9.7 How Does Facilitated Diffusion Occur? 273 (a) (b) (c) (d) (e) (f) (g) Subunit Composition Channel and Pore Structure Transported Ion MgEMg 2ϩ ASIC Na ϩ KcsA K ϩ NaK Na ϩ Glutamate Glutamate CorA Mg 2ϩ ClC Cl Ϫ Amt-1 NH 3 AmtB AQP1, AQP2, etc. H 2 O Glpf Glycerol TABLE 9.3 Composition of Membrane Channels FIGURE 9.40 The structures of channel proteins that transport (a) glycerol (pdb id ϭ 1FX8), (b) glutamate (pdb id ϭ 1XFH), (c) ammonia (pdb id ϭ 1U7G), (d) chloride (pdb id ϭ 1OTS), (e) potassium (pdb id ϭ 2A79), (f) water (pdb id ϭ 1J4N), and (g) proteins (pdb id ϭ 1RHZ).In all cases, the view is in the plane of the membrane. Note the transmembrane ␣-helices in the membrane-spanning part of each structure. 274 Chapter 9 Membranes and Membrane Transport ions across the cell membrane. Potassium channels are facilitated diffusion devices, conducting K ϩ down the electrochemical gradient for K ϩ . Whether found in bacte- ria, archaea, plants, or animals, all known potassium channels are members of a sin- gle protein family. Potassium channels have two important characteristics: They are highly selective for K ϩ ions over Na ϩ ions, and they conduct K ϩ ions at very high rates (almost as fast as any entity can diffuse in water—the so-called diffusion limit). All K ϩ channels have two essential structural features: (1) a selectivity filter, a structural element that allows K ϩ to pass through the channel but prevents passage of Na ϩ , and (2) a gate, a structure that opens and closes the channel. Some K ϩ channels are ligand-gated, such that an ion, a small organic molecule, or even an- other protein can open the gate by binding to the channel. Other K ϩ channels are voltage-gated, in which a portion of the channel protein is able to move (and open or close the channel) in response to a change in voltage across the membrane. The selectivity filter is conserved and nearly identical across all organisms, whereas gat- ing mechanisms are diverse and varied. The structure of KcsA, the K ϩ channel from Streptococcus lividans, is typical (Figure 9.41). The structure consists of four identical subunits, and, facing the cy- tosol, it has a water-filled pore that traverses more than half of the membrane bi- layer, ending at the selectivity filter. A hydrated K + ion is suspended in the center of the pore. Each subunit contributes three ␣-helices to the pore structure: two trans- membrane helices (M1 and M2, the outer and inner helices, respectively) and one helix that extends only halfway across the membrane, with its C-terminal end (with a partial negative charge) facing the center of the pore. The selectivity filter in KcsA consists of four pentapeptides, one from each sub- unit, with the sequence TVGYG. The backbone carbonyls of the first four residues and the threonine side-chain oxygen—evenly spaced—face the center of the pore. These oxygens create four possible K ϩ -binding sites. In each site, a bound, dehy- drated K ϩ is surrounded by eight oxygens from the protein: four above and four be- low. The arrangement of protein oxygens at each site is very similar to the arrange- (a) (d) (b) (c) 1 2 3 4 FIGURE 9.41 Structure of the KcsA potassium channel from Streptococcus lividans (pdb id ϭ 1K4C). (a) The four identical subunits of the channel, which surround a central pore, are shown in different colors. (b) Each subunit contributes three ␣-helices (blue, green, red) to the tetramer structure. (c) The selectivity filter is made from loops from each of the subunits, two of which are shown here. (d) The tetrameric channel, as viewed through the pore. K + K + K + K + K + K + K + FIGURE 9.42 Model for outward and inward transport through the KcsA potassium channel.The selectivity fil- ter in the channel contains four K ϩ -binding sites, only two of which are filled at any time. 9.7 How Does Facilitated Diffusion Occur? 