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• W2004157218 abstract "The goals of this study are to investigate the mechanism and site of action whereby a humanether-a-go-go-related gene (HERG)-specific scorpion peptide toxin, ErgTx, suppresses HERG current. We apply cysteine-scanning mutagenesis to the S5-P and P-S6 linkers of HERG and examine the resulting changes in ErgTx potency. Data are compared with the characteristics of charybdotoxin (ChTx, or its analogs) binding to the Shaker channel. ErgTx binds to the outer vestibule of HERG but may not physically occlude the pore. In contrast to ChTx·Shaker interaction, elevating [K]o (from 2 to 98 mm) does not affect ErgTx potency, and through-solution electrostatic forces only play a minor role in influencing ErgTx·HERG interaction. Cysteine mutations of three positions in S5-P linker (Trp-585, Gly-590, and Ile-593) and 1 position in P-S6 linker (Pro-632) induce profound changes in ErgTx binding (ΔΔG > 2 kcal/mol). We propose that the long S5-P linker of the HERG channel forms an amphipathic α-helix that, together with the P-S6 linker, forms a hydrophobic ErgTx binding site. This study paves the way for future mutant cycle analysis of interacting residues in the ErgTx·HERG complex, which, in conjunction with NMR determination of the ErgTx solution structure, will yield information about the topology of HERG's outer vestibule. The goals of this study are to investigate the mechanism and site of action whereby a humanether-a-go-go-related gene (HERG)-specific scorpion peptide toxin, ErgTx, suppresses HERG current. We apply cysteine-scanning mutagenesis to the S5-P and P-S6 linkers of HERG and examine the resulting changes in ErgTx potency. Data are compared with the characteristics of charybdotoxin (ChTx, or its analogs) binding to the Shaker channel. ErgTx binds to the outer vestibule of HERG but may not physically occlude the pore. In contrast to ChTx·Shaker interaction, elevating [K]o (from 2 to 98 mm) does not affect ErgTx potency, and through-solution electrostatic forces only play a minor role in influencing ErgTx·HERG interaction. Cysteine mutations of three positions in S5-P linker (Trp-585, Gly-590, and Ile-593) and 1 position in P-S6 linker (Pro-632) induce profound changes in ErgTx binding (ΔΔG > 2 kcal/mol). We propose that the long S5-P linker of the HERG channel forms an amphipathic α-helix that, together with the P-S6 linker, forms a hydrophobic ErgTx binding site. This study paves the way for future mutant cycle analysis of interacting residues in the ErgTx·HERG complex, which, in conjunction with NMR determination of the ErgTx solution structure, will yield information about the topology of HERG's outer vestibule. The rapid delayed rectifier (IKr) 1The abbreviations used are: IKrrapid delayed rectifierHERGhumanether-a-go-go-related geneErgTxHERG-specific peptide toxinChTxcharybdotoxinLQTlong QT syndromeWTwild-typeTEAtetraethylammonium1The abbreviations used are: IKrrapid delayed rectifierHERGhumanether-a-go-go-related geneErgTxHERG-specific peptide toxinChTxcharybdotoxinLQTlong QT syndromeWTwild-typeTEAtetraethylammonium is an important repolarizing current in many regions of the heart (1.Tseng G.-N. J. Mol. Cell Cardiol. 