Voltage-Gated Ion Channels and Electrical Excitability
Authors: Clay M Armstrong, Bertil Hille
Journal: Neuron (1998)

Our ability to do gymnastics, to perceive a colorful world, and to process language relies on rapid communication among neurons. Such signaling, the fastest in our bodies, involves electrical messages produced as ion channels in cell membranes open and close. Various ion channels mediate sensory transduction, electrical "computations," propagation over long distances, and synaptic transmission. Here, we focus on voltage-gated ion channels, the family of channels that includes the familiar Na+, K+, and Ca2+ channels of nerve and muscle excitability. In the computer metaphor of the brain, the voltage-gated ion channels are like the transistors of logical circuits, detecting, amplifying, and reshaping electrical messages. Our basic understanding of these proteins maintains the framework and rigor established 50 years ago by 27Hodgkin A.L. Huxley A.F. A quantitative description of membrane current and its application to conduction and excitation in nerve.J. Physiol. (Lond.). 1952; 117: 500-544Crossref Scopus (13123) Google Scholar, enriched by much new molecular information and by insights gained from patch-clamp methods. Although we have had full amino acid sequences of voltage-gated channels for over a decade, we still lack even modest resolution three-dimensional information. All three-dimensional diagrams in the literature derive from functional studies without the benefit of crystallography or NMR. Figure 1A represents widely accepted functional information, much of it deriving from early biophysical and pharmacological work that will be described in this article. An overriding conclusion is that ion channels are aqueous pores. Proceeding through the pore from the outside, an ion would find a wide outer vestibule, a narrow selectivity filter, a voluminous inner vestibule, and finally, at the cytoplasmic end, the gating machinery that closes the pore. Highly charged voltage sensors control activation of the channel but are less important for inactivation. All of the voltage-gated channels have a 4-fold symmetry, with the pore formed at the central line of contact of four channel-forming domains. Unlike ligand-gated channels of fast chemical synapses, much more of the mass of the voltage-gated channels lies on the intracellular side of the membrane than on the extracellular side. In this review, we first consider how this field was initiated six decades ago and then see how functions of voltage-gated ion channels have been uncovered and mapped onto the linear amino acid sequence of the protein. The rigor of the original analysis set the tone for a new discipline that now produces more than 5000 papers a year. The period from 1939 to 1952 was a heroic time in the study of membrane biophysics. During this period, Hodgkin and Huxley explained the propagated action potential. Their definitive description of ionic permeability changes in the axon membrane in 1952 was closely preceded by five important discoveries. Four of them occurred shortly before the Second World War. Hodgkin showed that local circuit currents from an excited region of nerve are needed to bring the next region into activity. This meant that depolarization is the natural stimulus for action potential propagation. J. Z. 66Young J.Z. Structure of nerve fibers and synapses in some invertebrates.Cold Spring Harbor Symp. Quant. Biol. 1936; 4: 1-6Google Scholar rediscovered the giant axon of the squid. It provided the first convenient way to place electrodes and even electrode arrays inside an excitable cell. 11Cole K.S. Curtis H.J. Electrical impedance of the squid giant axon during activity.J. Gen. Physiol. 1939; 22: 649-670Crossref PubMed Scopus (229) Google Scholar showed that the membrane of the squid giant axon increases its conductance 40-fold during an action potential, in apparent agreement with Bernstein's earlier theory of membrane breakdown. 25Hodgkin A.L. Huxley A.F. Action potentials recorded from inside a nerve fibre.Nature. 1939; 144: 710-711Crossref Scopus (234) Google Scholar discovered with intracellular electrodes that the peak of the action potential overshoots 0 mV by a significant margin. The overshoot was a serious problem for the Bernstein theory, and 26Hodgkin A.L. Katz B. The effect of sodium ions on the electrical activity of the giant axon of the squid.J. Physiol. (Lond.). 