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• W2020882032 abstract "Outward movement of the voltage sensor is coupled to activation in voltage-gated ion channels; however, the precise mechanism and structural basis of this gating event are poorly understood. Potential insight into the coupling mechanism was provided by our previous finding that mutation to Lys of a single residue (Asp540) located in the S4-S5 linker endowed HERG (human ether-a-go-go-related gene) K+ channels with the unusual ability to open in response to membrane depolarization and hyperpolarization in a voltage-dependent manner. We hypothesized that the unusual hyperpolarization-induced gating occurred through an interaction between Lys540 and the C-terminal end of the S6 domain, the region proposed to form the activation gate. Therefore, we mutated six residues located in this region of S6 (Ile662–Tyr667) to Ala in D540K HERG channels. Mutation of Arg665, but not the other five residues, prevented hyperpolarization-dependent reopening of D540K HERG channels. Mutation of Arg665 to Gln or Asp also prevented reopening. In addition, D540R and D540K/R665K HERG reopened in response to hyperpolarization. Together these findings suggest that a single residue (Arg665) in the S6 domain interacts with Lys540 by electrostatic repulsion to couple voltage sensing to hyperpolarization-dependent opening of D540K HERG K+ channels. Moreover, our findings suggest that the C-terminal ends of S4 and S6 are in close proximity at hyperpolarized membrane potentials. Outward movement of the voltage sensor is coupled to activation in voltage-gated ion channels; however, the precise mechanism and structural basis of this gating event are poorly understood. Potential insight into the coupling mechanism was provided by our previous finding that mutation to Lys of a single residue (Asp540) located in the S4-S5 linker endowed HERG (human ether-a-go-go-related gene) K+ channels with the unusual ability to open in response to membrane depolarization and hyperpolarization in a voltage-dependent manner. We hypothesized that the unusual hyperpolarization-induced gating occurred through an interaction between Lys540 and the C-terminal end of the S6 domain, the region proposed to form the activation gate. Therefore, we mutated six residues located in this region of S6 (Ile662–Tyr667) to Ala in D540K HERG channels. Mutation of Arg665, but not the other five residues, prevented hyperpolarization-dependent reopening of D540K HERG channels. Mutation of Arg665 to Gln or Asp also prevented reopening. In addition, D540R and D540K/R665K HERG reopened in response to hyperpolarization. Together these findings suggest that a single residue (Arg665) in the S6 domain interacts with Lys540 by electrostatic repulsion to couple voltage sensing to hyperpolarization-dependent opening of D540K HERG K+ channels. Moreover, our findings suggest that the C-terminal ends of S4 and S6 are in close proximity at hyperpolarized membrane potentials. The human ether-a-go-go-related gene (HERG) 1The abbreviations used are: HERGhuman ether-a-go-go-related geneKvvoltage-gated potassiumOopen stateOhhyperpolarization-induced open stateWTwild-typeMES2-(N-morpholino)ethanesulfonic acidHCNhyperpolarization-activated channel encodes the α-subunit of the ion channel underlying the cardiac delayed rectifier K+ current, IKr (1Sanguinetti M.C. Jiang C. Curran M.E. Keating M.T. Cell. 1995; 81: 299-307Abstract Full Text PDF PubMed Scopus (2152) Google Scholar, 2Trudeau M. Warmke J.W. Ganetzky B. Robertson G.A. Science. 1995; 269: 92-95Crossref PubMed Scopus (1099) Google Scholar). Similar to other voltage-gated K+ (Kv) channels, HERG channels are closed at negative transmembrane potentials and open or inactivate in response to membrane depolarization. Membrane depolarization causes outward displacement of the voltage-sensing S4 domain (3Stuhmer W. Conti F. Suzuki H. Wang X.D. Noda M. Yahagi N. Kubo H. Numa S. Nature. 1989; 339: 597-603Crossref PubMed Scopus (947) Google Scholar, 4Perozo E. Papazian D.M. Stefani E. Bezanilla F. Biophys. J. 1992; 62: 160-171Abstract Full Text PDF PubMed Scopus (68) Google Scholar, 5Stefani E. Toro L. Perozo E. Bezanilla F. Biophys. J. 1994; 66: 996-1010Abstract Full Text PDF PubMed Scopus (138) Google Scholar, 6Bezanilla F. Perozo E. Stefani E. Biophys. J. 1994; 66: 1011-1021Abstract Full Text PDF PubMed Scopus (236) Google Scholar), which precedes opening of the activation gate. Although the structural basis of the activation gate is not clear, substantial evidence implicates a critical role for the S6 α-helices (7Doyle D.A. Morais Cabral J. Pfuetzner R.A. Kuo A. Gulbis J.M. Cohen S.L. Chait B.T. MacKinnon R. Science. 