275 ment of water molecules around a hydrated K ϩ . This simple structure is strikingly selective for K ϩ . The physical basis for selection between K ϩ and Na ϩ is the atomic radius—1.33 Å for K ϩ and 0.95 Å for Na ϩ . Still, K ϩ channels select for K ϩ over Na ϩ by a factor of more than a thousand! As K ϩ moves through the KcsA channel, there are, on average, two K ϩ ions bound in the selectivity filter at any given time, either in positions 1 and 3 or positions 2 and 4 (with water molecules occupying the other positions). Ions can move in either di- rection across the channel, depending on the existing electrochemical gradient. One K ϩ enters the channel from one side as a different ion exits on the other side. The cycle of steps for inward or outward movement is shown in Figure 9.42. High selectivity, along with high conduction rates, seems at first paradoxical. If K ϩ ions bind too tightly in the filter, they could not move quickly through the pore. Two factors keep the binding just tight enough, but not too tight: (1) repulsion be- tween the closely spaced K ϩ ions at their two sites and (2) a conformational change induced by K ϩ binding. At low K ϩ concentrations, the filter conformation is very dif- ferent and only one K ϩ can bind at a time. When K ϩ concentration increases, some ion-binding energy is used to induce the conformation change that creates a more symmetric pore, weakening K ϩ binding. Weaker binding makes higher conduction rates possible. The KcsA channel is gated by intracellular pH. It is closed at neutral pH and above, and it opens at acidic pH. What is the conformational change that opens this and other K ϩ channels? After comparing the closed pore conformation of KcsA with the opened pore conformation of the related MthK channel (Figure 9.43), MacKinnon has proposed that helix bending and rearrangement deep in the mem- brane opens K ϩ channels. The inner helices obstruct the central pore in the closed conformation. However, bending at a glycine residue near the center of the mem- brane splays the inner helices outward from the channel center, allowing free access for ions between the cytosol and the selectivity filter. This critical Gly residue is con- served in most K ϩ channel sequences, making this a likely gating mechanism for most K ϩ channels. The B. cereus NaK Channel Uses a Variation on the K ؉ Selectivity Filter Could the K ϩ channel selectivity filter be modified to accommodate other ions, for instance Na ϩ ? Comparison of amino acid sequences from a variety of ion channels (Figure 9.44) shows that this is indeed the case. Variations on the TVGYG filter se- quence are found in ion channels with a range of selectivities for K ϩ , Na ϩ , and even Ca 2ϩ . Bacillus cereus contains an ion channel with equal preference for Na ϩ and K ϩ that is similar to the transient receptor potential (TRP) channels found widely in eu- karyotes. The structure of this channel (Figure 9.45) is similar in many ways to the K ϩ channels, but the selectivity filter sequence of this NaK channel is TVGDG. The substitution of D for Y changes the filter in several ways. Binding sites 1 and 2, the sites most selective for K ϩ , are eliminated, leaving a “pore vestibule” that can accommodate an ion but not bind it tightly. The remaining sites, binding sites 3 and Closed Open FIGURE 9.43 Comparison of the closed (pdb id ϭ 1K4C) and open (pdb id ϭ 1LNQ) states of the potassium channel. T V G Y G DLYP T V G DG NFSP L K + Na + , K + Ca 2+ T G E DWN S V Sequence of selectivity filter Selective for FIGURE 9.44 Ion selectivity in cation channels is a func- tion of peptide sequence of the selectivity filter. Conserved glycines in the TVGYG motif of the KcsA potassium channel are shown in red. Amino acids that are chemically similar are yellow. From top to bottom, the selectivity for K ϩ over Na ϩ decreases and the selec- tivity for Ca 2ϩ increases. (Adapted from Zagotta,W. N.,2006. Permutations of permeability. Nature 440:427–428.) 