1949; 108: 37-77Crossref Scopus (1726) Google Scholar eventually provided the crucial resolution: the overshoot is determined by the Na+ equilibrium potential and must be due to Na+ entry during the action potential. With this background, Hodgkin and Huxley sought to understand how excitation regulates the entry of Na+ ions. They developed the voltage clamp to measure ion movements as electric currents. The clamp records revealed an inward current followed by an outward current during step depolarizations. In a major conceptual leap27Hodgkin A.L. Huxley A.F. A quantitative description of membrane current and its application to conduction and excitation in nerve.J. Physiol. (Lond.). 1952; 117: 500-544Crossref Scopus (13123) Google Scholar deduced that these membrane currents could be assigned to Na+ and K+ permeability mechanisms whose conductances are functions of time and membrane potential. The assumption of separable permeability components and the realization that membrane potential is the controlling variable were the paradigm shifts that opened a new field of inquiry. Their five seminal papers systematically extracted kinetic constants for an empirical description of the conductance changes, showed that this detailed kinetic description—the Hodgkin-Huxley model—suffices to explain all of the classical properties of action potential excitation and propagation, and even offered a plausible physical basis for the control by membrane potential. The stage was brilliantly set. Consider a few of the findings of these papers. Today, we like to emphasize that ion channels have two major properties: permeation and gating. This separation was clear in the original papers. With respect to permeation, the principal emphasis was that each component of current obeyed Ohm's law with a reversal potential at the Nernst potential for Na+ and K+ ions. When extracellular Na+ concentration was changed, they argued that the flux in either direction across the membrane was proportional to the concentrations on either side. The clear implication was that ion movement was strictly diffusion down an electrochemical gradient without additional forces. With respect to gating, much more was discovered. Notably, the permeabilities to Na+ and to K+ had quite different time courses. The Na+ conductance rose and inactivated quickly during a depolarization, and in the axon the K+ conductance rose more slowly without inactivation. Changing ion concentrations changed the direction of current flow but not the time course: "The changes in membrane permeability appear to depend on membrane potential, and not on membrane current." In addition, Hodgkin and Huxley succeeded in describing the permeability changes as a set of chemical reactions whose rate constants are a function of voltage. The rate of change of each permeability depended quite steeply on the membrane potential, and a 10-fold increase in opening occurred with membrane potential depolarizations as small as 7–12 mV. What did Hodgkin and Huxley say about mechanism? About permeation, they said very little except that flux was downhill. They did not mention the concept of an aqueous pore, nor did they use the word channel. About gating, a word they also did not use, they argued, "Details of the mechanism will probably not be settled for some time, but it seems difficult to escape the conclusion that the changes in ionic permeability depend on the movement of some component of the membrane which behaves as though it had a large charge or dipole moment." These charged controlling "particles" were envisioned to move under the influence of the electric field in the membrane and to "allow ions to pass when they occupy particular sites in the membrane." The distribution of the particles was compared to a Boltzmann distribution, exactly as is done today, and the steepness of the voltage dependence required that as many as six electronic charges move fully across the membrane during activation. To account for the sigmoid time course of activation of the conductances, the model supposed the movement of three gating particles for Na+ and four for K+. These hypothetical particles combined the functions that we now separately assign to the voltage sensors, the gates, and the conducting pore. Their multiplicity in the model was a harbinger of the modern finding of four structurally separate voltage sensors in each channel. The novelty, depth, and durability of these insights are a monument to the powerful