1998; 280: 69-77Crossref PubMed Scopus (5745) Google Scholar, 8Liu Y. Holmgren M. Jurman M.E. Yellen G. Neuron. 1997; 19: 175-184Abstract Full Text Full Text PDF PubMed Scopus (432) Google Scholar, 9Yellen G. Q. Rev. Biophys. 1998; 31: 239-295Crossref PubMed Scopus (401) Google Scholar, 10Perozo E. Cortes D.M. Cuello L.G. Science. 1999; 285: 73-78Crossref PubMed Scopus (496) Google Scholar). The equivalent domains of the KcsA channel (inner helices) line the channel pore and crisscross near the cytoplasmic side of the membrane to create a narrow aperture (7Doyle D.A. Morais Cabral J. Pfuetzner R.A. Kuo A. Gulbis J.M. Cohen S.L. Chait B.T. MacKinnon R. Science. 1998; 280: 69-77Crossref PubMed Scopus (5745) Google Scholar). Narrowing or widening of the aperture is hypothesized to mediate channel closure or opening, respectively. Activation of KcsA channels by protons involves counterclockwise rotation of the inner helices, which presumably widens the diameter of the aperture to allow ion permeation (10Perozo E. Cortes D.M. Cuello L.G. Science. 1999; 285: 73-78Crossref PubMed Scopus (496) Google Scholar). Substituted cysteine accessibility mutagenesis experiments have identified specific residues in the S6 domain of Shaker channels that are located on either side of the putative activation gate (8Liu Y. Holmgren M. Jurman M.E. Yellen G. Neuron. 1997; 19: 175-184Abstract Full Text Full Text PDF PubMed Scopus (432) Google Scholar). human ether-a-go-go-related gene voltage-gated potassium open state hyperpolarization-induced open state wild-type 2-(N-morpholino)ethanesulfonic acid hyperpolarization-activated channel What is the structural basis for coupling the movement of the voltage sensors with opening of the activation gate? Displacement of S4 could be transmitted to the activation gate via the concerted movement of domains S4-S6 or via specific interactions between more limited regions of the channel. For example, the intracellular S4-S5 linker may provide a direct link between the S4 domain and the activation gate (9Yellen G. Q. Rev. Biophys. 1998; 31: 239-295Crossref PubMed Scopus (401) Google Scholar, 11Durell S.R. Hao Y. Guy H.R. J. Struct. Biol. 1998; 121: 263-284Crossref PubMed Scopus (94) Google Scholar). Voltage-dependent modification of the S4-S5 linker by thiol-reactive reagents suggests that conformational changes in the linker are coupled to movement of the S4 domain (12Holmgren M. Jurman M.E. Yellen G. J. Gen. Physiol. 1996; 108: 195-206Crossref PubMed Scopus (98) Google Scholar). Mutations in the S4-S5 linker influence the activation gating properties of Kv2.1 and Kv3.1 channels (13Shieh C.-C. Klemic K.G. Kirsch G.E. J. Gen. Physiol. 1997; 109: 767-778Crossref PubMed Scopus (65) Google Scholar). Finally, a point mutation located in the S4-S5 linker (D540K) fundamentally alters the gating properties of HERG channels (14Sanguinetti M.C. Xu Q.P. J. Physiol. (Lond.). 1999; 514: 667-675Crossref Scopus (142) Google Scholar). D540K HERG channels have the unique ability to open in response to membrane hyperpolarization while retaining the ability to activate and inactivate in response to membrane depolarization. Although these studies suggest an important role for the S4-S5 linker in channel gating, specific interactions between the linker and the activation gate have not been demonstrated. The unique gating properties of D540K HERG provide the opportunity to characterize specific interactions that mediate coupling between voltage sensing and channel activation. We hypothesized that the closed state of D540K HERG channels is destabilized by the interaction of Lys540 with a specific amino acid(s) of the S6 domain. In this study we focused our attention on the C-terminal region of S6 because of the proposed role of the homologous region in KcsA channel activation. Six adjacent residues in S6 of D540K HERG were individually mutated to Ala, and the ability of the channels to open in response to membrane hyperpolarization was assessed. This approach identified a single residue (Arg665) that interacted with Lys540 to permit hyperpolarization-induced channel opening. Further mutagenesis suggested that the mechanism of this unique gating was electrostatic repulsion between basic residues at positions 540 and 665. Mutations were introduced into wild-type (WT) HERG using site-directed mutagenesis as previously described (15Sarkar G. Sommer S.S. BioTechniques. 