Structure of the HERG K+ Channel S5P Extracellular Linker
2003; Elsevier BV; Volume: 278; Issue: 43 Linguagem: Inglês
10.1074/jbc.m212824200
ISSN1083-351X
AutoresAllan M. Torres, Paramjit S. Bansal, Margaret Sunde, Catherine E. Clarke, Jane A. Bursill, David J. Smith, Asne R. Bauskin, Samuel N. Breit, Terence J. Campbell, Paul F. Alewood, Philip W. Kuchel, Jamie I. Vandenberg,
Tópico(s)Receptor Mechanisms and Signaling
ResumoThe HERG K+ channel has very unusual kinetic behavior that includes slow activation but rapid inactivation. These features are critical for normal cardiac repolarization as well as in preventing lethal ventricular arrhythmias. Mutagenesis studies have shown that the extracellular peptide linker joining the fifth transmembrane domain to the pore helix is critical for rapid inactivation of the HERG K+ channel. This peptide linker is also considerably longer in HERG K+ channels, 40 amino acids, than in most other voltage-gated K+ channels. In this study we show that a synthetic 42-residue peptide corresponding to this linker region of the HERG K+ channel does not have defined structural elements in aqueous solution; however, it displays two well defined helical regions when in the presence of SDS micelles. The helices correspond to Trp585–Ile593 and Gly604–Tyr611 of the channel. The Trp585–Ile593 helix has distinct hydrophilic and hydrophobic surfaces. The Gly604–Tyr611 helix corresponds to an N-terminal extension of the pore helix. Electrophysiological studies of HERG currents following application of exogenous S5P peptides show that the amphipathic helix in the S5P linker interacts with the pore region of the channel in a voltage-dependent manner. The HERG K+ channel has very unusual kinetic behavior that includes slow activation but rapid inactivation. These features are critical for normal cardiac repolarization as well as in preventing lethal ventricular arrhythmias. Mutagenesis studies have shown that the extracellular peptide linker joining the fifth transmembrane domain to the pore helix is critical for rapid inactivation of the HERG K+ channel. This peptide linker is also considerably longer in HERG K+ channels, 40 amino acids, than in most other voltage-gated K+ channels. In this study we show that a synthetic 42-residue peptide corresponding to this linker region of the HERG K+ channel does not have defined structural elements in aqueous solution; however, it displays two well defined helical regions when in the presence of SDS micelles. The helices correspond to Trp585–Ile593 and Gly604–Tyr611 of the channel. The Trp585–Ile593 helix has distinct hydrophilic and hydrophobic surfaces. The Gly604–Tyr611 helix corresponds to an N-terminal extension of the pore helix. Electrophysiological studies of HERG currents following application of exogenous S5P peptides show that the amphipathic helix in the S5P linker interacts with the pore region of the channel in a voltage-dependent manner. HERG (human ether-a-go-go related gene) encodes the pore-forming α-subunit of the rapid delayed rectifier potassium channel, IKr (1Sanguinetti M.C. Jiang C. Curran M.E. Keating M.T. Cell. 1995; 81: 299-307Abstract Full Text PDF PubMed Scopus (2183) Google Scholar). The channel is an important contributor to repolarization of the cardiac action potential (1Sanguinetti M.C. Jiang C. Curran M.E. Keating M.T. Cell. 1995; 81: 299-307Abstract Full Text PDF PubMed Scopus (2183) Google Scholar, 2Jurkiewicz N.K. Sanguinetti M.C. Circ. Res. 1993; 72: 75-83Crossref PubMed Google Scholar, 3Tseng G.N. J. Mol. Cell. Cardiol. 2001; 33: 835-849Abstract Full Text PDF PubMed Scopus (170) Google Scholar). Furthermore, mutations in HERG cause congenital long QT syndrome type 2 (4Curran M.E. Splawski I. Timothy K.W. Vincent G.M. Green E.D. Keating M.T. Cell. 1995; 80: 795-803Abstract Full Text PDF PubMed Scopus (2022) Google Scholar), which results in a markedly increased risk of ventricular arrhythmias and sudden cardiac death (5Chiang C.E. Roden D.M. J. Am. Coll. Cardiol. 