential is impossible until the Na⁺ channels (their states are shown below) return to the closed but not inactivated state (see Fig. 6-58). Until then, the membrane remains refractory to stimulation. The dashed line shows the relaxation of the membrane potential under weak stimuli that do not lead to channel opening.

The process of action potential generation described above applies to only a small patch of the plasma membrane. However, the self-amplifying depolarization of this region is sufficient to depolarize neighboring areas of the membrane, which are then drawn into the same cycle of action potential generation. In this way, the action potential propagates from the initial site of depolarization along the entire plasma membrane. A more detailed Discussion of the functions and Properties of the action potential is provided in Chapter 19.

Neurons and muscle cells contain several thousand voltage-gated Na⁺ channels. The current flowing through the membrane is the sum of all microcurrents through each individual channel. The total current can be recorded using a microelectrode (see Section 19.2.3). However, it is also possible to record currents flowing through individual channels. This is done using a special technique that allows isolating a very small patch of the membrane containing only a few channels and then recording the currents flowing through it. This technique (patch-clamp) provides a more detailed picture of how these channels function.

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6.4.17. Recording currents passing through an isolated membrane patch shows that individual Na⁺ channels open in an "all-or-none" fashion [34].

The Development of the patch-clamp technique has significantly advanced The Study of ion channels. Using this method, it is possible to analyze transport through a single channel protein molecule located in a small patch of membrane (Fig. 6-60) and to record signals from ion channels in any cell type, including electrically non-excitable ones. Many of these cells, such as Yeast, are too small to be studied by traditional electrophysiological Methods involving the insertion of intracellular electrodes.

Patch-clamp recordings have shown that individual Na⁺ channels open in an "all-or-none" fashion. In the open state, their conductance does not change, and the times of opening and closing are random. Therefore, the total current flowing through a large population of Na⁺ channels in the whole-cell membrane reflects not the degree of opening of an individual channel, but rather the average probability that it is open (Fig. 6-61).

The phenomenon of voltage-gated opening and closing can be understood from simple physical principles. Inside a resting nerve or muscle cell, the electrical potential is 50 to 100 mV lower than outside. Such a potential difference across the membrane might seem insignificant, but considering that the membrane is only about 5 nm thick, the resulting gradient is approximately 100,000 V/cm. Consequently, membrane proteins experience an extremely strong electrical field. Naturally, membrane proteins, like all others, have A number of charged groups on their surface. The electrical field exerts forces on the molecular structure. Many membrane proteins are not significantly affected by changes in the electrical field across the membrane. Ion channels, however, have evolved an exquisitely balanced sensitivity to the electrical field: they can adopt several alternative conformations, the stability of which depends on the strength of the electrical field. Small perturbations do not affect the conformation of the channels, but sufficiently strong influences, such as random thermal motions of surrounding molecules, can trigger a transition to another conformation (see Fig. 6-58).

The functions of voltage-gated Na⁺ channels are specifically blocked by two paralytic poisons: tetrodotoxin (TTX), obtained from pufferfish, and saxitoxin, which is isolated from certain species of marine dinoflagellates. Due to their high affinity and specificity, these toxins have proven indispensable for pharmacological studies, counting the number of Na⁺ channels in the membrane, and purifying these channels. It has been shown that the plasma membrane of Skeletal Muscle cells contains only a few hundred Na⁺ channels per 1 µm², i.e., one channel per 10,000 phospholipid molecules. Despite such a low channel density, these membranes are electrically excitable because each channel has a high conductance, passing more than 8,000 ions per millisecond.

In 1984, the DNA nucleotide sequence determining the STRUCTURE OF THE voltage-gated Na⁺ channel (from electric eel) was determined. It was found to encode a single long polypeptide chain (about 1800 amino acid residues) containing four homologous transmembrane domains (each of which contains six predicted α-helices spanning the membrane). These helices apparently interact with one another to form the walls of a water-filled pore. More recently, the gene encoding a voltage-gated Ca²⁺ channel was sequenced. It turned out to be another long polypeptide whose Primary Structure is highly homologous to that of the Na⁺ channel. It is highly likely that voltage-gated ion channels belong to a family of evolutionarily and structurally related proteins. In each of these channels, one of the predicted transmembrane segments contains positively charged amino acid residues spaced at regular intervals. It is possible that these residues collectively function as a voltage sensor, causing the channel to open in response to sufficient membrane depolarization (see Fig. 6-58).

Fig. 6-60. Recording signals using the patch-clamp technique. Due to the tight seal between the micropipette and the membrane, current can flow only through the channels present in the membrane patch covering the pipette tip. The current flow can be recorded either as in case A (in an intact cell) or as in case B (with the membrane patch excised). The advantages of option B lie in the ease of changing conditions on either side of the membrane to study the effects of different solutions on channel behavior. The orientation of the excised membrane patch can also be reversed (see also Figs. 4-33 and 4-34).