2001; 33: 835-849Abstract Full Text PDF PubMed Scopus (167) Google Scholar). The major subunit that forms the IKr channel in human heart is HERG (2.Sanguinetti M.C. Jiang C. Curran M.E. Keating M.T. Cell. 1995; 81: 299-307Abstract Full Text PDF PubMed Scopus (2118) Google Scholar). Inherited mutations in HERG have been identified and linked to congenital long QT syndrome (LQT2) (2.Sanguinetti M.C. Jiang C. Curran M.E. Keating M.T. Cell. 1995; 81: 299-307Abstract Full Text PDF PubMed Scopus (2118) Google Scholar, 3.Roden D.M. Balser J.R. Cardiovasc. Res. 1999; 44: 242-246Crossref PubMed Scopus (49) Google Scholar). Furthermore, drugs that suppress IKr have been linked to “acquired LQT” (3.Roden D.M. Balser J.R. Cardiovasc. Res. 1999; 44: 242-246Crossref PubMed Scopus (49) Google Scholar). These all point to the critical role played by IKr in maintaining cardiac electrical stability. Therefore, information about the structure of the IKr/HERG channel is important. Such information will help in rational drug design for agents that are useful for combating some forms of LQT syndrome (IKr/HERG agonists), or for agents with zero or low risk for inducing acquired LQT syndrome (no effects on IKr/HERG). rapid delayed rectifier humanether-a-go-go-related gene HERG-specific peptide toxin charybdotoxin long QT syndrome wild-type tetraethylammonium rapid delayed rectifier humanether-a-go-go-related gene HERG-specific peptide toxin charybdotoxin long QT syndrome wild-type tetraethylammonium One of the most critical factors in determining IKr/HERG function is its C-type inactivation process. This is the basis for the “inward rectification” property of IKr/HERG, which is important for shaping the action potentials in the heart. Inward rectification dictates that there is little or no outward IKr at positive plateau voltages (important for maintaining the plateau phase) but large outward IKr during phase 3 (ensuring efficient repolarization back to the resting membrane potential). Furthermore, the C-type inactivation process in the IKr/HERG channel appears to be intimately related to channel sensitivity to many different drugs (4.Ficker E. Jarolimek W. Kiehn J. Baumann A. Brown A.M. Circ. Res. 1998; 82: 386-395Crossref PubMed Scopus (262) Google Scholar, 5.Wang S. Morales M.J. Liu S. Strauss H.C. Rasmusson R.L. FEBS Lett. 1997; 417: 43-47Crossref PubMed Scopus (104) Google Scholar, 6.Numaguchi H. Mullins F.M. Johnson J.P.J. Johns D.C. Po S.S. Yang I.C.H. Tomaselli G.F. Balser J.R. Circ. Res. 2000; 87: 1012-1018Crossref PubMed Scopus (84) Google Scholar). C-type inactivation results from conformational changes in the outer mouth region of the channel, which prevent current flow through the pore (7.Smith P.L. Baukrowitz T. Yellen G. Nature. 1996; 379: 833-836Crossref PubMed Scopus (654) Google Scholar). Therefore, an important target for structural analysis is the outer mouth region of the IKr/HERG channel. There is a general consensus that the outer vestibules of various potassium channels share a common architecture (8.MacKinnon R. Reinhart P.H. White M.M. Neuron. 1988; 1: 997-1001Abstract Full Text PDF PubMed Scopus (111) Google Scholar, 9.Doyle D.A. Cabral J.M. Pfuetzner R.A. Kuo A. Gulbis J.M. Cohen S.L. Chait B.T. MacKinnon R. Science. 1998; 280: 69-77Crossref PubMed Scopus (5649) Google Scholar, 10.Rauer H. Lanigan M.D. Pennington M.W. Aiyar J. Ghanshani S. Cahalan M.D. Norton R.S. Chandy K.G. J. Biol. Chem. 2000; 275: 1201-1208Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Indeed, homology modeling of outer vestibules of mammalian voltage-gated K+ (Kv) channels based on the crystal structure of KcsA (9.Doyle D.A. Cabral J.M. Pfuetzner R.A. Kuo A. Gulbis J.M. Cohen S.L. Chait B.T. MacKinnon R. Science. 1998; 280: 69-77Crossref PubMed Scopus (5649) Google Scholar), a bacterial proton-gated potassium channel, has been successful in several cases (10.Rauer H. Lanigan M.D. Pennington M.W. Aiyar J. Ghanshani S. Cahalan M.D. Norton R.S. Chandy K.G. J. Biol. Chem. 2000; 275: 1201-1208Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). However, such a strategy may not be applicable to the HERG channel. First, the C-type inactivation in the IKr/HERG channel is unique among all potassium channels that have this gating process: C-type inactivation in IKr/HERG is extremely fast in onset and in reversal and is strongly voltage-dependent (11.Spector P.S. Curran M.E. Zou A. Keating M.T. Sanguinetti M.C. J. Gen. Physiol. 