276 Chapter 9 Membranes and Membrane Transport 4, bind Na ϩ and K ϩ equally well. In addition, the D for Y substitution creates a Ca 2ϩ - binding site at the extracellular entrance to the selectivity filter (see Figure 9.45c). It appears likely that variations of the selectivity filter sequence can “tune” it to ac- commodate and select for a variety of transported cations (Figure 9.45). CorA Is a Pentameric Mg 2؉ Channel The transport of Mg 2ϩ in bacteria and archaea is accomplished primarily by the CorA family of membrane channels. Its pentameric structure (Figure 9.46) contrasts with the tetrameric K ϩ and NaK channels in several ways. With a large N-terminal cytosolic domain and C-terminal transmembrane domain, it resembles a funnel or cone. One of the two transmembrane ␣-helices extends 100 Å into the cytosol and is the longest continuous ␣-helix in any known protein. Remarkable features of the structure include a ring of 20 lysine residues around the outside of the structure near the membrane–cytosol interface and a cluster of 50 aspartate and glutamate residues (on so-called willow helices) adjacent to the lysines and extending into the cytosol. The available structures of CorA are closed-pore structures. A ring of five Asn 314 residues blocks the opening to the pore on the periplasmic face, a ring of Met 291 residues narrows the pore to 3.3 Å at the center of the membrane, and a ring of Leu 294 residues reduce the pore diameter to 2.5 Å at the cytosolic face of the mem- brane. Gating of the pore must overcome these obstacles, as well as the repulsive ring of positive Lys side chains. It is tempting to imagine that the long ␣-helix and the negatively charged willow helices could act as a lever to pry apart the repulsive ring of lysines and open the Mg 2+ pore. Chloride, Water, Glycerol, and Ammonia Flow Through Single-Subunit Pores Membrane channels can also be formed within a single subunit of a protein. The ClC channels (ubiquitous in cells from bacteria to animals) are homodimeric, each subunit having two similar halves, with opposite orientation in the mem- brane. The ClC pore is hourglass-shaped, with a 15-Å-long selectivity filter in the middle (Figure 9.47a). The filter contains 3 Cl Ϫ -binding sites, with coordination from Tyr and Ser hydroxyls and several peptide backbone NH groups. The Cl Ϫ binding site nearest the extracellular solution can be occupied either by a Cl Ϫ ion or by a glutamate carboxyl group. With the glutamate carboxyl in place, the pore is closed, but an increase in Cl Ϫ concentration can displace the Glu side chain, with Cl Ϫ binding to this position and opening the pore. Thus, this Cl Ϫ channel is chloride-gated. (b) (c)(a) Extracellular Ca 3 4 Intracellular FIGURE 9.45 Structure of the channel from Bacillus cereus (pdb id ϭ 2AHY), which has equal preference for Na ϩ and K ϩ . (a) One subunit of the tetramer has been removed to reveal the five ion binding sites in the center of the channel. A Ca 2ϩ ion is bound at the extracellular entrance to the channel (aqua, top), and K ϩ is bound to the other four sites.(b) The tetrameric channel, as viewed through the pore. (c) Substitution of D for Y in the selectivity filter eliminates binding sites 1 and 2, leaving a pore vestibule that binds a K ϩ with low affinity. Sites 3 and 4 are preserved, but bind Na ϩ and K ϩ equally well.The bottom site contains a fully hydrated K ϩ , in a manner similar to the KcsA channel (Figure 9.41). (a) (b) Periplasm Axial view Asn 314 Leu 294 Met 291 Cytosol Basic sphincter Willow helices Asp 89 Asp 253 FIGURE 9.46 The structure of the pentameric CorA Mg 2ϩ channel from Thermus maritima (pdb id ϭ 2HN2). The transmembrane pore is formed from five short ␣-helices (red) and stabilized by four longer ␣-helices (aqua).The pore entrance from the periplasm is gated closed by a ring of 5 Asn residues (gold), and a ring of 5 Leu (orange) and 5 Met (beige) residues narrows the pore to 2.5 Å. The large cytosolic domain includes a basic sphincter of 20 Lys residues (blue), and a ring of 50 Asp and Glu residues (green) at the tips of the willow helices. Asp 89 and Asp 253 (purple) participate in Mg 2ϩ binding. 