physical intuition of these two great scientists. Cloning of the pore-forming α subunits of voltage-gated Na+, K+, and Ca2+ channels was reported in 1984–1988. It showed that, fundamentally, these channels are all members of the same gene superfamily with the same overall structure. This good news confirmed that there should be much mechanistic similarity among the channels and that we were free to generalize—as we had already been doing in the biophysical work of the previous decades. The Na+ and Ca2+ channel clones, first reported by the Numa laboratory, predict large peptides (>2000 amino acids) containing four homologous repeats (domains I–IV), each of which has a motif with six putative transmembrane segments, S1–S6 (Figure 1B). The cloned K+ channel was first reported by the laboratories of Jan and Jan (57Tempel B.L. Papazian D.M. Schwarz T.L. Jan Y.N. Jan L.Y. Sequence of a probable potassium channel component encoded at Shaker locus of Drosophila.Science. 1987; 237: 770-775Crossref PubMed Scopus (469) Google Scholar), Tanouye (Kamb et al., 1987), and Pongs (49Pongs O. Kecskemethy N. Muller R. Krah-Jentgens I. Baumann A. Kiltz H.H. Canal I. Llamazares S. Ferrus A. Shaker encodes a family of putative potassium channel proteins in the nervous system of Drosophila.EMBO J. 1988; 7: 1087-1096Crossref PubMed Scopus (309) Google Scholar). It is about one fourth as large as an Na+ channel and contains only one copy of the S1–S6 motif. As might be expected, K+ channels were later shown to be tetramers of the pore-forming subunit (43MacKinnon R. Determination of the subunit stoichiometry of a voltage-activated potassium channel.Nature. 1991; 350: 232-235Crossref PubMed Scopus (748) Google Scholar), making them structurally quite similar to Na+ and Ca2+ channels. In all of three clones, segments S1, S2, S3, S5, and S6 are quite hydrophobic, and each is long enough to span the membrane as a helix. In the S4 segments, however, every third residue is a positively charged arginine or lysine, for a total of seven positive charges in KV1.1 and five to eight positive charges in the various S4 domains of Na+ and Ca2+ channels. The S4 segment was almost immediately recognized as a candidate for the voltage sensor—one of Hodgkin and Huxley's controlling particles. After a brief consignment to the cytoplasm, it joined the membrane-crossing segments where it could experience the electric field of the membrane as required. The great importance of another amphipathic loop between segments S5 and S6 (called P or H5 and shown in red in Figure 1A) was recognized only later. It was first assigned its present status as part of the outer vestibule and the conducting pore on the basis of thoughtful molecular modeling (Conti and Guy, 1990). The folding topology in Figure 1B has much to support it. To mention only some of the evidence, the N terminus is known from the experiments of Aldrich and colleagues to be cytoplasmic, and in Shaker B (ShB) K+ channels this terminus contains the molecular machinery for fast "N-type" inactivation gating (30Hoshi T. Zagotta W.N. Aldrich R.W. Biophysical and molecular mechanisms of Shaker potassium channel inactivation.Science. 1990; 250: 533-538Crossref PubMed Scopus (1238) Google Scholar). The sidedness of many residues of these channels has been identified by mutating an individual residue to cysteine and then testing whether one of Karlin's thiol-reactive compounds, e.g., MTSET (1Akabas M.H. Stauffer D.A. Xu M. Karlin A Acetylcholine receptor channel structure probed in cysteine-substitution mutants.Science. 1992; 258: 307-310Crossref PubMed Scopus (591) Google Scholar), must be applied to the extracellular or the cytoplasmic face to label the cysteine. One particularly illuminating set of experiments showed that S4 extends across the membrane: a cysteine introduced into the N-terminal end of S4 can be labeled only from the outside, and a cysteine introduced into the other end of S4 can be labeled only from the inside, both in Na+ (62Yang N. Horn R. Evidence for voltage-dependent S4 movement in sodium channels.Neuron. 1995; 15: 213-218Abstract Full Text PDF PubMed Scopus (344) Google Scholar, 63Yang N. George A.L. Horn R. Molecular basis of charge movement in voltage-gated sodium channels.Neuron. 1996; 16: 113-122Abstract Full Text Full Text PDF PubMed Scopus (519) Google Scholar) and K+ channels (37Larsson H.P. Baker O.S. Dhillon D.S. Isacoff E.Y. Transmembrane movement of the shaker K+ channel S4.Neuron. 