Fig. 6-61. Recording of the current flowing through a single voltage-gated Na⁺ channel in a tiny patch of the plasma membrane of an embryonic rat muscle cell (see Fig. 6-60). The membrane is depolarized by a step (A). Three current records (B) are from three experiments with the same membrane patch. Each major shift in current corresponds to the opening and closing of a single channel. Comparison shows that the open and closed times vary widely, whereas the rate of charge flow through the open channel is nearly constant. The small fluctuations in the current records are electrical noise from the recording apparatus. The aggregate current recorded in 144 repeated trials is shown in C. It is equivalent to the Na⁺ current through a relatively large patch of membrane containing 144 channels. Comparison of B and C shows that the aggregate current reflects the probability that an individual channel will open. This probability decreases with time as the channels in the depolarized membrane transition to an inactivated conformation. The kinetics of channel opening and inactivation in embryonic muscle cells are much slower than in a typical nerve cell. (From J. Patlak and R. Horn, J. Gen. Physiol., 79, 333-351, 1982, by permission of the Rockefeller University Press.)

Much more is known about the structure of another class of ion channels, which open in response to the binding of specific neurotransmitters rather than to changes in membrane potential. These transmitter-gated ion channels also belong to a family of related proteins. However, unlike voltage-gated Na⁺ and Ca²⁺ channels, each of which is formed by a single long polypeptide chain, all transmitter-gated ion channels studied to date are constructed from several homologous subunits.

6.4.18. The Acetylcholine Receptor Is a Transmitter-Gated Cation Channel [35]

Transmitter-gated ion channels are specialized for converting extracellular chemical signals into electrical signals. They are typically located at specialized junctions (called chemical synapses) between nerve cells and their target cells. These channels are concentrated in the plasma membrane of the target cell in the synaptic region. The channels can open transiently in response to the binding of a neurotransmitter released from the nerve terminal, thereby altering the permeability of the postsynaptic membrane of the target cell (Fig. 6-62). Unlike voltage-gated channels, which are responsible for generating action potentials, transmitter-gated channels are relatively insensitive to the membrane potential and are therefore incapable of self-amplifying excitation. Instead, they alter membrane permeability and thereby influence the membrane potential. The magnitude of this change depends on The amount of transmitter released at the synapse and the duration of its presence there. Clearly, an action potential can only be generated if voltage-gated channels are also present in the same target cell membrane.

In addition to its characteristic ion selectivity, each transmitter-gated channel has a highly specific binding site for its neurotransmitter. The best-studied example of a transmitter-gated channel is the acetylcholine receptor of skeletal muscle cells. This channel opens transiently in response to acetylcholine, a neurotransmitter released from the nerve terminal at the Neuromuscular Junction (see Section 19.3.1). The acetylcholine receptor holds a special place in The history of ion channel research. It was the first ion channel to be purified, the first to have its complete Amino Acid Sequence determined, the first to be shown to function after reconstitution in a synthetic lipid bilayer, and the first for which the electrical signal of a single opening channel was recorded. The gene for this channel was also the first channel protein gene to be isolated, cloned, and sequenced. Success in studying this receptor was possible for at least two reasons. First, there is an extraordinarily rich source for its isolation: the electric Organs of electric fish and rays. These organs are modified Muscles adapted to deliver an electric Shock to prey. Second, certain neurotoxins, such as α-bungarotoxin from the venom of certain snakes, bind to this receptor with high affinity (K_a = 10⁹ liters/mole) and specificity, and can be used to purify it by Affinity Chromatography. Using fluorescently or radioactively labeled α-bungarotoxin, it has been shown that acetylcholine receptors are densely packed in the plasma membrane of muscle cells at the neuromuscular junction (about 20,000 receptors per μm²), whereas only a few such receptors are found elsewhere in the same membrane.

Fig. 6-62. A chemical synapse. An arriving action potential at the nerve terminal stimulates the release of neurotransmitter, which is stored in secretory vesicles and released from the cell when the vesicles fuse with the plasma membrane of the nerve terminal. The released neurotransmitter binds to transmitter-gated ion channels concentrated in the plasma membrane of the postsynaptic cell, opening them. The resulting ion flow alters the membrane potential of the target cell, thereby transmitting the nerve signal.

Occupied and closed Occupied and open

Fig. 6-63. Three conformations of the acetylcholine receptor. The binding of two acetylcholine molecules opens the gate of this transmitter-gated ion channel. However, even with acetylcholine bound, the receptor apparently remains open for only a short time before closing. Acetylcholine then dissociates from the receptor, returning it to its original state.