1996; 107: 611-619Crossref PubMed Scopus (373) Google Scholar). These unique kinetic features suggest that conformational changes in the outer mouth region of HERG during membrane depolarization may be different from those of the other channels. Second, HERG has an unusually long S5-P linker (43 amino acids), much longer than the 14- to 18-amino acid S5-P linker seen in most other Kv channels, as well as the so-called “turret” of KcsA (see Fig. 1 A). We have shown that mutations in the middle of HERG's S5-P linker, far away from the P (pore) loop in the one-dimensional sequence, can have profound effects on the outer mouth properties of the channel (12.Dun W. Jiang M. Tseng G.-N. Pflugers Archiv. 1999; 439: 141-149Crossref PubMed Scopus (38) Google Scholar). There have been no reports on similar effects of mutations in the middle of S5-P linker for other Kv channels. Thus, the S5-P linker of HERG may play a unique structural and functional role. Understanding such a role may provide answers to questions such as why the C-type inactivation in the HERG channel has such unique kinetic properties and how C-type inactivation is related to the channel's sensitivity to various drugs. Peptide toxins have been very useful tools in structural analysis of potassium channels, well before the data of KscA crystal structure were available (13.Possani L.D. Selisko B. Gurrola G.B. Perspect. Drug Disc. Des. 1999; 15/16: 15-40Crossref Scopus (77) Google Scholar, 14.Garcia M.L. Gao Y.-D. McManus O.B. Kaczorowski G.J. Toxicon. 2001; 39: 739-748Crossref PubMed Scopus (103) Google Scholar). Many short peptide scorpion toxins (α-KTx) are available that can block various potassium channels with differences in specificity and potency (13.Possani L.D. Selisko B. Gurrola G.B. Perspect. Drug Disc. Des. 1999; 15/16: 15-40Crossref Scopus (77) Google Scholar, 14.Garcia M.L. Gao Y.-D. McManus O.B. Kaczorowski G.J. Toxicon. 2001; 39: 739-748Crossref PubMed Scopus (103) Google Scholar, 15.Tytgat J. Chandy K.G. Garcia M.L. Gutman G.A. Martin-Eauclaire M.-F. van der Walt J.J. Possani L.D. Trends Pharmacol. Sci. 1999; 20: 444-447Abstract Full Text Full Text PDF PubMed Scopus (354) Google Scholar). Many α-KTx toxins have six cysteines, forming three disulfide bridges (15.Tytgat J. Chandy K.G. Garcia M.L. Gutman G.A. Martin-Eauclaire M.-F. van der Walt J.J. Possani L.D. Trends Pharmacol. Sci. 1999; 20: 444-447Abstract Full Text Full Text PDF PubMed Scopus (354) Google Scholar). They have a well-conserved positive residue (Lys-27 in ChTx) that serves to plug the channel pore (Fig. 1 B) (16.Goldstein S.A.N. Miller C. Biophys. J. 1993; 65: 1613-1619Abstract Full Text PDF PubMed Scopus (166) Google Scholar). ErgTx is purified fromCentruroides noxius Hoffmann (GenBank™accession number CnErg1). It has 42 amino acids and 4 disulfide bonds (17.Scaloni A. Bottiglieri C. Ferrara L. Corona M. Gurrola G.B. Batista C. Wanke E. Possani L.D. FEBS Lett. 2000; 479: 155-157Crossref PubMed Scopus (23) Google Scholar). Its amino acid sequence and disulfide bond pattern are not homologous to those of representative α-KTx toxins shown in Fig. 1 B. Previous work has shown that ErgTx is a potent blocker of HERG expressed in mammalian cells and native IKr in guinea pig ventricular myocytes (IC50 in the low nanomolar range) (18.Gurrola G.B. Rosati B. Rocchetti M. Pimienta G. Zaza A. Arcangeli A. Olivotto M. Possani L.D. Wanke E. FASEB J. 1999; 13: 953-962Crossref PubMed Scopus (94) Google Scholar). It does not block IKs, IK, ATP, IRK1, or EAG at 1 μm (18.Gurrola G.B. Rosati B. Rocchetti