1990; 8: 404-407PubMed Google Scholar). Before use in expression experiments, the constructs were characterized by restriction mapping and DNA sequence analyses. cRNA for injection into oocytes was prepared with SP6 Capscribe (Roche Molecular Biochemicals) following linearization with EcoRI. Xenopus laevis frogs were anesthetized by immersion in 0.2% tricaine for 15 min. Ovarian lobes were digested with 2 mg/ml Type 1A collagenase (Sigma) in Ca2+-free ND96 solution for 1.5 h to remove follicle cells. Stage IV and V oocytes were injected with WT or mutant HERG cRNA (5–15 ng) and then cultured in Barth's solution supplemented with 50 μg/ml gentamycin and 1 mm pyruvate at 18 °C. Barth's solution contained (in mm): 88 NaCl, 1 KCl, 0.4 CaCl2, 0.33 Ca(NO3)2, 1 MgSO4, 2.4 NaHCO3, 10 HEPES (pH 7.4). The two-microelectrode voltage clamp technique (16Stuhmer W. Methods Enzymol. 1992; 207: 319-339Crossref PubMed Scopus (262) Google Scholar) was used to record membrane currents in oocytes 1–3 days after cRNA injection. To attenuate endogenous chloride currents, Cl− was replaced with MES in the external solution containing (in mm) 96 NaMES, 2 KMES, 2 CaMES2, 5 HEPES, 1 MgCl2, adjusted to pH 7.6 with methanesulfonic acid. Currents were recorded at room temperature (21–23 °C). Glass microelectrodes were filled with 3 m KCl, and their tips were broken to obtain tip resistances of 0.8–1.5 megaohms. Oocytes were voltage-clamped with a GeneClamp 500 amplifier (Axon Instruments, Foster City, CA). Voltage commands were generated using pClamp8 software (Axon Instruments), a personal computer, and a DigiData 1200 series interface (Axon Instruments). The oocyte membrane potential was held at −80 mV between test pulses, unless noted otherwise. The isochronal voltage dependence of activation of the hyperpolarization-induced open state (Oh) and the normal depolarization-induced open state (O) of D540K HERG channels were determined from tail currents measured at −70 mV following 2-s hyperpolarizing or depolarizing voltage steps, respectively. Tail current amplitude (It) was determined by fitting the decaying portion of tail currents to a bi-exponential function and extrapolating to the beginning of the repolarizing step. It was plotted versus test potential (Vt) and fitted to the sum of two Boltzmann distributions using ORIGIN software (Northampton, MA). I=ImaxOh/(1+exp[(Vt−V1/2Oh)/kOh])+Equation 1 ImaxO/(1+exp[(V1/2O−Vt)/kO]) V1/2 Oh and V1/2 O are the voltages at which the Oh and O state currents are half-activated. kOh and kO are the slope factors for the biphasic activation curve.Imax Oh and Imax O are the maximum Oh and O state tail current values. For this analysis we assume that the relative probability for the Oh and O state approaches zero at extreme voltages. For WT HERG (no Oh state) the first half of Equation 1 was set to zero. The time course of deactivation was obtained by fitting decaying tail currents to a bi-exponential function. Data are expressed as the means ± S.E. (n = number of oocytes). The properties of WT and D540K HERG have been described previously (1Sanguinetti M.C. Jiang C. Curran M.E. Keating M.T. Cell. 1995; 81: 299-307Abstract Full Text PDF PubMed Scopus (2152) Google Scholar). Briefly, WT HERG current activated very slowly in response to weak depolarizations (e.g. −20 mV), whereas the activation rate was relatively fast at stronger depolarizations (e.g. +20 mV) (Fig. 1 A, left panel). Fast inactivation predominated at depolarized potentials, resulting in a decrease in steady state current magnitude at potentials positive to −10 mV (Fig. 1 B). Membrane hyperpolarization to potentials negative to −90 mV failed to induce inward current (Fig. 1, A and B). Repolarization to −70 mV after a depolarizing test pulse induced a tail current with a rapid rising phase followed by a slow decay, as channels that were inactivated during the test pulse rapidly recovered from inactivation and then slowly deactivated (Fig. 1 A, left panel). As reported previously (14Sanguinetti M.C. Xu Q.P. J. Physiol. (Lond.). 