2000; 36: 1-12Crossref PubMed Scopus (281) Google Scholar, 6Keating M.T. Sanguinetti M.C. Cell. 2001; 104: 569-580Abstract Full Text Full Text PDF PubMed Scopus (870) Google Scholar). A wide range of drugs that block the HERG K+ channel also result in drug-induced long QT syndrome, the most common cause of serious drug-induced arrhythmia and death (7Haverkamp W. Breithardt G. Camm A.J. Janse M.J. Rosen M.R. Antzelevitch C. Escande D. Franz M. Malik M. Moss A. Shah R. Eur. Heart J. 2000; 21: 1216-1231Crossref PubMed Scopus (412) Google Scholar, 8Vandenberg J. Walker B. Campbell T. Trends Pharmacol. Sci. 2001; 22: 240-246Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar). Therefore, there is considerable interest in gaining a better understanding of the structure and structure-function relationships in the HERG K+ channel. HERG is a member of the family of voltage-gated K+ channels (VGK) 1The abbreviations used are: VGK, voltage-gated K+ channel; NMDG, N-methyl-d-glucamine; NOESY, nuclear Overhauser enhancement spectroscopy; CHO, Chinese hamster ovary. that contain six transmembrane domains, denoted by S1–S6, and a pore helix that is interposed between S5 and S6. The positively charged S4 acts as the voltage sensor for activation (9Warmke J.W. Ganetzky B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3438-3442Crossref PubMed Scopus (870) Google Scholar). Unlike other members of the VGK family, the HERG channel also undergoes very rapid voltage-dependent inactivation and recovery from inactivation (1Sanguinetti M.C. Jiang C. Curran M.E. Keating M.T. Cell. 1995; 81: 299-307Abstract Full Text PDF PubMed Scopus (2183) Google Scholar, 10Spector P.S. Curran M.E. Zou A. Keating M.T. Sanguinetti M.C. J. Gen. Physiol. 1996; 107: 611-619Crossref PubMed Scopus (379) Google Scholar, 11Smith P.L. Baukrowitz T. Yellen G. Nature. 1996; 379: 833-836Crossref PubMed Scopus (672) Google Scholar, 12Schonherr R. Heinemann S.H. J. Physiol. (Lond.). 1996; 493: 635-642Crossref Scopus (267) Google Scholar). Consequently the HERG K+ channel functions as an inward rectifier, i.e. it passes little current at depolarized potentials but large currents during the terminal repolarization phase of the cardiac action potential (13Hancox J.C. Levi A.J. Witchel H.J. Pfluegers Arch. Eur. J. Physiol. 1998; 436: 843-853Crossref PubMed Scopus (117) Google Scholar, 14Zhou Z. Gong Q. Ye B. Fan Z. Makielski J.C. Robertson G.A. January C.T. Biophys. J. 1998; 74: 230-241Abstract Full Text Full Text PDF PubMed Scopus (639) Google Scholar). This rapid inactivation is also critical for the role of the channel in suppressing arrhythmias initiated by ectopic electrical excitation (11Smith P.L. Baukrowitz T. Yellen G. Nature. 1996; 379: 833-836Crossref PubMed Scopus (672) Google Scholar, 15Lu Y. Mahaut-Smith M.P. Varghese A. Huang C.L. Kemp P.R. Vandenberg J.I. J. Physiol. (Lond.). 2001; 537: 843-851Crossref Scopus (99) Google Scholar). Inactivation of the HERG K+ channel results from conformational changes in the outer pore region of the channel (11Smith P.L. Baukrowitz T. Yellen G. Nature. 1996; 379: 833-836Crossref PubMed Scopus (672) Google Scholar, 12Schonherr R. Heinemann S.H. J. Physiol. (Lond.). 1996; 493: 635-642Crossref Scopus (267) Google Scholar, 16Fan 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 (48) Google Scholar, 17Ficker E. Jarolimek W. Kiehn J. Baumann A. Brown A.M. Circ. Res. 1998; 82: 386-395Crossref PubMed Scopus (268) Google Scholar, 18Herzberg I.M. Trudeau M.C. Robertson G.A. J. Physiol. (Lond.). 1998; 511: 3-14Crossref Scopus (105) Google Scholar, 19Liu J. Zhang M. Jiang M. Tseng G.N. J. Gen. Physiol. 2002; 120: 723-737Crossref PubMed Scopus (103) Google Scholar) and involves so-called "collapse of the pore" (20Yellen G. Q. Rev. Biophys. 