M. Pimienta G. Zaza A. Arcangeli A. Olivotto M. Possani L.D. Wanke E. FASEB J. 1999; 13: 953-962Crossref PubMed Scopus (94) Google Scholar). Recently, we showed that ErgTx sensitivity may be determined by the S5-P linker of the HERG channel (19.Pardo-Lopez L. Garcia-Valdes J. Gurrola G.B. Robertson G.A. Possani L.D. FEBS Lett. 2002; 510: 45-49Crossref PubMed Scopus (31) Google Scholar). Due to the four disulfide bonds, ErgTx should have a compact and rigid structure, amenable to NMR analysis of its solution structure. Our ultimate goal is to map the ErgTx binding site on HERG and to identify amino acid pairs that interact with each other across the toxin-channel interface (20.Hidalgo P. MacKinnon R. Science. 1995; 268: 307-310Crossref PubMed Scopus (423) Google Scholar, 21.Stocker M. Miller C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9509-9513Crossref PubMed Scopus (86) Google Scholar). In this way, we hope to obtain a “footprint” of the interaction surface of ErgTx on HERG and to derive the three-dimensional arrangement of relevant residues on the outer vestibule of this channel. This study is a first step toward that goal. We applied cysteine-scanning mutagenesis, replacing all residues in the outer vestibule region of HERG by cysteine one at a time, and studied how these mutations affect ErgTx binding to the channel. We chose cysteine, instead of alanine (22.Ranganathan R. Lewis J.H. MacKinnon R. Neuron. 1996; 16: 131-139Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar) or lysine (23.Goldstein S.A.N. Pheasant D.J. Miller C. Neuron. 1994; 12: 1377-1388Abstract Full Text PDF PubMed Scopus (292) Google Scholar), for two reasons. First, the cysteine side chain is small, hydrophobic, and usually well tolerated. Thus, it increases our chance of studying more mutant channels. Second, cysteine side chains can be specifically modified by methanethiosulfonate reagents. This will allow us to use different strategies to estimate the distances between channel residues of interests and the toxin binding site (21.Stocker M. Miller C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9509-9513Crossref PubMed Scopus (86) Google Scholar, 24.Gross A. MacKinnon R. Neuron. 1996; 16: 399-406Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). Based on these data, we propose that the central region of HERG's S5-P linker forms an amphipathic α-helix that interacts with the pore of the channel to form the ErgTx binding site. Venom was purified from scorpionsCentruroides noxius as described previously (17.Scaloni A. Bottiglieri C. Ferrara L. Corona M. Gurrola G.B. Batista C. Wanke E. Possani L.D. FEBS Lett. 2000; 479: 155-157Crossref PubMed Scopus (23) Google Scholar, 18.Gurrola G.B. Rosati B. Rocchetti M. Pimienta G. Zaza A. Arcangeli A. Olivotto M. Possani L.D. Wanke E. FASEB J. 1999; 13: 953-962Crossref PubMed Scopus (94) Google Scholar) with minor modifications. Briefly, crude venom was dissolved in water and centrifuged at 10,000 × g for 15 min. The supernatant was lyophilized and kept at −20 °C until final purification. After Sephadex G-50 gel filtration, fraction II was directly applied to a semi-preparative C18 reverse-phase column (Vydac, Hesperia, CA) and eluted with a linear gradient from 5% of solvent A (0.12% trifluoroacetic acid in water) to 60% solvent B (0.10% trifluoroacetic acid in acetonitrile) over 90 min. The component eluted at ∼30 min was further chromatographed by high performance liquid chromatography using an analytical C18 reverse-phase column. This gave a major pure component (ErgTx), whose primary structure was obtained by direct amino acid sequencing and by mass spectrometry (Fig. 1 B) (17.Scaloni A. Bottiglieri C. Ferrara L. Corona M. Gurrola G.B. Batista C. Wanke E. Possani L.D. FEBS