1999; 514: 667-675Crossref Scopus (142) Google Scholar), D540K HERG current activated nearly instantaneously upon depolarization from a holding potential of −80 mV. Hyperpolarization to potentials negative to −90 mV induced a slowly activating current that attained a steady state amplitude in 2 s (Fig. 1 A, right panel). The resulting current-voltage (I-V) relationship showed that inward current magnitude was much greater than outward current for equivalent electrochemical driving forces (Fig. 1 B). Repolarization to −70 mV after a hyperpolarizing test pulse (e.g. −160 mV) induced a tail current whose magnitude was larger and decay slower than tail currents induced after depolarizing test pulses (Fig. 1 A, inset). Tail currents after depolarizing test pulses (e.g. +40 mV) initially increased as channels recovered from inactivation and then decayed as channels deactivated into the closed state (Fig. 1 A, inset). The voltage dependence for activation of the Oh and O states for D540K HERG was determined by plotting peak tail current at −70 mV versus test potential and fitting the relationship to the sum of two Boltzmann functions. The normalized voltage dependence of the D540K channel opening had an inverted bell-shaped relationship (Fig. 1 C). The V1/2 values for O and Oh state activation were −15.5 ± 2.5 and −109.8 ± 1.8 mV, respectively. Therefore, the voltage dependence of depolarization- and hyperpolarization-induced channel opening was sufficiently separated to result in a voltage range near −60 mV where most channels were in a closed state. We tested the hypothesis that the Oh state is dependent upon interactions between Lys540 and a specific residue(s) in the C-terminal region of S6. Amino acids 662–667 (Fig. 2) of D540K HERG were individually mutated to Ala, and the resulting double mutant channel function was assayed using the voltage pulse protocol illustrated in Fig. 1 A. An amino acid mutation that prevented hyperpolarization-induced channel activation was considered a candidate residue for direct interaction with Lys540. The double mutant channels could be classified into three distinct groups based upon the relative magnitudes of inward and outward currents and the rate of deactivation from the Oh state. Group 1, comprising D540K/I663A, D540K/Q664A, and D540K/Y667A HERG channels, were most similar to D540K HERG. These mutant channels were characterized by large inward currents activated by hyperpolarization and extremely fast activation of outward current (Fig. 3, A–C). Unlike D540K HERG channels, Group 1 mutant channels exhibited a relatively large instantaneous current relative to the time-dependent component of inward current in response to hyperpolarization. In addition, deactivation of channels from the Oh state was slower than D540K HERG (Fig. 4). Group 2, consisting of D540K/I662A and D540K/L666A HERG channels, had relatively small hyperpolarization-activated inward currents compared with depolarization-activated outward currents (Fig. 3, D and E). Furthermore, deactivation of current after hyperpolarizing pulses was fast compared with deactivation after depolarizing pulses, and this rate was faster than D540K HERG channels (Fig. 4). In contrast to Groups 1 and 2, D540K/R665A HERG channels did not open in response to hyperpolarizing pulses as negative as −160 mV (Fig. 3 F). The gating kinetics of D540K/R665A HERG channels were also different from the other double mutant channels. The rates of current activation and deactivation were much slower (Fig. 3 F).Figure 4Time constants for Oh state current deactivation at −70 mV following a pulse to −140 mV (n = 4–10).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The voltage dependence of channel activation was determined by plotting the relative amplitude of tail currents at −70 mV as a function of test potential. The currents were normalized to the largest tail current amplitude. For Group 1 channels, currents were normalized to the tail current measured after a pulse to −160 mV (Fig. 5 A). For Group 2 and D540K/R665A HERG, currents were normalized to the tail current measured after a depolarizing pulse (Fig. 5 B). The voltage dependence of channel activation was fit with the sum of two Boltzmann functions, one describing the hyperpolarization-dependent channel opening and another describing the depolarization-dependent channel opening. The V1/2 for Oh state activation varied between −65 and −112 mV, and the slope factor varied between 9 and 31 mV for the different double mutant channels, compared with a V1/2 of −110 mV and a slope factor of 9 mV for D540K (Table I). The V1/2 for O state activation varied between −11 and −37 mV with a slope factor between 12 and 22 mV for the different double mutant channels, compared with −16 mV and 12 mV for D540K (Table I). The O state activation parameters of D540K/I663A HERG channels could not be accurately determined because the relative