News Physiol Sci 13: 177-182, 1998;
1548-9213/98 $5.00
News in Physiological Sciences, Vol. 13, No. 4, 177-182,
August 1998
© 1998 Int. Union Physiol. Sci./Am. Physiol. Soc.
Fast Inactivation of Voltage-Gated K+ Channels: From Cartoon to Structure
Christoph Antz and
Bernd Fakler
C. Antz and B. Fakler are in the Dept. of Physiology II, Univ. of Tübingen,Ob dem Himmelreich 7, 72074 Tübingen, Germany.
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Abstract
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Fast inactivation of voltage-gated potassium (Kv) channels is the best understood gating transition in ion channels and is brought about by an NH2-terminal domain (ball domain) of the channel's 
-subunit, which physically blocks the open pore. Recent analysis by nuclear magnetic resonance spectroscopy showed that ball domains from various Kv channels exhibit well-defined but distinct structures in aqueous solution.
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Introduction
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Electrical signals transferring information in biological systems are generated by flow of the inorganic ions Na+, K+, Ca2+, and Cl– through large transmembrane proteins: the ion channels. To operate sensibly, these proteins must rapidly open and close their ion-permeable pores in response to biological signals such as changes in transmembrane voltage or concentration of ligands (6). This process has been referred to as "gating" and occurs through conformational changes within the channel protein. "Activation gating" leads to opening of the channel pore, whereas "inactivation gating" results in closure of the ion pathway. Fast inactivation of voltage-gated transient outward-rectifier K+ channels (so-called A-type K+ channels) is the best understood gating transition in ion channels. Together with an activation in the subthreshold range, inactivation of these channels governs the firing rate (spiking) of neurons by controlling excitability in the interspike interval (6). This review focuses on the fast inactivation gating of A-type K+ channels as it developed from a phenomenological model of channel function to a molecular understanding of the process, including the three-dimensional structure of the inactivation gate.
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Definition of the inactivation gate
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In the first quantitative description of Na+ and K+ channel gating, Hodgkin and Huxley (7) postulated that multiple gating particles existed and the time course of conductivity was explained by the position of these particles. In voltage-dependent ion channels, gating processes are controlled and powered by the membrane potential. On depolarization, the channels first open by transition of the activation gate(s) and then enter a long-living nonconducting state, the inactivated state (Fig. 1
). The latter results from transition of the inactivation gate, which occludes the ion pore as long as the membrane is depolarized. On repolarization, the inactivation gate removes from the pore and allows for the next activation of the channel. In the Hodgkin-Huxley formalism, both activation and inactivation are driven by the transmembrane voltage, implying that the gating apparatus must carry charges that, by its movement during gating transitions, should generate a so-called gating current. Later on, such gating-currents were detected by Armstrong (2) in voltage-clamp experiments. However, it was only during activation of the channels that charge movement (so-called ON-charge movement) was measured. No component of the gating current could be associated with the inactivation process, as if inactivation occurred outside the membrane and did not involve movement of any charged particle through the transmembrane electrical field (2). This view was further supported by the fact that inactivation could be selectively removed by proteolytic agents (pronase or N-bromoacetamide) when applied to the cytoplasmic side of the channels. External perfusion with the same agents did not affect inactivation gating. Additionally, a number of pharmacological agents [tetraethylammonium (TEA), pancuronium, or N-propylguanidinium] were found to compete with the inactivation process when applied to the intracellular but not the extracellular face of the membrane (2).
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FIGURE 1. Fast inactivation in A-type K+ channels: the ball-and-chain mechanism. The cartoon represents functional states of a channel with respect to position of inactivation particle and activation gate: C, closed or activatable state; O, open or conducting state; I , inactivated state. [Modified from Armstrong (2) and Miller (10).]
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The findings that inactivation 1) was voltage independent, 2) removed by proteolytic agents, and 3) mimicked by various pharmacological agents led Armstrong (2) and Bezanilla to propose a novel mechanism for channel inactivation: the "ball-and-chain" model (Fig. 1
). In this model, inactivation is brought about by an inactivation particle (ball) that is tethered to the cytoplasmic face of the channel protein by a protease-cleavable domain (chain). The inactivation ball binds to its receptor, once exposed by channel activation, and thereby physically occludes the ion pore. Thus the ball-and-chain model assumes strict coupling between activation and inactivation in that only activated or open channels can inactivate. As a consequence, voltage dependence of inactivation is not intrinsic to this gating transition but rather results from the activation process.
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Localization of the inactivation gate
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The ball-and-chain model was proposed in the "premolecular era" and was based exclusively on electrophysiological and pharmacological data from "native" channels, mainly Na+ but also K+ channels. The challenge for this model was on when K+ channels entered the era of molecular cloning with the isolation of the Shaker gene from Drosophila melanogaster (14). This gene coded for a multiplicity of voltage-gated K+ channels through alternative splicing at the protein's NH2 terminus. Expression of these splice variations in Xenopus oocytes resulted in A-type K+ channels whose time course of inactivation varied significantly and could be removed by intracellular trypsin (Fig. 2A). These findings, together with the presence of multiple potential trypsin cleavage sites in the NH2 terminus of the Shaker B (ShB) amino acid sequence, led Aldrich and co-workers (8) to focus on this part of the K+ channel -subunit. From a number of deletion and insertion mutations (Fig. 2B), these authors learned that the NH2-terminal 83 amino acids (aa) were involved in inactivation and could be divided into two functional domains. Mutations in the first domain, which constitutes about the first 20 aa, slowed or completely removed fast inactivation. Deletions in the second domain extending from about aa 20 to at least aa 83 speeded up inactivation, whereas insertions slowed it down (8). Although these findings were very reminiscent of ball-and-chain domains, they did not tell too much about the physical nature of the inactivation gate.
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FIGURE 2. Localization of the inactivation gate in the primary sequence of cloned A-type K+ channels. A: single-channel openings and ensemble currents of Shaker B (ShB) channels before (control) and after application of trypsin to cytoplasmic side of an inside-out patch. Currents were elicited by a voltage step to 20 mV after a 1-s prepulse to –100 mV; holding potential was –70 mV. Scale bars are as indicated. B: single-channel openings of deletion mutations in the NH2-terminal region of ShB. Amino acid (aa) sequence (given in 1-letter code) is shown at top, with the first 20 aa marked by a box; bars indicate deletions. Filled bars, deletions that disrupted inactivation; open bars, deletions that left inactivation intact. Single-channel openings were elicited by the same pulse protocol as in A. C: restoration of inactivation in ShB 6-46 channels induced by cytoplasmic application of the NH2-terminal inactivation peptides derived from ShB (top) and Raw3 (Kv3.4; bottom). Currents were recorded in response to voltage steps to 50 mV (ShB) or to –25, 0, 25, and 50 mV (Raw3) from a holding potential of –110 mV. Peptide concentrations and scale bars are as indicated. D: ball-and-chain model after integration of structural data as obtained from site-directed mutagenesis. [Modified from Hoshi et al. (8), Zagotta et al. (15), and Murrell-Lagnado and Aldrich (11).]
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However, in a second set of experiments, the same authors showed that inactivation could be completely reconstituted in a noninactivating mutant (ShB6–46, Fig. 2B) by cytoplasmic application of a synthetic peptide corresponding to aa 1–20 of ShB (15) (Fig. 2C). This peptide-induced inactivation was indeed specific because the rate of inactivation linearly depended on the concentration of the applied peptide and inactivation could not be restored by peptides whose sequence mimicked that of noninactivating NH2-terminal point mutations (15).
Moreover, NH2 terminus and the NH2-terminal peptide of ShB behaved like an open-channel blocker as predicted by the ball-and-chain model: 1) inactivation was almost voltage independent, 2) recovery from inactivation was speeded up by increased external K+ concentrations just as blockade can be relieved by permeant ions (4), 3) the pore blocker TEA competes with and prevents the inactivation gate from closing the channel, and 4) the inactivated channel reopens on repolarization, indicating that the inactivation gate leaves an open channel when released from its receptor (4, 13).
This receptor is constituted at least in part by aa residues in the internal mouth of the channel. Isacoff et al. (9) showed that mutations in the S4-S5 linker (Fig. 2D) affected both the single-channel conductance and the inactivation process. All these aa residues, two of which were charged (the others were hydrophobic), influenced the stability of the channel-gate complex. This is in line with the extensive work of Murrel-Lagnado and Aldrich (11) in uncovering that inactivation induced by the ShB-inactivation peptide is determined by two types of channel-gate interaction: long-range electrostatic interactions, attracting the positively charged gate to the receptor, and hydrophobic interactions, which stabilize the gate in the receptor-bound state.
Together, all the results obtained from the cloned K+ channels were entirely consistent with the ball-and-chain mechanism of inactivation. In extension to its original version (Fig. 1C), the "postcloning" model (Fig. 2D) contains the information about where in the channel protein the functional domains are localized: 1) the NH2 terminus of Shaker and mammalian voltage-gated K+ (Kv) channels [Kv3.4 (Raw3) and Kv1.4 (RCK4)] represents the entity of the inactivation ball domain (8, 11, 13, 15; Fig. 2C), 2) the chain is formed by the rest of the NH2 terminus up to the beginning of the transmembrane segment S1, and 3) the S4-S5 linker forms part of the ball receptor (Fig. 2D). As a consequence of localization of the gate in the NH2 terminus, fast inactivation is currently also known as N-type inactivation.
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Structure of inactivation gates
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Although the ball-and-chain model as it emerged from the "molecular mutagenesis machinery" (Fig. 2D) was closer to physical reality, the questions remained, What does an inactivation gate look like? and What do we really know without a high-resolution molecular structure? (10).
As a first step toward structural understanding of the inactivation process, our lab analyzed all identified inactivation ball domains with high-resolution nuclear magnetic resonance (NMR) spectroscopy in aqueous solution (1). This analysis showed that the mammalian ball peptides derived from Kv1.4 and Kv3.4 (constituted of aa 1–37 and aa 1–30, respectively) exhibited well-defined and compact structures (Fig. 3), whereas the Shaker peptide (aa 1–20) revealed a dynamic equilibrium of locally nonrandom structures rather than a stable overall structure (1).
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FIGURE 3. Structure of inactivation gates derived from the mammalian Kv channels, Kv1.4 (RCK4) and Kv3.4 (Raw3). Backbone (left) and surface structure (right) of the Kv1.4 peptide (A) and Kv3.4 peptide (B). Backbone representation shows 8th lowest-energy structures for each peptide. N and C, NH2 and COOH terminals, respectively. For Kv1.4 peptide, secondary structural motifs are highlighted in blue (-helix, aa 21–34) and green (ß-turn type II, aa 19–21). In the surface illustration, colors indicate the surface charge, which was scaled arbitrarily and ranged from blue (negatively charged) to red (positively charged). Both peptides are arranged with respect to their dipole vectors (red arrows).
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Although well defined, the structures of the two mammalian inactivation peptides, which share almost no homology in primary sequence, were rather different in backbone and surface (Fig. 3). Thus the backbone of the Kv1.4 peptide consisted of a highly flexible NH2 terminus and an -helix capped by a ß-turn motif, whereas the Kv3.4 peptide showed compact folding throughout the molecule but did not exhibit any typical secondary structure (1) (Fig. 3). Despite these differences, however, the spatial distribution of charged and hydrophobic surface domains as well as the dipole moment were similar in both inactivation peptides (Fig. 3, left).
These similarities and differences in structural characteristics are in line with the functional properties observed for these peptides. On one hand, all three peptides were basically able to induce inactivation in noninactivating K+ channels. On the other hand, when investigated in the same channel, differentially structured ball peptides revealed different functional properties: the Kv3.4-derived peptide completely blocked noninactivating Kv1.1 channels, whereas the Kv1.4 peptide did not completely inactivate these channels, even at a 10-fold higher concentration (Fig. 4, A and B). Thus binding to the ball receptor is obviously different for ball peptides with different structures. Complete understanding of this fact, however, requires structural determination of both partners, ball and receptor. As a consequence, the ball-and-chain model as presented in its latest version in Fig. 4C must still remain preliminary and awaits completion by further structural work.