Lett. 2000; 479: 155-157Crossref PubMed Scopus (23) Google Scholar). HERG in a vector pGH19 was a kind gift of Dr. Gail Robertson (University of Wisconsin-Madison). We subcloned the HERG cDNA sequence (GenBank™ accession number U04270) into the KpnI/XbaI site of a vector, pAlterMax, which was required for the oligonucleotide-directed mutagenesis procedure with a commercial kit (Altered Site II in vitromutagenesis system, Promega). Cysteine substitution mutations were confirmed by direct DNA sequencing around the mutation sites. In most cases, two separate colonies from each mutant were used for cRNA transcription and oocyte expression. No differences were seen in the phenotype of channels translated from the two cRNAs. For transcription, wild-type (WT) and mutant HERG sequences in the pAlterMax vector were linearized with NotI and transcribed using the T7 RNA polymerase and a commercial kit (mMESSAGE mMACHINE, Ambion, Austin, TX). All cRNA samples were quantified by denaturing RNA gels using densitometry (ChemiImager model 4400, α-Innotech Corp.). Oocyte isolation and cRNA injection were as described previously (25.Tseng-Crank J.C.L. Tseng G.-N. Schwartz A. Tanouye M.A. FEBS Lett. 1990; 268: 63-68Crossref PubMed Scopus (127) Google Scholar). Briefly, stage V oocytes were isolated from follicular cell layer after mild collagenase digestion and injected with cRNA solution using a Drummond digital microdispenser. The injection volume was ∼40 nl/oocyte, equivalent to cRNA of 12–18 ng/oocyte. Three to five days after cRNA injection, channel function was studied using the two-microelectrode voltage clamp method as previously described (26.Schreibmayer W. Lester H.A. Dascal N. Pflugers Archiv. 1994; 426: 453-458Crossref PubMed Scopus (147) Google Scholar). During recordings, oocytes were superfused with a low-Cl ND96 solution at room temperature. For experiments shown in Fig. 5, oocytes were not superfused but were placed in the bath solution of a fixed volume (1 ml). ErgTx was dissolved in sterile bovine serum albumin (0.1 mg/ml) solution at 2 μm and frozen in small aliquots. An aliquot was thawed and used for experiments in <2 days without refreezing. After control data were obtained, 5 μl of the ErgTx stock solution was added to the bath solution (1 ml) to reach a final concentration of 10 nm. Repetitive pipetting was needed to ensure complete equilibration of ErgTx in the bath solution. Toxin effects were evaluated when steady state was reached (4–10 min). Voltage clamp protocol generation and data acquisition were controlled by pClamp 5.5 (Axon Instruments). Data analysis was performed with pClamp 6 or 8, Excel (Microsoft) and PeakFit (Jandel Scientific). Specific protocols and methods of data analysis are described in the figure legends. Where appropriate, data are presented as mean ± S.E. Statistical analysis was performed with one-way analysis of variance, followed by Dunn's test (SigmaStat 2.0, SPSS). Fig. 2 A(panels a and c) shows the hallmark of wild-type (WT) HERG currents: strong inward rectification due to rapid onset and reversal of C-type inactivation in a voltage-dependent manner. A depolarization pulse to +60 mV elicited little outward current because of C-type inactivation. Subsequent repolarization to +40 to −60 mV induced outward tail currents with a distinct rising phase (recovery from C-type inactivation). This led to a prominent negative slope in the tail I–V relationship (+60 to −60 mV, Fig. 2 A, panel c). At more negative voltages, tail currents became less outward and reversed at −100 mV (reversal potential or E rev), close to the Nernst K+ equilibrium potential (E K ∼ −105 mV in 2 mm [K]o). Replacing histidine at position 587 to lysine (H587K, Fig. 2,top), disrupted both the C-type