1996; 16: 387-397Abstract Full Text Full Text PDF PubMed Scopus (441) Google Scholar). The general hairpin topology of the P region was worked out by mutating residues that altered sensitivity to pore blockers. The key was that a threonine residue in the middle of the P region is crucial for block by intracellular tetraethylammonium ion (TEA+), whereas residues both before and after this threonine determine the sensitivity to block by extracellular TEA+ and charybdotoxin (64Yellen G. Jurman M.E. Abramson T. MacKinnon R. Mutations affecting internal TEA blockade identify the probable pore-forming region of a K+ channel.Science. 1991; 215: 939-942Google Scholar; and see18Hartmann H.A. Kirsch G.E. Drewe J.A. Taglialatela M. Joho R.H. Brown A.M. Exchange of conduction pathways between two related K+ channels.Science. 1991; 215: 942-945Google Scholar). Many other insightful experiments support the proposed topology (e.g.,53Shih T.M. Goldin A.L. Topology of the Shaker potassium channel probed with hydrophilic epitope insertions.J. Cell Biol. 1997; 136: 1037-1045Crossref PubMed Scopus (42) Google Scholar), and we are aware of no contradictions. Hodgkin and Huxley conceptually separated ion conduction from what we now call gating, but the nature of the conducting path, whether ion carrier or ion pore, lipid or protein, did not become clear for many years. Selective block and channel conductance estimates from experiments with the channel blockers tetrodotoxin (TTX+) and TEA+ led each of us to conclude by the early seventies that Na+ and K+ channels must be separate aqueous pores and that transport based on a carrier such as valinomycin was too slow to be considered. The final proof was made possible 10 years later by the the patch clamp (Hamill et al., 1981), which conferred the ability to measure current through a single ion channel (Figure 2). We found this beautiful and exactly as anticipated. What is the nature of the conducting pore? Because of its simple tetrameric structure, the K+ channel has been a favorite for experimentation. This channel's main task is letting K+ ions out and keeping Na+ ions from going in. To explain the >50:1 selectivity, one can invoke only the geometry of the pore and the energetics of interaction of the ions with water and with the residues of the pore. We both found by ion substitution studies in the early seventies that the pore narrows somewhere to a bore of only 3.0 Å—just accommodating Rb+ ions. As the crystal radius of Na+ ions is smaller than that of K+ ions, Na+ would fit through any hole that K+ can. Therefore, the discrimination has to be blamed on the unfavorable energetics of stripping most of the water from the more strongly hydrated Na+ ion in a small hole without providing much favorable interaction with the channel in return. This constriction of the pore is now attributed to the four 20-residue P regions, which are thought to dip partway into the membrane from the extracellular side and then loop back out (red loops in Figure 1B). These residues narrow the pore, forming the lining of the outer vestibule and the narrow selectivity filter (Figure 3A) but not the inner vestibule. Within each P region is a remarkably conserved "signature sequence", -TXXTXGYG- (-Thr-X-X-Thr-X-Gly-Tyr-Gly ), found in at least 50 cloned K+-selective channels, including the much smaller inward rectifier channels. The signature sequence is thought to be the heart of the selectivity filter. How does it work? Thus far, the most illuminating experiment has been negative. The sequence of the Shaker K+ channel P region (TMTTGYG) contains three threonine residues whose -OH groups seemed prime candidates for complexing with and selecting K+ ions. However, all hydroxyl groups except the one on the first threonine were found to be unnecessary for a selective channel, and evidence regarding the first was inconclusive (19Heginbotham L. Lu Z. Abramson T. MacKinnon R. Mutations in the K+ channel signature sequence.Biophys. J. 1994; 66: 1061-1067Abstract Full Text PDF PubMed Scopus (654) Google Scholar). 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The outer part of the P region is a funnel that reaches its narrowest part near the signature sequence. External TEA+ blocks the wider part of the funnel, and its binding is strongly enhanced by a mutation in the outer pore region (T449Y; Figure 3A). The part of the pore internal to the selectivity filter is formed by the S5 and S6 segments (10Choi K.L. Mossman C. Aube J. Yellen G. The internal quaternary ammonium receptor site of Shaker potassium channels.Neuron. 