9.8 How Does Energy Input Drive Active Transport Processes? 277 Channel proteins often solve chemical and thermodynamic problems in innova- tive ways. Ion selectivity, for example, requires that ions be dehydrated in the chan- nel, and dehydration is energetically expensive. Binding sites have to compensate for the energetic cost of dehydration by providing favorable compensatory interac- tions between the ion and the binding amino acid residues. The ammonia transport channel solves a different problem. Ammonia is a gas, but the protonated ammo- nium ion, NH 4 ϩ , is the species that diffuses to the channel opening. The transport channel in this case is a hydrophobic pore 20 Å in length (Figure 9.47b). The hydrophobic character of the channel lowers the pK a of ammonium from its nor- mal 9.25 to less than 6, facilitating the transport of NH 3 but not the monovalent cation, NH 4 ϩ . In the narrow hydrophobic channel, His 168 and His 318 line the pore and stabilize three NH 3 molecules through hydrogen bonding. On either side of the narrow pore, broad vestibules contain NH 3 in equilibrium with NH 4 ϩ . Another example of adaptation to the transported species is the tetrameric glyc- erol channel GlpF from E. coli, with a transport pore in each monomer. Six trans- membrane helices and two half-membrane-spanning helices form a right-handed helical bundle around each channel (Figure 9.47c). Glycerol molecules taken up by an E. coli cell first enter a 15-Å-wide vestibule in the transport protein, becoming progressively dehydrated before entry into a 28-Å amphipathic channel and selec- tivity filter. The channel accommodates three glycerol molecules, lined up in a sin- gle file, with their alkyl backbones wedged against a hydrophobic corner and their hydroxyl groups forming hydrogen bonds with the side chains of channel residues. The aquaporin water channels are closely related to the GlpF glycerol channel, with tetrameric structures and similar right-handed helical bundles forming trans- port channels. Selection for water or glycerol in these proteins is based on subtle differences in the selectivity filters within the transport channels. For example, the Phe and Trp residues that comprise the hydrophobic corner surrounding the alkyl moiety of the middle glycerol site in GlpF are replaced by a His residue in the cor- responding water-binding site in aquaporin Apq1. 9.8 How Does Energy Input Drive Active Transport Processes? Passive and facilitated diffusion systems are relatively simple, in the sense that the transported species flow downhill energetically, that is, from high concentration to low concentration. However, other transport processes in biological systems must be driven in an energetic sense. In these cases, the transported species move from low concentration to high concentration, and thus the transport requires energy input. As such, it is considered active transport. The most common energy input is ATP hydrolysis, with hydrolysis being tightly coupled to the transport event. Other energy sources also drive active transport processes, including light energy and the energy stored in ion gradients. The original ion gradient is said to arise from a primary active transport process, and the transport that depends on the ion gradient for its energy input is referred to as a secondary active transport process (see later discussion of (a) (b) (c) FIGURE 9.47 Structures of channels for (a) chloride, (b) ammonia, and (c) glycerol. All structures are axial views. (a) The ClC chloride channel from E.coli (pdb id ϭ 1OTS). (b) The AmtB ammonia channel from E. coli with four bound NH 4 ϩ (pdb id ϭ 1U7G). (c) The GlpF glycerol channel from E.coli, with bound glycerol (pdb id ϭ 1FX8). 