inactivation process and the K+ selectivity of the pore. Current traces from H587K were elicited by the same voltage clamp protocol as that used for WT HERG (Fig. 2 A, panel b). The step to +60 mV induced a prominent outward current. Subsequent repolarization steps elicited smaller outward currents that reversed between −10 and −20 mV. The I–V relationship of H587K was almost linear in the voltage range between −140 and 0 mV, with an upward turn at more positive voltages (Fig. 2 A, panel c). Therefore, the C-type inactivation process was disrupted in H587K. Furthermore, removing extracellular Na+ ions shifted theE rev of H587K in the negative direction to about −60 mV (Fig. 2 A, panel d), indicating that external Na+ ions contributed significantly to currents through the H587K channel pore. The calculated K+ to Na+ permeability ratio (P K:P Na) for H587K was 1.5 ± 0.1, much lower than that of the WT HERG (191 ± 91). These changes in H587K were not due to the added permanent positive charge, because increasing the protonation of H587 in WT HERG (by changing the extracellular pH from 8.5 to 6.5) did not affect the C-type inactivation process or the K:Na selectivity of the pore (27.Jiang M. Dun W. Tseng G.-N. Am. J. Physiol. 1999; 277: H1283-H1292PubMed Google Scholar). Furthermore, replacing His-587 with proline creates the same phenotype as H587K (12.Dun W. Jiang M. Tseng G.-N. Pflugers Archiv. 1999; 439: 141-149Crossref PubMed Scopus (38) Google Scholar). These observations indicate that the extracellular S5-P linker of HERG, or at least the middle of this linker including position 587, participates in conformational changes that determine the channel's outer mouth properties. There has been no report on similar effects of mutations made in the S5-P linker of the Shaker channel on the channel's outer mouth properties. Fig. 2 B illustrates another example of differences between HERG and Shaker in their outer mouth properties. Position 449 in the Shaker channel is located at the external entrance to the pore, corresponding to position 631 of the HERG channel (Fig. 1 A). It is an important determinant of channel sensitivity to external TEA (28.Heginbotham L. MacKinnon R. Neuron. 1992; 8: 483-491Abstract Full Text PDF PubMed Scopus (376) Google Scholar). Replacing the threonine residue at this position with an aromatic residue (T449Y and T449F) greatly enhances TEA binding (due to a stabilizing interaction between positively charged TEA and π-electrons of the aromatic ring at 449), while replacing Thr-449 with a positively charged residue (T449K) has the opposite effect (due to electrostatic repulsion) (28.Heginbotham L. MacKinnon R. Neuron. 1992; 8: 483-491Abstract Full Text PDF PubMed Scopus (376) Google Scholar). We tested the effects of mutating the equivalent residue in the HERG channel (Ser-631) on TEA potency (29.Fan J.-S. Jiang M. Dun W. McDonald T.V. Tseng G.-N. Biophys. J. 1999; 76: 3128-3140Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). TEA blocked the outer mouth of the WT HERG channel with an IC50 of ∼50 mm. This is reflected by the decrease of outward tail current at −80 mV in Fig. 2 B (panel a). The appearance of a prominent outward peak and the higher level of outward current at +20 mV in the presence of TEA was due to an interference of C-type inactivation by TEA bound to the outer mouth of the channel (7.Smith P.L. Baukrowitz T. Yellen G. Nature. 