1993; 10: 533-541Abstract Full Text PDF PubMed Scopus (221) Google Scholar, 34Kirsch G.E. Shieh C.C. Drewe J.A. Vener D.F. Brown A.M. Segmental exchanges define 4-aminopyridine binding and the inner mouth of K+ pores.Neuron. 1993; 11: 503-512Abstract Full Text PDF PubMed Scopus (82) Google Scholar, 41Lopez G.A. Jan Y.N. Jan L.Y. Evidence that the S6 segment of the Shaker voltage-gated K+ channel comprises part of the pore.Nature. 1994; 367: 179-182Crossref PubMed Scopus (147) Google Scholar, 29Holmgren M. Smith P.L. 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Thus TEA+, which is about the size of a hydrated K+ ion, can occupy the outer and inner vestibules but cannot enter the selectivity filter. Similar studies have been done with Na+ channels. To generate the upstroke of the action potential, these channels have three selectivity tasks: letting Na+ go in, keeping K+ from going out, and preventing Ca2+ ions from getting stuck in the pore and interfering with Na+ permeation. Early studies defined an outer vestibule as the receptor for potent block by extracellular tetrodotoxin and an inner vestibule as the receptor for block by local anesthetics. These studies also provided clear arguments for an acid group—a negative charge—within the pore that could interact with permeant cations and with tetrodotoxin. The initial evidence was that the conductance of Na+ channels drops sharply when the bathing pH is lowered, as if an essential acid group (21Hille B. Charges and potentials at the nerve surface divalents and pH.J. Gen. Physiol. 1968; 51: 221-236Crossref PubMed Scopus (408) Google Scholar) located quite near the extracellular end of the pore (61Woodhull A.M. Ionic blockage of sodium channels in nerve.J. Gen. Physiol. 1973; 61: 687-708Crossref PubMed Scopus (1210) Google Scholar) becomes neutralized, preventing cations from passing. The narrow part of the pore admits cations up to the size of the aminoguanidinium ion (requiring at least a 3 × 5 Å rectangular hole; Figure 3B), yet K+ ions permeate only 1/12 as well as Na+ (22Hille B. The permeability of the sodium channel to organic cations in myelinated nerve.J. Gen. Physiol. 1971; 58: 599-619Crossref PubMed Scopus (382) Google Scholar, 23Hille B. The permeability of the sodium channel to metal cations in myelinated nerve.J. Gen. Physiol. 1972; 59: 637-658Crossref PubMed Scopus (311) Google Scholar). Why don't K+ ions (2.7 Å diameter) glide through such an aperture as easily? Hille's working hypothesis was that as ions passing through the aperture are partially dehydrated, they are stabilized by compensatory direct interaction with the negative charge in the selectivity filter. In this view, small ions like Li+ and Na+ are well stabilized by the negative charge because they can get close to it, and larger ions like K+ and Rb+ cannot. Subsequent cloning showed that acid groups are indeed present in the P regions of all Na+ channels (Figure 3C), and mutagenesis proved that they contribute significantly to conductance, selectivity, and tetrodotoxin binding (58Terlau H. Heinemann S.H. Stühmer W. Pusch M. Conti F. Imoto K. Numa S. Mapping the site of block by tetrodotoxin and saxitoxin of sodium channel II.FEBS Lett. 1991; 293: 93-96Crossref PubMed Scopus (354) Google Scholar, 20Heinemann S.H. Terlau H. Stühmer W. Imoto K. Numa S. Calcium channel characteristics conferred on the sodium channel by single mutations.Nature. 1992; 356: 441-443Crossref PubMed Scopus (612) Google Scholar). Nevertheless, thus far we don't know how to make quantitative tests of the selectivity hypothesis. The P regions of Na+ and Ca2+ channels have similar sequences, and illuminating experiments have been performed to determine the basis for their Na+ or Ca2+ preference. Both channels have two rings of charge encircling the pore, each ring containing four residues, one from each homologous domain (Figure 3C). The outer ring is entirely negative and is thought to be relatively distant from the pore axis. The inner ring, two to three residues deeper into the pore, is composed of DEKA (Asp Glu Lys Ala) in the Na+ channel and EEEE (Glu Glu Glu Glu) in the Ca2+ channel. In general, it seems appropriate that the channel conducting doubly charged Ca2+ should have more negative charge. With this in mind20Heinemann S.H. Terlau H. Stühmer W. Imoto K. Numa S. Calcium channel characteristics conferred on the sodium channel by single mutations.Nature. 