278 Chapter 9 Membranes and Membrane Transport the E. coli proton–drug exchanger). When transport results in a net movement of electric charge across the membrane, it is referred to as electrogenic transport. If no net movement of charge occurs during transport, the process is electrically neutral. All Active Transport Systems Are Energy-Coupling Devices Hydrolysis of ATP is essentially a chemical process, whereas movement of species across a membrane is a mechanical process (that is, movement). An active transport process that depends on ATP hydrolysis thus couples chemical free energy to me- chanical (translational) free energy. The bacteriorhodopsin protein in Halobacterium halobium couples light energy and mechanical energy. Oxidative phosphorylation (see Chapter 20) involves coupling between electron transport, proton transloca- tion, and the capture of chemical energy in the form of ATP synthesis. Similarly, the overall process of photosynthesis (see Chapter 21) amounts to a coupling between captured light energy, proton translocation, and chemical energy stored in ATP. Many Active Transport Processes are Driven by ATP Monovalent Cation Transport: Na ؉ ,K ؉ -ATPase All animal cells actively extrude Na ϩ ions and accumulate K ϩ ions. These two transport processes are driven by Na ؉ ,K ؉ -ATPase, also known as the sodium pump, an integral protein of the plasma membrane. Most animal cells maintain cytosolic concentrations of Na ϩ and K ϩ of 10 mM and 100 mM, respectively. The extracellular milieu typically contains about 100 to 140 mM Na ϩ and 5 to 10 mM K ϩ . Potassium is required within the cell to ac- tivate a variety of processes, whereas high intracellular sodium concentrations are inhibitory. The transmembrane gradients of Na ϩ and K ϩ and the attendant gradi- ents of Cl Ϫ and other ions provide the means by which neurons communicate. They also serve to regulate cellular volume and shape. Animal cells also depend upon these Na ϩ and K ϩ gradients to drive transport processes involving amino acids, sug- ars, nucleotides, and other substances. In fact, maintenance of these Na ϩ and K ϩ gradients consumes large amounts of energy in animal cells—20% to 40% of total metabolic energy in many cases and up to 70% in neural tissue. The Na ϩ - and K ϩ -dependent ATPase comprises three subunits: an ␣-subunit of 1016 residues (120 kD), a 35-kD -subunit, and a 6.5-kD ␥-subunit. The sodium pump actively pumps three Na ϩ ions out of the cell and two K ϩ ions into the cell per ATP hydrolyzed: ATP 4Ϫ ϩ H 2 O ϩ 3 Na ϩ (inside) ϩ 2 K ϩ (outside)⎯⎯→ADP 3Ϫ ϩ H 2 PO 4 Ϫ ϩ 3 Na ϩ (outside) ϩ 2 K ϩ (inside) (9.3) ATP hydrolysis occurs on the cytoplasmic side of the membrane (Figure 9.48), and the net movement of one positive charge outward per cycle makes the sodium pump electrogenic in nature. The ␣-subunit of Na ϩ ,K ϩ -ATPase consists of ten transmembrane ␣-helices, with three cytoplasmic domains, denoted A, P, and N. A large cytoplasmic loop between transmembrane helices 4 and 5 forms the P (phosphorylation) and N (nucleotide- binding) domains. The enzyme is covalently phosphorylated at Asp 369 during ATP hydrolysis. The crystal structure of the enzyme reveals two rubidium ions bound to putative K + -binding sites in the center of the protein (Figure 9.48). A minimal mechanism for Na ϩ ,K ϩ -ATPase postulates that the enzyme cycles be- tween two principal conformations, denoted E 1 and E 2 (Figure 9.49). E 1 has a high affinity for Na ϩ and ATP and is rapidly phosphorylated in the presence of Mg 2ϩ to form E 1 -P, a state that contains three occluded Na ϩ ions (occluded in the sense that they are tightly bound and not easily dissociated from the enzyme in this con- formation). A conformation change yields E 2 -P, a form of the enzyme with relatively low affinity for Na ϩ but a high affinity for K ϩ . This state presumably releases 3 Na ϩ ions and binds 2 K ϩ ions on the outside of the cell. Dephosphorylation leaves E 2 K 2 , a form of the enzyme with two occluded K ϩ ions. A conformation change, which ap- 9.8 How Does Energy Input Drive Active Transport Processes? 