1996; 379: 833-836Crossref PubMed Scopus (654) Google Scholar). Surprisingly, replacing Ser-631 with an aromatic residue (S631Y) reduced the sensitivity to external TEA (Fig. 2 B, panel b). Furthermore, replacing Ser-631 with a negatively charged residue (S631E) or a positively charged residue (S631K) reduced the sensitivity to TEA. There is no difference in TEA sensitivity between these two mutants. These data suggest that the outer mouth configuration in the HERG channel differs significantly from that of the Shaker channel so that the side chain at position 631 is shielded from bound TEA. Fig. 3 shows that ErgTx potently suppressed the HERG current amplitude. The suppressing effect could be detected at 1 nm ErgTx. However, ErgTx did not affect Kv1.4, Kv4.3, Kv2.1, or KvLQT1 even at 50 nm. This apparent selectivity, by itself, is not conclusive evidence for a unique outer vestibule structure in the HERG channel among the potassium channels examined here. Therefore, we further investigated the mechanism by which ErgTx suppresses HERG current, and positions in HERG that are important for ErgTx binding. These features are compared with those of ChTx (or its analogs) blockade of the Shaker (or Shaker-like) channel. In the latter cases, the mechanism and site of action of the toxins have been well characterized (16.Goldstein S.A.N. Miller C. Biophys. J. 1993; 65: 1613-1619Abstract Full Text PDF PubMed Scopus (166) Google Scholar, 22.Ranganathan R. Lewis J.H. MacKinnon R. Neuron. 1996; 16: 131-139Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar, 23.Goldstein S.A.N. Pheasant D.J. Miller C. Neuron. 1994; 12: 1377-1388Abstract Full Text PDF PubMed Scopus (292) Google Scholar, 24.Gross A. MacKinnon R. Neuron. 1996; 16: 399-406Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). Such a comparison will help us deduce how and where ErgTx binds to the HERG channel and reduces the current. Fig. 4 A illustrates a representative time course of changes in HERG current amplitude when the oocyte was exposed to increasing concentrations of ErgTx (1–100 nm) and after wash out of the toxin. Currents were elicited by repetitive depolarization pulses from V h −80 to +20 mV for 1 s applied once every minute. The peak tail current amplitudes before ErgTx application (Ic) and that at the steady state of ErgTx effect (Itx) were used to measure the fraction of unblocked channels (Itx/Ic). ErgTx suppressed HERG current in a concentration-dependent manner. The effect was totally reversible. The data points can be well fit with Equation 1, Itx/Ic=Amax/(1+[ErgTx]/Kd)+(1−Amax)Equation 1 where A max is the fraction of current sensitive to ErgTx (93 ± 3%), andK d is the dissociation constant (6.45 ± 1.03 nm). It is important to note that there is a residual current (on average ∼10% of control) not suppressed by high concentrations of ErgTx. This is clearly shown in Fig. 4 A: increasing [ErgTx] from 50 to 100 nm induced little further suppression of the current. In the presence of 100 nm ErgTx, the current's waveform resembled that of the control current (showing C-type inactivation and a high K+ selectivity, trace dof Fig. 4 A). Therefore, this cannot be due to an ErgTx-insensitive background or “leak” conductance. Instead, it suggests that ErgTx did not totally occlude the HERG pore. This is different from ChTx blockade of the Shaker channel: Lys-27 of ChTx binds and plugs the channel pore completely (22.Ranganathan R. Lewis J.H. MacKinnon R. Neuron. 1996; 16: 131-139Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar, 23.Goldstein S.A.N. Pheasant D.J. Miller C. Neuron. 1994; 12: 1377-1388Abstract Full Text PDF PubMed Scopus (292) Google Scholar). The difference between ErgTx suppression of HERG and ChTx blockade of Shaker is consistent with the lack of a “Lys-27-equivalent” in the ErgTx sequence (Fig. 1 B). Fig. 4 B also shows that ErgTx potency was not affected by elevating [K]o from 2 to 98 mm. This is distinctly different from the situation of ChTx blockade of the Shaker channel. In this case, Lys-27 of ChTx is critical for Ko sensitivity (16.Goldstein S.A.N. Miller C. Biophys. J. 1993; 65: 1613-1619Abstract Full Text PDF PubMed Scopus (166) Google Scholar, 22.Ranganathan R. Lewis J.H. MacKinnon R. Neuron. 