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FIGURE 4. Differential structural and functional properties of Kv3.4 (left) and Kv1.4 (right) inactivation gates. A: surface structure of the peptides with color coding as in Fig. 3. B: inactivation of Kv1.1 channels induced by fast application of the peptides. Application is indicated by horizontal bar; concentration and scale bars are as indicated. C: ball-and-chain mechanism at its latest stage. Channel core represents latest version of a model of the Shaker channel originally given in Ref. 5; inactivation gate is Kv3.4 peptide. Only 1 of the 4 ball domains is shown.
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Concluding remarks
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As long as crystals of the entire channel molecule are missing, the goal for the near future is to continue work on the functionally well-defined ball domains for which, for the first time, structural data are available. Further goals on that path are the structural and functional backgrounds of the inactivation regulation by protein phosphorylation (3) or that of inactivation transferred to noninactivating -subunits by an associated ß-subunit (12). These pieces of work, together with the structure of the channel's core, will someday allow a complete and final understanding of the inactivation process of voltage-dependent K+ channels.
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References
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Antz, C., M. Geyer, B. Fakler, M. K. Schott, H. R. Guy, R. Frank, J. P. Ruppersberg, and H. R. Kalbitzer. NMR-structure of inactivation gates of mammalian voltage-dependent potassium channels. Nature 385: 272–275, 1997.[Medline]
-
Armstrong, C. M. Sodium channels and gating currents. Physiol. Rev. 61: 644–683, 1981.[Free Full Text]
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Covarrubias, M., A. Wei, L. Salkoff, and T. B. Vyas. Elimination of rapid potassium channel inactivation by phosphorylation of the inactivation gate. Neuron 13: 1403–1412, 1994.[Medline]
-
Demo, S. D., and G. Yellen. The inactivation gate of the Shaker K+ channel behaves like an open-channel blocker. Neuron 7: 743–753, 1991.[Medline]
-
Guy, H. R., and S. R. Durell. Atomic scale structure and functional model of a voltage-gated potassium channel. Biophys. J. 62: 238–250, 1992.
-
Hille, B. Ionic Channels of Excitable Membranes. Sunderland, MA: Sinauer
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Hodgkin, A. L., and A. F. Huxley. A quantitative description of membrane current and its application to conduction and excitation in nerve. J. Physiol. (Lond.) 117: 500–544, 1952.
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Hoshi, T., W. N. Zagotta, and R. W. Aldrich. Biophysical and molecular mechanisms of Shaker potassium channel inactivation. Science 250: 533–538, 1990.[Abstract/Free Full Text]
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Isacoff, E. Y., Y. N. Jan, and L. Y. Jan. Putative receptor for the cytoplasmic inactivation gate in the Shaker K+ channel. Nature 353: 86–90, 1991.[Medline]
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Miller, C. M. 1990: Annus mirabilis of potassium channels. Science 252: 1092–1096, 1991.[Medline]
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Murrell-Lagnado, R. D., and R. W. Aldrich. Interactions of amino terminal domains of Shaker K channels with a pore blocking site studied with synthetic peptides. J. Gen. Physiol. 102: 949–975, 1993.[Abstract/Free Full Text]
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Rettig, J., S. H. Heinemann, F. Wunder, C. Lorra, D. N. Parcej, J. O. Dolly, and O. Pongs. Inactivation properties of voltage-gated K+ channels altered by presence of beta-subunit. Nature 369: 289–294, 1994.[Medline]
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Ruppersberg, J. P., R. Frank, O. Pongs, and M. Stocker. Cloned neuronal IK(A) channels reopen during recovery from inactivation. Nature 353: 657–660, 1991.[Medline]
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Schwarz, T. L., B. L. Tempel, D. M. Papazian, Y. N. Jan, and L. Y. Jan. Multiple potassium channel components are produced by alternative splicing at the Shaker locus in Drosophila. Nature 331: 137–142, 1988.[Medline]
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Zagotta, W. N., T. Hoshi, and R. W. Aldrich. Restoration of inactivation in mutants of Shaker potassium channels by a peptide derived from ShB. Science 250: 568–571, 1990.[Abstract/Free Full Text]
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Introduction
Definition of the inactivation...