The acetylcholine receptor is a glycoprotein composed of five transmembrane Polypeptides, two of one type and three others, encoded by four different genes. Because these four genes show close homology, they are thought to have arisen from a single ancestral gene. The two identical polypeptides in the pentamer contain the acetylcholine-binding sites. When two transmitter molecules bind to the pentameric complex, they induce a conformational change that opens the channel. The channel remains open for about 1 millisecond and then closes. Apparently, as with the voltage-gated Na⁺ channel, the open state is short-lived and rapidly transitions to a closed state of lower free energy (Fig. 6-63). Acetylcholine molecules then dissociate from the receptor complex and are hydrolyzed by a specific enzyme (acetylcholinesterase). Once freed of the bound neurotransmitter, the acetylcholine receptor returns to its original resting state.

Fig. 6-64. A model for the structure of the water-filled transmembrane pore formed by the five homologous subunits (α, α, β, γ, δ) of the acetylcholine receptor (A). Note that both α subunits contain an acetylcholine-binding site and that the bulk of the receptor is located in the extracellular space. Each subunit consists of ~500 amino acid residues. The M_r of the receptor is ~300,000 Da. The polypeptide chain of each subunit is thought to cross the lipid bilayer as four α-helices (B). One of these helices (shown in color) contains more polar amino acid residues than the others and is thought to line the water-filled pore when the five subunits assemble (A).

Electron Microscopy and low-angle X-Ray Diffraction have been used to study the structure of the acetylcholine receptor, but a definitive answer to how the transmembrane hydrophilic channel is formed is still lacking. Several models have been proposed, based primarily on the Amino acid sequences of the subunits. One model is shown in Fig. 6-64. The presence of clusters of negatively charged amino acid residues lining the channel opening probably explains the well-known fact that negatively charged ions cannot pass through the channel, whereas positively charged ions up to 0.65 nm in size can. The channel is penetrated mainly by Na⁺ and K⁺ ions, along with some Ca²⁺. There is no strict selectivity among cations, so the flux of each ion through the channel is determined mainly by its concentration and electrochemical driving force. Because the voltage gradient balances the concentration gradient of K⁺ across the membrane at the resting potential, the driving force for K⁺ ions is close to zero (see Scheme 6-2). In contrast, for Na⁺ ions, both the voltage gradient and the concentration gradient act in the same direction, driving the ions into the cell. The same is true for Ca²⁺, but its extracellular concentration is much lower than that of sodium ions, and thus THE CONTRIBUTION OF Ca²⁺ to the total current is negligible. Therefore, the opening of acetylcholine receptor channels leads to a large influx of Na²⁺ ions (with a maximum rate of about 30,000 ions per channel per millisecond). This current depolarizes the membrane, which serves as a signal for Muscle contraction, as described below.

The DNA nucleotide sequences encoding the subunits of several different transmitter-gated ion channels have also been determined. The deduced amino acid sequences are homologous to one another and to the corresponding subunits of the acetylcholine receptor, indicating an evolutionary relationship among these ion channels.

6.4.19. Neuromuscular Transmission Involves the Sequential Activation of at Least Four Different Sets of Gated Channels [36]

The crucial role of gated ion channels in the function of electrically excitable cells can be illustrated by the stimulation of a muscle cell to contract by an incoming Nerve Impulse. This seemingly simple response involves the sequential opening and closing of at least four different sets of gated channels, all occurring in less than 1 second (Fig. 6-65).

1. The process begins when a nerve impulse reaches the nerve terminal and depolarizes its plasma membrane. This depolarization transiently opens voltage-gated Ca²⁺ channels in this membrane. Because the concentration of Ca²⁺ outside the cell is more than 1000 times higher than the concentration of free Ca²⁺ inside, calcium ions rush into the nerve terminal. The resulting increase in Ca²⁺ concentration in the cytosol of the nerve terminal stimulates the local release of acetylcholine into the synaptic cleft.

2. The released acetylcholine binds to acetylcholine receptors on the plasma membrane of the postsynaptic muscle cell, transiently opening their cation channels. The resulting influx of Na⁺ causes a local depolarization of the muscle cell membrane.

3. The depolarization of the muscle cell plasma membrane opens voltage-gated Na⁺ channels in this membrane, allowing even more Na⁺ ions to enter. This amplifies the membrane depolarization, which in turn opens neighboring voltage-gated Na⁺ channels, ultimately generating a wave of depolarization (an action potential) that propagates to cover the entire muscle membrane.

4. The generalized depolarization of the muscle cell plasma membrane causes Ca²⁺ channels in the sarcoplasmic reticulum membrane to open transiently, releasing Ca²⁺ into the cytosol. This increases the intracellular Ca²⁺ concentration, triggering the contraction of myofibrils in the muscle cell (see Section 11.1.11). It is not yet fully understood how voltage changes in the muscle plasma membrane signal the opening of voltage-gated Ca²⁺ channels in the sarcoplasmic reticulum membrane. Another possibility is that depolarization of the muscle plasma membrane activates Inositol phospholipid-mediated signaling pathways, as discussed in Chapter 12.