1996; 16: 131-139Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar). It is suggested that Lys-27 of ChTx plugs the potassium channel pore by binding to a site close to K+ binding site within the pore (16.Goldstein S.A.N. Miller C. Biophys. J. 1993; 65: 1613-1619Abstract Full Text PDF PubMed Scopus (166) Google Scholar, 22.Ranganathan R. Lewis J.H. MacKinnon R. Neuron. 1996; 16: 131-139Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar). Elevating [K]o increases K+ occupancy inside the pore, and the resulting electrostatic repulsion between K+ ions and Lys-27 of ChTx destabilizes toxin binding. The lack of Kosensitivity in ErgTx suppression of HERG is again consistent with the lack of a Lys-27-equivalent residue in ErgTx and with the suggestion that ErgTx does not physically plug the HERG pore. Studies of toxin binding to potassium channels have implicated the S5-P linker (turret of KcsA, Fig. 1 A) and P-S6 linker as important components of toxin binding site (23.Goldstein S.A.N. Pheasant D.J. Miller C. Neuron. 1994; 12: 1377-1388Abstract Full Text PDF PubMed Scopus (292) Google Scholar, 24.Gross A. MacKinnon R. Neuron. 1996; 16: 399-406Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). Therefore, we performed cysteine-scanning mutagenesis by replacing all residues in the S5-P linker (positions 571–613, 43 residues) and the P-S6 linker (positions 631 to 638, 8 residues) of HERG with cysteine one at a time and studied the resulting effects on current suppression by ErgTx. Out of 51 positions mutated, six mutants were poorly or not expressed (N573C, K595C, P605C, N633C, E637C, and K638C). Of the remaining 45 mutants, the ErgTx potency was evaluated in 98 mm[K]o using the same voltage clamp protocol as shown in Fig. 4 A. One ErgTx concentration (10 nm) was used in all measurements, and the K d values were calculated using the following modified Equation 2,Itx/Ic=0.9/(1+[ErgTx]/Kd)+0.1Equation 2 The assumption is that the maximal effects in all cases were a 90% suppression (Fig. 4). This is valid because WT HERG has an IC50 of 7.2 ± 1.1 nm when estimated using Equation 2 based on data obtained with 10 nm ErgTx, very close to the IC50 determined from the complete concentration-response relationship (6.5 ± 1.0 nm, Fig. 4). The data are summarized in Fig. 5. Most mutants (30 out of 45) showed little or no change in ErgTx binding (changes in binding free energy, ΔΔG, less than 0.5 kcal/mol). Of the remaining 15 mutants, three in the S5-P linker (W585C, G590C, and I593C), and one in the P-S6 linker (P632C) caused outstanding changes in the binding free energy (>2 kcal/mol). For the remaining 11 mutants, the changes in binding free energy were modest although statistically significant (0.5–1 kcal/mol). Fig. 5, top, shows a sequence alignment between Shaker and HERG in the outer vestibule region. We compare the positions known to be important for ChTx (or its analog) binding to Shaker or Shaker-like channels (20.Hidalgo P. MacKinnon R. Science. 1995; 268: 307-310Crossref PubMed Scopus (423) Google Scholar, 22.Ranganathan R. Lewis J.H. MacKinnon R. Neuron. 1996; 16: 131-139Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar, 23.Goldstein S.A.N. Pheasant D.J. Miller C. Neuron. 1994; 12: 1377-1388Abstract Full Text PDF PubMed Scopus (292) Google Scholar, 24.Gross A. MacKinnon R. Neuron." @default.
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• W2004157218 title "Mapping the Binding Site of a Humanether-a-go-go-related Gene-specific Peptide Toxin (ErgTx) to the Channel's Outer Vestibule" @default.
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