279 pears to be accelerated by the binding of ATP (with a relatively low affinity), releases the bound K ϩ inside the cell and returns the enzyme to the E 1 state. Enzyme forms with occluded cations represent states of the enzyme with cations bound in the transport channel. The alternation between high and low affinities for Na ϩ , K ϩ , and ATP serves to tightly couple the hydrolysis of ATP and ion binding and transport. Na ؉ ,K ؉ -ATPase Is Inhibited by Cardiotonic Steroids Certain plant and animal steroids such as ouabain (Figure 9.50) specifically inhibit Na ϩ ,K ϩ -ATPase and ion transport. These substances are traditionally referred to as cardiac glycosides or cardiotonic steroids, both names derived from the potent effects of these mole- cules on the heart. These molecules all possess a cis-configuration of the C-D ring junction, an unsaturated lactone ring (five- or six-membered) in the -configura- tion at C-17, and a -OH at C-14. There may be one or more sugar residues at C-3. The sugars are not required for inhibition, but do contribute to water solu- bility of the molecule. Cardiotonic steroids bind exclusively to the extracellular surface of Na ϩ ,K ϩ -ATPase when it is in the E 2 -P state, forming a very stable E 2 -P(cardiotonic steroid) complex. Ouabain 3 Na + 2 K + Na + ,K + - ATPase + ATP H 2 O ADP + P i + H + (a) (b) N A P ANIMATED FIGURE 9.48 (a) A schematic diagram of the Na ϩ ,K ϩ -ATPase of the mammalian plasma membrane. ATP hydrolysis occurs on the cytoplasmic side of the membrane, Na ϩ ions are transported out of the cell, and K ϩ ions are transported in. The transport stoichiometry is 3 Na ϩ out and 2 K ϩ in per ATP hydrolyzed. Ouabain and other cardiac glycosides inhibit Na ϩ ,K ϩ -ATPase by binding on the extracellular surface of the pump protein. (b) Structure of the Na ϩ ,K ϩ -ATPase, showing the ␣-subunit, residues 28–73 of the -subunit (gray) and the transmembrane helix (residues 23–51, yellow) of the ␥-subunit (pdb id ϭ 3B8E). See this figure animated at www.cengage.com/login. H 2 O P 2 K + 3 Na + 2 Na + Na + 2 K + ATP K 2 E 1 ATP Na 3 E 1 – P Na 3 E 1 E 2 - P K 2 E 2 K 2 E 2 - P E 2 - P Na 3 ATP E 1 ATP ADP ANIMATED FIGURE 9.49 A mechanism for Na ϩ ,K ϩ -ATPase.The model assumes two principal conformations, E 1 and E 2 . Binding of Na ϩ ions to E 1 is fol- lowed by phosphorylation and release of ADP. Na ϩ ions are transported and released, and K ϩ ions are bound before dephosphorylation of the enzyme.Transport and release of K ϩ ions complete the cycle. See this figure animated at www.cengage.com/login. 280 Chapter 9 Membranes and Membrane Transport Medical researchers studying high blood pressure have consistently found that people with hypertension have high blood levels of an endogenous Na ϩ ,K ϩ -ATPase inhibitor. In such patients, inhibition of the sodium pump in the cells lining the blood vessel wall results in accumulation of sodium and calcium in these cells and the narrowing of the vessels to create hypertension. An 8-year study aimed at the iso- lation and identification of the agent responsible for these effects by researchers at the University of Maryland Medical School and the Upjohn Laboratories in Michi- gan yielded a surprising result. Mass spectrometric analysis of compounds isolated from many hundreds of gallons of blood plasma has revealed that the hypertensive agent is ouabain itself or a closely related molecule! Calcium Transport: Ca 2؉ -ATPase Calcium, an ion acting as a cellular signal in vir- tually all cells (see Chapter 32), plays a special role in muscles. It is the signal that stimulates muscles to contract (see Chapter 16). In the resting state, the levels of Ca 2ϩ near the muscle fibers are very low (approximately 0.1 M), and nearly all of the calcium ion in muscles is sequestered inside a complex network of vesicles called the sarcoplasmic reticulum, or SR (see Figure 16.1). Nerve impulses induce the SR membrane to quickly release large amounts of Ca 2ϩ , with cytosolic levels ris- ing to approximately 10 M. At these levels, Ca 2ϩ stimulates contraction. Relaxation of the muscle requires that cytosolic Ca 2ϩ levels be reduced to their resting values. This is accomplished by an ATP-driven Ca 2ϩ transport protein known as the Ca 2؉ - ATPase, which bears many similarities to the Na ϩ ,K ϩ -ATPase. It has an ␣-subunit of the same approximate size, it forms a covalent E-P intermediate during ATP hy- drolysis, and its mechanism of ATP hydrolysis and ion transport is similar in many ways to that of the sodium pump. The structure of the Ca 2ϩ -ATPase includes a transmembrane (M) domain con- sisting of ten ␣-helical segments and a large cytoplasmic domain that itself consists of a nucleotide-binding (N) domain, a phosphorylation (P) domain, and an actua- tor (A) domain (Figure 9.51). The calcium transport cycle begins with binding of two Ca 2ϩ ions. Subsequent ATP binding causes a 90° rotation of N and a 30° rotation of A, thus joining all three cytoplasmic domains (N, A, and P), and pulling a transmembrane helix partly out of the membrane. Phosphorylation of Asp 351 , dissociation of ADP, and conversion of the E 1 -P state to E 2 -P induce a 110° rotation of A and a rearrangement of the trans- membrane domain, which acts like a piston to release Ca 2ϩ inside the SR. A TGES sequence in A (residues 181 to 184) then guides nucleophilic attack of water on E 2 -P, releasing phosphate and restoring the original structures of both the trans- membrane and the cytoplasmic domains of the enzyme. The Gastric H ؉ ,K ؉ -ATPase Production of protons is a fundamental activity of cel- lular metabolism, and proton production plays a special role in the stomach. The highly acidic environment of the stomach is essential for the digestion of food in all HO HCO OH OH CH 3 Strophanthidin HO O H OH CH 3 Digitoxigenin CH 3 O O O O O OH HO CH 3 Ouabain HOH 2 C O OH H O HH H HO HO OH CH 3 H OH FIGURE 9.50 The structures of several cardiotonic steroids.The lactone rings are yellow. 9.8 How Does Energy Input Drive Active Transport Processes? 281 animals. The pH of the stomach fluid is normally 0.8 to 1. The pH of the parietal cells of the gastric mucosa in mammals is approximately 7.4. This represents a pH gradient across the mucosal cell membrane of 6.6, the largest known transmembrane gradient in eukaryotic cells. This enormous gradient must be maintained constantly so that food can be digested in the stomach without damage to the cells and organs adjacent to the stomach. The gradient of H ϩ is maintained by an H ؉ ,K ؉ -ATPase, E 1 • ATPE 1 • 2Ca 2+ E 1 -P • ADP ATP ADP P i 2Ca 2+ 2Ca 2+ E 1 E 2 • P i E 2 E 1 -PE 2 -P 2H + 2H + N P A ACTIVE FIGURE 9.51 The transport cycle of the sarcoplasmic reticulum Ca 2ϩ -ATPase involves at least five different conformations of the pro- tein.The states shown here are E 1 и2Ca 2ϩ (pdb id ϭ 3B9B); E 1 иATP (pdb id ϭ 1SU4); E 1 -PиADP (pdb id ϭ 1T5C); E 2 иP i (pdb id ϭ 2ZBD); and E 2 (pdb id ϭ 2EAR). Blue-shaded states in the reaction sequence correspond to adjacent structures. Test yourself on the concepts in this figure at www.cengage.com/login. 282 Chapter 9 Membranes and Membrane Transport which uses the energy of hydrolysis of ATP to pump H ϩ out of the mucosal cells and into the stomach interior in exchange for K ϩ ions. This transport is electrically neu- tral, and the K ϩ that is transported into the mucosal cell is subsequently pumped back out of the cell together with Cl Ϫ in a second electroneutral process (Figure 9.52). Thus, the net transport effected by these two systems is the movement of HCl into the interior of the stomach. (Only a small amount of K ϩ is needed, because it is recycled.) The H ϩ ,K ϩ -ATPase bears many similarities to the plasma membrane Na ϩ ,K ϩ -ATPase and the SR Ca 2ϩ -ATPase described earlier. It has a similar molecular weight, it forms an E-P intermediate, and many parts of its peptide sequence are ho- mologous with the Na ϩ ,K ϩ -ATPase and Ca 2ϩ -ATPase. Bone Remodeling by Osteoclast Proton Pumps Other proton-translocating ATPases exist in eukaryotic and prokaryotic systems. Vacuolar ATPases (V-type ATPases) are found in vacuoles, lysosomes, endosomes, Golgi, chromaffin granules, and coated vesicles. Various H ϩ -transporting ATPases occur in yeast and bacteria as well. H ϩ -transporting ATPases found in osteoclasts (multinucleate cells that break down bone during normal bone remodeling) provide a source of circulating cal- cium for soft tissues such as nerves and muscles. About 5% of bone mass in the hu- man body undergoes remodeling at any given time. Once growth is complete, the body balances formation of new bone tissue by cells called osteoblasts with resorp- A DEEPER LOOK Cardiac Glycosides: Potent Drugs from Ancient Times The cardiac glycosides have a long and colorful history. Many species of plants producing these agents grow in tropical regions and have been used by natives in South America and Africa to pre- pare poisoned arrows used in fighting and hunting. Zulus in South Africa, for example, have used spears tipped with cardiac glycoside poisons. The sea onion, found commonly in southern Europe and northern Africa, was used by the Romans and the Egyptians as a cardiac stimulant, diuretic, and expectorant. The Chinese have long used a medicine made from the skins of certain toads for sim- ilar purposes. Cardiac glycosides are also found in several species of domestic plants, including the foxglove, lily of the valley, olean- der (figure part a), and milkweed plants. Monarch butterflies (figure part b) acquire these compounds by feeding on milkweed and then storing the cardiac glycosides in their exoskeletons. Car- diac glycosides deter predation of monarch butterflies by birds, which learn by experience not to feed on monarchs. Viceroy but- terflies (figure part c) mimic monarchs in overall appearance. Al- though viceroys contain no cardiac glycosides and are edible, they are avoided by birds that mistake them for monarchs. In 1785, the physician and botanist William Withering de- scribed the medicinal uses for agents derived from the foxglove plant. In modern times, digitalis (a preparation of dried leaves prepared from the foxglove, Digitalis purpurea) and other purified cardiotonic steroids have been used to increase the contractile force of heart muscle, to slow the rate of beating, and to restore normal function in hearts undergoing fibrillation (a condition in which heart valves do not open and close rhythmically but rather remain partially open, fluttering in an irregular and ineffective way). Inhibition of the cardiac sodium pump increases the intra- cellular Na ϩ concentration, leading to stimulation of the Na ϩ -Ca 2ϩ exchanger, which extrudes sodium in exchange for inward move- ment of calcium. Increased intracellular Ca 2ϩ stimulates muscle contraction. Careful use of digitalis drugs has substantial thera- peutic benefit for patients with heart problems. (a) Cardiac glycoside inhibitors of Na ϩ ,K ϩ -ATPase are produced by many plants, including foxglove, lily of the valley, milkweed, and oleander (shown here). (b) The monarch butterfly, which concentrates cardiac glycosides in its exoskeleton, is shunned by predatory birds. (c) Predators also avoid the viceroy, even though it contains no cardiac glycosides, because it is similar in appearance to the monarch. (a) Oleander (b) Monarch butterfly (c) Viceroy butterfly Patti Murray/Animals Animals e.r.degginger/Animals Animals Arthur Hill/Visuals Unlimited Net: out H + H + K + K + Cl – Cl – Gastric mucosal cell Stomach ACTIVE FIGURE 9.52 The H ϩ ,K ϩ -ATPase of gastric mucosal cells mediates proton transport into the stomach. Potassium ions are recycled by means of an associated K ϩ /Cl Ϫ cotransport system. The action of these two pumps results in net transport of H ϩ and Cl Ϫ into the stomach. Test yourself on the concepts in this figure at www.cengage.com/login.