amplitude of tail currents was too small. D540K/R665A HERG channels did not reopen with hyperpolarization. Unlike the other mutants, the voltage dependence of O state activation for D540K/R665A HERG was best described by the sum of two Boltzmann functions (Table I).Table IVoltage dependence of Oh and O current activationChannelOh activationO activationnV1/2kV1/2kmVmVWT HERGN/AN/A−11.4 ± 0.79.0 ± 0.48 D540K−109.8 ± 1.811.7 ± 0.9−15.5 ± 2.512.4 ± 0.47 D540R−96.0 ± 1.012.7 ± 1.2−7.6 ± 2.715.0 ± 1.38D540K/I663A−95 ± 213.1 ± 1.3N.D.N.D.7D540K/Q664A−104.6 ± 2.514.9 ± 1.9−30.7 ± 3.114.6 ± 0.74D540K/Y667A−86.9 ± 3.925.5 ± 1.4−26.5 ± 2.012.4 ± 1.38D540K/I662A−65.0 ± 4.731.0 ± 2.5−36.7 ± 3.022.3 ± 1.14D540K/L666A−108 ± 1.29.6 ± 0.3−10.6 ± 1.516.2 ± 0.510D540K/R665AN/AN/A−57.4 ± 0.67.4 ± 0.54−15.5 ± 4.49.7 ± 1.0The voltage dependence of Oh and O current activation was determined by plotting peak tail current amplitude at −70 mV versus test potential and fitting the data to the sum of two Boltzmann functions. V1/2, potential of half-maximal Oh or O state current activation; k, slope factor; n, number of oocytes. N/A (not applicable), no Oh current detected. N.D. (not determined), unable to accurately determine O current activation parameters due to small tail current amplitude. Open table in a new tab The voltage dependence of Oh and O current activation was determined by plotting peak tail current amplitude at −70 mV versus test potential and fitting the data to the sum of two Boltzmann functions. V1/2, potential of half-maximal Oh or O state current activation; k, slope factor; n, number of oocytes. N/A (not applicable), no Oh current detected. N.D. (not determined), unable to accurately determine O current activation parameters due to small tail current amplitude. In summary, mutation of Arg665 to Ala, but not the other five residues in the scanned region of the S6 domain, rescued WT channel function by preventing the hyperpolarization-dependent reopening of D540K HERG channels. Although D540K-I662A and D540K-L666A HERG channels reopened with membrane hyperpolarization, the relative magnitude of inward current was small and deactivation from the Oh state was rapid compared with D540K HERG, consistent with destabilization of the Oh state. These findings demonstrate that specific residues in S6 can influence the stability of the D540K HERG channel Oh state and identify a critical role for Arg665 in hyperpolarization-dependent channel gating. To further investigate the role of the amino acid at position 665 in mediating hyperpolarization-induced reopening of D540K HERG channels, we mutated Arg665 to an acidic residue (Asp), a polar residue (Gln), and a basic residue (Lys). Neither D540K-R665D nor D540K-R665Q channels reopened with hyperpolarizing pulses (Fig. 6, A, B, and E). In contrast, substitution of Arg665 with another basic residue, Lys, induced channels to reopen in response to hyperpolarization (Fig. 6 C). The biophysical properties of D540K-R665K HERG channels were similar to D540K HERG channels, with the exceptions that depolarization-induced activation was shifted by −18 mV and the slope of the Oh activation curve was less steep (Table I). To confirm the importance of a basic residue at position 540, we substituted another basic residue, Arg, at this position in WT HERG. Like D540K HERG, D540R HERG channels also reopened in a time- and voltage-dependent manner with membrane hyperpolarization (Fig. 6 D). The V1/2 for the Oh and O state activation curves for D540R were shifted by about +10 mV compared with D540K HERG (Table I). These findings underscore the requirement for basic residues at position 665 in S6 and position 540 in the S4-S5 linker to permit hyperpolarization-induced channel opening and suggest that electrostatic repulsion might mediate this unusual gating. If an electrostatic interaction between residues 540 and 665 was the sole mechanism of hyperpolarization-induced channel opening, then substitution of Arg665 with an acidic residue in WT HERG (D540) might also produce a channel with properties similar to D540K HERG. We therefore substituted Glu or Asp for Arg at position 665 in WT HERG. R665E HERG channels did not functionally express. R665D HERG channels expressed but did not reopen with membrane hyperpolarization, even when cells were pulsed to −200 mV (data not shown). The R665D mutation did, however, affect the gating properties in that the transition from the depolarized O state to the closed state was markedly slow compared with WT HERG (Fig. 