Fig. 6-65. Schematic diagram of a neuromuscular junction, showing how several gated channels participate in stimulating muscle contraction by a nerve impulse. The channels are numbered in the sequence in which they open (see text). The Mechanism of opening of Ca²⁺ channels in the sarcoplasmic reticulum is unknown.

6.4.20. Ionophores Increase the Ion Permeability of Membranes [37]

Ionophores are small hydrophobic molecules that dissolve in lipid bilayers and increase their permeability to ions. Most ionophores are synthesized by microorganisms (likely as weapons against competitors), and some are used as Antibiotics. Ionophores are widely used in cell biology to increase membrane permeability to specific ions in studies on synthetic bilayers, cells, and cellular organelles. There are two classes of ionophores: mobile ion carriers and channel-forming ionophores (Fig. 6-66). Both types of ionophores work by shielding the charge of the transported ion so that it can pass through the hydrophobic interior of the lipid bilayer. Since ionophores are not coupled to any energy source, they merely permit ions to move down their electrochemical gradients.

An example of a mobile ion carrier is valinomycin. It is a polymer that increases membrane permeability to K⁺ ions. Valinomycin has a ring-like structure. The outer hydrophobic portion of its molecule consists of valine side chains and contacts the hydrocarbon core of the lipid bilayer. The inner polar region is just the right size to accommodate a single potassium ion (Fig. 6-67). Valinomycin transports K⁺ down its electrochemical gradient; it binds the ion on one side of the membrane, diffuses with it across the bilayer, and releases it on the other side.

Another example of a mobile ion carrier is the ionophore A23187, which transports divalent cations such as Ca²⁺ and Mg²⁺. This ionophore typically acts as an ion-exchange shuttle: for every divalent cation it imports into the cell, it exports two H⁺ ions from the cell. When cells are exposed to ionophore A23187, Ca²⁺ ions rush into the cytosol down a steep electrochemical gradient. Therefore, ionophore A23187 is widely used in cell biology to increase the concentration of free Ca²⁺ in the cytosol, thereby mimicking certain mediator-driven signal Transduction pathways in the cell (see Section 12.3.10).

If the membrane Temperature drops below its freezing point, mobile carriers can no longer diffuse through the lipid bilayer, and ion transport ceases. The presence of such a temperature dependence indicates that the ionophore in question is a mobile carrier. Conversely, if ion transport continues even in a frozen bilayer, it can be concluded that it is mediated by a channel-forming ionophore.

An example of this type of ionophore is gramicidin A. It is a linear polypeptide consisting of 15 amino acid residues, all of which have hydrophobic side chains. Two gramicidin molecules are thought to dimerize in the bilayer to form a transmembrane channel that allows monovalent cations (H⁺ most easily, K⁺ less so, and Na⁺ with difficulty) to flow down their electrochemical gradients. Such dimers are unstable: they constantly form and dissociate, so the average open time of the channel is about 1 s. In the presence of a large electrochemical gradient, gramicidin A can conduct about 20,000 cations per open channel in 1 millisecond, which is 1,000 times more than a single mobile carrier molecule can transport in the same time. Gramicidin is an antibiotic produced by certain bacterial strains to kill other microorganisms. Its antibacterial effect is based on disrupting the normal concentration gradients of H⁺, Na⁺, and K⁺, which are vital for cell survival.

Fig. 6-66. Mobile ion carrier and channel-forming ionophore. In both cases, the ion flow passes through the membrane only down the electrochemical gradient.

Fig. 6-67. A valinomycin molecule coordinated with a K⁺ ion located in the center of the ring structure by six oxygen atoms.

Summary

Lipid bilayers are largely impermeable to most polar molecules. To transport small water-soluble molecules into or out of the cell, plasma membranes contain a large variety of transport proteins, each responsible for transferring a specific substance across the membrane. There are two classes of membrane transport proteins: transporters (carriers) and channels. Both form continuous pathways across the lipid bilayer.

Carrier proteins bind specific solutes and transport them across the bilayer by undergoing a series of conformational changes that expose the solute-binding sites sequentially, first on one side of the membrane and then on the other. Some carrier proteins transport solutes only "downhill," whereas others, undergoing conformational changes driven by ATP hydrolysis or ion binding, can function as pumps, actively pumping their bound solute "uphill" against its electrochemical gradient.

Channel proteins form water-filled pores in the bilayer, thereby allowing inorganic ions of appropriate size and charge to move across the membrane down their electrochemical gradients. The rate of passage in this case is at least 1000 times faster than transport mediated by carrier proteins. These ion channels are gated and typically open briefly in response to specific stimuli in the membrane, such as the binding of neurotransmitters (transmitter-gated channels) or changes in membrane potential (voltage-gated channels).