1992; 356: 441-443Crossref PubMed Scopus (612) Google Scholar succeeded in converting a Na+ channel into a Ca2+-preferring channel by point mutations that increased the negativity in the inner ring. From their experiments, the importance of the inner ring was immediately apparent. The mutated channels lost Na+/K+ discrimination (see also14Favre I. Moczydlowski E. Schild L. On the structural basis for ionic selectivity among Na+, K+, and Ca2+ in the voltage-gated sodium channel.Biophys. J. 1996; 71: 3110-3125Abstract Full Text PDF PubMed Scopus (182) Google Scholar) and became quite Ca2+ permeable. In addition, Ca2+ interference with monovalent permeability became severe, because Ca2+ binds tightly to the added negative charge. The importance of the EEEE ring in Ca2+ channels was examined in detail by Tsien and colleagues (13Ellinor P.T. Yang J. Sather W.A. Zhang J.F. Tsien R.W. Ca2+ channel selectivity at a single locus for high-affinity Ca2+ interactions.Neuron. 1995; 15: 1121-1132Abstract Full Text PDF PubMed Scopus (238) Google Scholar), yielding a detailed mechanism for Ca2+/Na+ discrimination by calcium channels. Divalent/monovalent selection thus seems well understood, whereas monovalent/monovalent selectivity is at best understood in principle. Voltage-gated channels are exquisitely sensitive to small changes in membrane potential. Hodgkin and Huxley realized that Na+ and K+ channel opening or activation must result from movement of charges within the membrane. Their work predicted "gating current," a small charge movement generated (in more recent terms) by the voltage-driven conformational changes that open the channels. Theoretically speaking, there was no alternative, and in due course the expected current was detected, first in connection with excitation-contraction coupling in muscle (51Schneider M.F. Chandler W.K. Voltage-dependent charge movement of skeletal muscle, a possible step in excitation-contraction coupling.Nature. 1973; 242: 244-246Crossref PubMed Scopus (624) Google Scholar) and subsequently in nerve membranes (Figure 4A; 5Armstrong C.M. Bezanilla F. Currents related to movement of the gating particles of the sodium channels.Nature. 1973; 242: 459-461Crossref PubMed Scopus (425) Google Scholar, 33Keynes R.D. Rojas E. Kinetics and steady-state properties of the charged system controlling sodium conductance in the squid giant axon.J. Physiol. (Lond.). 1974; 239: 393-434Google Scholar). By 1980, it seemed probable that the channels were composed of protein, leading one of us to propose that the gating particles of Hodgkin and Huxley were in fact charged membrane helices—specifically, a negatively charged helix that moved inward relative to a positively charged helix, yielding a lot of charge movement, and hence voltage sensitivity, with relatively little physical motion (4Armstrong C.M. Sodium channels and gating currents.Physiol. Rev. 1981; 61: 644-683Crossref PubMed Scopus (346) Google Scholar). Each of four or five proposed subunits would contain such a pair of helices. When the Na+ channel was cloned a few years later, the positively charged helix—the S4 segment—was immediately visible. The expected negatively charged helix does not exist, and it is still not entirely clear how the necessary counter charges for stabilizing the amphipathic S4 helix in the membrane are provided. The likelihood that S4 was the long-sought voltage sensor was immediately apparent, but experimental proof has come more slowly. A first strategy was to create mutant channels with neutral residues replacing some of the positive charges in S4 (56Stühmer W. Conti F. Suzuki H. Wang X.D. Noda M. Yahagi N. Kubo H. Numa S. Structural parts involved in activation and inactivation of the sodium channel.Nature. 1989; 339: 597-603Crossref PubMed Scopus (924) Google Scholar). These experiments provided suggestive support, but they were complicated by the failure of channels with many neutralized residues to express and by drastic and often unanticipated changes in gating properties when this sensitive helix was altered in any way. Recently, cysteine mutagenesis and cysteine labeling with thiol-reactive compounds has provided good evidence that the S4 segments in both Na+ and K+ channels move as expected following voltage changes (62Yang N. Horn R. 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