7). To further characterize the role of Arg665 in WT HERG channel gating, we studied the effects of substitution of Arg665with the hydrophobic residue Ala or the polar residue Gln. R665A, and to a lesser extent R665Q, HERG channels deactivated more slowly than WT HERG (Fig. 7). Mutations at position 665 also shifted the voltage dependence of O state activation to more hyperpolarized potentials compared with WT or D540K HERG (R665D,V1/2 = −35.6 ± 1.4 mV, k = 10.6 ± 0.6 mV; R665A, V1/2 = −30.9 ± 0.6 mV, k = 6.5 ± 0.1 mV; and R665Q HERG,V1/2 = −19.4 ± 1.2 mV, k = 7.1 ± 0.3 mV). Taken together, these data indicate that mutation of Arg665 slows deactivation, providing further evidence for the critical role of this residue in HERG channel gating. Despite recent advances in the understanding of the structural basis of the voltage sensor and the activation gate, little is known about how voltage sensing is coupled to channel opening. Potassium channel activation likely involves rotation of the S4 (17Cha A. Snyder G.E. Selvin P.R. Bezanilla F. Nature. 1999; 402: 809-813Crossref PubMed Scopus (435) Google Scholar, 18Glauner K.S. Mannuzzu L.M. Gandhi C.S. Isacoff E.Y. Nature. 1999; 402: 813-817Crossref PubMed Scopus (246) Google Scholar) and S6 (10Perozo E. Cortes D.M. Cuello L.G. Science. 1999; 285: 73-78Crossref PubMed Scopus (496) Google Scholar) domains. S6 rotation presumably opens the channel by increasing the diameter of the aperture formed by crisscrossing of the S6 domains (10Perozo E. Cortes D.M. Cuello L.G. Science. 1999; 285: 73-78Crossref PubMed Scopus (496) Google Scholar, 19Liu Y.S. Sompornpisut P. Perozo E. Nat. Struct. Biol. 2001; 8: 883-887Crossref PubMed Scopus (185) Google Scholar). The coupling between voltage sensing and channel opening could be mediated via a concerted movement of the S4-S6 domains or transduced by a discrete region of the protein (e.g. the S4-S5 linker) (9Yellen G. Q. Rev. Biophys. 1998; 31: 239-295Crossref PubMed Scopus (401) Google Scholar). The coupling mechanism is usually vectorial; membrane depolarization induces opening of the Kv channels, whereas hyperpolarization induces opening of the HCN channels. In contrast, opening of D540K HERG channels is coupled to both depolarization and hyperpolarization. The single channel conductance of the O and Oh states is identical (20Mitcheson J.S. Chen J. Sanguinetti M.C. J. Gen. Physiol. 2000; 115: 229-240Crossref PubMed Scopus (224) Google Scholar), implying that D540K affects the activation gate but not the selectivity filter. A simplified gating scheme for D540K HERG channels is shown (Scheme FSI). In the same manner that depolarization-induced outward movement of the S4 domain is coupled to normal channel opening (3Stuhmer W. Conti F. Suzuki H. Wang X.D. Noda M. Yahagi N. Kubo H. Numa S. Nature. 1989; 339: 597-603Crossref PubMed Scopus (947) Google Scholar, 4Perozo E. Papazian D.M. Stefani E. Bezanilla F. Biophys. J. 1992; 62: 160-171Abstract Full Text PDF PubMed Scopus (68) Google Scholar, 5Stefani E. Toro L. Perozo E. Bezanilla F. Biophys. J. 1994; 66: 996-1010Abstract Full Text PDF PubMed Scopus (138) Google Scholar, 6Bezanilla F. Perozo E. Stefani E. Biophys. J. 1994; 66: 1011-1021Abstract Full Text PDF PubMed Scopus (236) Google Scholar), we propose that the voltage dependence of the Oh state derives from hyperpolarization-induced inward movement of S4. We hypothesized that the Lys540 point mutation in HERG causes channels to reopen in response to membrane hyperpolarization because of a direct interaction with the activation gate. Therefore, we sequentially mutated to Ala residues 662–667 located in the C-terminal region of S6. Of the six Ala-substituted double mutants examined, only D540K-R665A HERG channels failed to reopen upon hyperpolarization, suggesting a direct interaction between these two residues was necessary to mediate channel reopening. Mutation of the other five S6 residues to Ala had a variable effect on the ability of the D540K HERG channel to reopen at negative potentials. Mutation of Ile663, Gln664, or Tyr667 (Group 1) had the least effect on channel properties compared with D540K HERG. Mutation of Ile662 or Tyr666 (Group 2) reduced the relative magnitude of inward current activated by hyperpolarization and accelerated the rate of deactivation from the Oh state. Thus, mutation of Ile662 or Tyr666 to Ala reduced the functional consequence of the presumed interaction between D540K and Arg665. A possible explanation for these findings is the location of the Group 2 versus the Group 1 residues relative to Arg665. Using the crystal structure of KcsA, a homology model of HERG was constructed (21Mitcheson J.S. Chen J. Lin M. Culberson C. Sanguinetti M.C. Proc. Natl. Acad. Sci. U. S. A. . 2000; 97: 12329-12333Crossref PubMed Scopus (860) Google Scholar). According to this model, Arg665 faces away from the central cavity of the channel. The side groups of Arg665, Ile662, and Tyr666 are clustered together (Fig. 8). In contrast, the Group 1 residues Ile663, Gln664, and Tyr667 project away from Arg665. Mutation of Ile662 or Tyr666 to the smaller Ala may alter the orientation of Arg665 in a manner that results in a diminished interaction with Lys540, whereas mutation of any one of the Group 1 amino acids has no significant effect on the Lys540-Arg665 interaction.Figure 8Putative location of Arg665 and nearby residues in the S6 domain of a HERG channel subunit. The homology model of HERG pore helix-S6 domain was based on the crystal structure of the KcsA potassium channel (7Doyle D.A. Morais Cabral J. Pfuetzner R.A. Kuo A. Gulbis J.M. Cohen S.L. Chait B.T. MacKinnon R. Science. 1998; 280: 69-77Crossref PubMed Scopus (5745) Google Scholar). The subunit was rotated by 90o (middle panel) and 180o (right panel). The S5 and S6 domains are colored yellow, and the pore helix is colored green. The six amino acids in S6 that were mutated to Ala are shown in colored space-filling mode: Ile662(blue); Ile663 (orange); Gln664 (cyan); Arg665(red); Leu666 (green); Tyr667 (white). Note that the side chains of Ile662 and Leu666 face toward position Arg665, whereas the side chains Ile663, Gln664, and Tyr667 face away (see text for details).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Substitution of Arg665 with a basic residue (Lys), but not an acidic (Asp) or polar residue (Gln), restored the ability of D540K HERG channels to enter the Oh state. Likewise, substitution of Asp540 with a basic amino acid, Arg (Fig. 6 D), but not Ala (14Sanguinetti M.C. Xu Q.P. J. Physiol. (Lond.). 1999; 514: 667-675Crossref Scopus (142) Google Scholar) resulted in channels that were capable of hyperpolarization-induced opening. Thus, basic residues are required at position 540 in the S4-S5 linker and position 665 in the C-terminal region of S6 to enable hyperpolarization-induced channel opening. This finding suggests that electrostatic repulsion repulsion mediates this channel reopening and implies that the C-terminal ends of the S4 and S6 domains are situated in close proximity at negative transmembrane potentials. However, our data do not provide evidence for or against the possibility that charge pairing between Asp540 and Arg665 stabilizes the closed state of WT HERG channels. To further explore the potential importance of electrostatic repulsion between specific residues in the S4-S5 linker and S6 we tested whether introduction of an acidic residue at position 665 would repel Asp540 and allow channels to reopen with hyperpolarization. We were unable to reproduce the property of hyperpolarization-induced channel gating by engineering acidic residues at positions 540 and 665. A possible explanation for this negative result relates to the relative length of the side chains of the residues at these positions. The side chains of Arg and Lys are substantially longer than Asp. Perhaps the side chains of Asp540 and Asp665 are not close enough or favorably oriented for electrostatic repulsion to occur and cause channel reopening. Although R665D HERG channels did not reopen with hyperpolarization, we did observe a dramatic effect on channel gating. Deactivation was markedly slower for R665D HERG compared with WT HERG channels, far in excess than what could be explained by the shift in voltage dependence of activation. Thus, once the open conformation is achieved by depolarization, the transitions to the closed conformation are inhibited by Asp665. Other amino acid substitutions at position 665 (R665A, R665Q) resulted in qualitatively similar but less dramatic effects. In a previous study, we individually mutated residues 646–667 to Ala in the S6 domain of HERG (21Mitcheson J.S. Chen J. Lin M. Culberson C. Sanguinetti M.C. Proc. Natl. Acad. Sci. U. S. A. . 2000; 97: 12329-12333Crossref PubMed Scopus (860) Google Scholar). Channel deactivation was not significantly affected with the exception of V659A and R665A. V659C channels were also reported to deactivate slowly (22Gillian A.L. Wang J. Tanty T. McCarthy E. Robertson G.A. Biophys. J. 2001; 80: 840AGoogle Scholar). It is possible that amino acid substitutions at specific residues in the S6 domain of HERG slow channel deactivation by impeding rotation of the helical bundle back into its resting conformation. It is conceivable that mutations at positions 540 and 665 may exert independent effects on channel gating. For example, mutation of Asp540 to Lys or Arg could enable inward movement of the S4 domain and cause channel reopening without interacting specifically with Arg665. An additional mutation of Arg665to a nonbasic residue might independently stabilize the closed configuration and prevent the D540K-induced channel reopening. Another possible explanation for our results is that Lys540 and Arg665 may interact by an allosteric mechanism. How is membrane hyperpolarization coupled to channel opening for D540K HERG but not WT HERG? One possibility is that the S4 domain in WT HERG moves inward with hyperpolarization but does not couple to channel opening at negative potentials unless Asp540 is mutated to Lys. This seems unlikely because inward gating currents are not measurable below −120 mV in other Kv channels, including ether-a-go-go channels that share high homology with HERG (5Stefani E. Toro L. Perozo E. Bezanilla F. Biophys. J. 1994; 66: 996-1010Abstract Full Text PDF PubMed Scopus (138) Google Scholar, 6Bezanilla F. Perozo E. Stefani E. Biophys. J. 1994; 66: 1011-1021Abstract Full Text PDF PubMed Scopus (236) Google Scholar, 23Tang C.Y. Bezanilla F. Papazian D.M. J. Gen. Physiol. 2000; 115: 319-338Crossref PubMed Scopus (47) Google Scholar). More likely, inward movement of the S4 domain is enabled by the D540K mutation. The time and voltage dependence of hyperpolarization-induced activation of D540K HERG is qualitatively similar to activation of HCN pacemaker channels. This suggested to us that an interaction between the S4-S5 linker and the S6 domain could mediate activation of HCN channels. Indeed, several mutations in the S4-S5 linker of HCN channels disrupted normal channel closure as though the link between voltage-sensing and channel opening was uncoupled (24Chen J. Mitcheson J., S. Tristani-Firouzi M. Lin M. Sanguinetti M.C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11277-11282Crossref PubMed Scopus (133) Google Scholar). Thus, the S4-S5 linker may physically couple S4 movement with the opening of both HERG and HCN channels. Further support for the importance of interactions between the S4-S5 linker and the C-terminal portion of S6 in channel gating comes from studies of chimeric Shaker and KcsA channels. Chimeric channels containing the pore module of KcsA (inner helix, pore domain, outer helix, and C terminus) inserted into the background of the voltage-sensing module of Shaker (S1-S4 and S4-S5 linker) traffic normally but do not gate in response to membrane depolarization (25Caprini M. Ferroni S. Planells-Cases R. Rueda J. Rapisarda C. Ferrer-Montiel A. Montal M. J. Biol. Chem. 2001; 276: 21070-21076Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). However, if the C terminus of Shaker is included in the chimera, the resulting channel opens in a voltage-dependent fashion (26Lu Z. Klem A.M. Ramu Y. Nature. 2001; 413: 809-813Crossref PubMed Scopus (274) Google Scholar). The specific residues of the C terminus that perform a critical role in transduction of voltage sensing to chimeric channel activation remain to be determined but may be analogous to the interactions described here for D540K HERG. In summary, the hyperpolarization-dependent reopening of D540K HERG channels is mediated by charge repulsion between Lys540 located in the S4-S5 linker and Arg665 located in the C-terminal end of the S6 domain. The bell-shaped voltage dependence of activation suggests that the S4 domain of D540K HERG can move inward in response to membrane hyperpolarization. We speculate that inward movement of the S4 domain with hyperpolarization is normally restricted by a stabilizing interaction between the S4-S5 linker and the C-terminal region of S6. Finally, our findings that Lys540 interacts with Arg665 puts constraints on the possible conformation of the HERG channel with respect to the location of the C-terminal ends of the S4 and S6 domains. We thank Peter Westenskow and Monica Lin for valuable technical support." @default.
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• W2020882032 title "Interactions between S4-S5 Linker and S6 Transmembrane Domain Modulate Gating of HERG K+ Channels" @default.
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