1998; 31: 239-295Crossref PubMed Scopus (405) Google Scholar). The pore region of HERG, including the pore helix, a selectivity filter, and S6, is highly homologous to that of other members of the VGK family (8Vandenberg J. Walker B. Campbell T. Trends Pharmacol. Sci. 2001; 22: 240-246Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar, 9Warmke J.W. Ganetzky B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3438-3442Crossref PubMed Scopus (870) Google Scholar) as well as to the bacterial K+ channel KcsA (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 (868) Google Scholar, 22Doyle 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 (5848) Google Scholar). This feature has enabled homology models of HERG K+ channels to be constructed based on the KcsA structure (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 (868) Google Scholar). However, the extracellular loop connecting the pore helix to the top of S5 (S5P loop) in HERG is very different from that in other VGK family members. First, the S5P loop in HERG is about 40 amino acids long, compared with 10–15 in most other members of the VGK family (9Warmke J.W. Ganetzky B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3438-3442Crossref PubMed Scopus (870) Google Scholar, 23Pardo-Lopez L. Zhang M. Liu J. Jiang M. Possani L.D. Tseng G.N. J. Biol. Chem. 2002; 277: 16403-16411Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar) (also see Fig. 1). Second, many mutations in the S5P loop disrupt the inactivation process in HERG (19Liu J. Zhang M. Jiang M. Tseng G.N. J. Gen. Physiol. 2002; 120: 723-737Crossref PubMed Scopus (103) Google Scholar, 23Pardo-Lopez L. Zhang M. Liu J. Jiang M. Possani L.D. Tseng G.N. J. Biol. Chem. 2002; 277: 16403-16411Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 24Dun W. Jiang M. Tseng G.N. Pfluegers Arch. Eur. J. Physiol. 1999; 439: 141-149Crossref PubMed Scopus (39) Google Scholar). Third, toxins that bind to the S5P loop of other VGK channels, e.g. agitoxin and charybdotoxin (25Garcia M.L. Gao Y. McManus O.B. Kaczorowski G.J. Toxicon. 2001; 39: 739-748Crossref PubMed Scopus (104) Google Scholar), do not bind to HERG. Conversely, toxins that bind to this region of HERG, e.g. ErgToxin and BeKm-1, do not bind to other members of the VGK family (26Gurrola 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 (95) Google Scholar, 27Korolkova Y.V. Kozlov S.A. Lipkin A.V. Pluzhnikov K.A. Hadley J.K. Filippov A.K. Brown D.A. Angelo K. Strobaek D. Jespersen T. Olesen S.P. Jensen B.S. Grishin E.V. J. Biol. Chem. 2001; 276: 9868-9876Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). The S5P loop of HERG therefore appears to be a critical region of the protein, but at present there is little specific information known about its three-dimensional structure. Following the determination of the structure of a number of prokaryotic K+ channels (22Doyle 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 (5848) Google Scholar, 28Jiang Y. Lee A. Chen J. Cadene M. Chait B.T. MacKinnon R. Nature. 2002; 417: 515-522Crossref PubMed Scopus (1222) Google Scholar, 29Jiang Y. Lee A. Chen J. Ruta V. Cadene M. Chait B.T. MacKinnon R. Nature. 2003; 423: 33-41Crossref PubMed Scopus (1657) Google Scholar, 30Kuo. A Gulbis J.M. Antcliff J.F. Rahman T. Lowe E.D. Zimmer J. Cuthbertson J. Ashcroft F.M. Ezaki T. Doyle D.A. Science. 2003; 300: 1922-1926Crossref PubMed Scopus (738) Google Scholar), great progress has been made in the understanding of the spatial arrangement of amino acid side chains in the pore region of K+ channels. Nevertheless, the crystallization and determination of the structure of ion channels remains a tremendously difficult task (31Rosenbusch J.P. J. Struct. Biol. 2001; 136: 144-157Crossref PubMed Scopus (69) Google Scholar), and to date no structures of mammalian ion channels have been determined (32Choe S. Nat. Rev. Neurosci. 2002; 3: 115-121Crossref PubMed Scopus (157) Google Scholar). For this reason, CD spectropolarimetry (33Halsall A. Dempsey C.E. J. Mol. Biol. 1999; 293: 901-915Crossref PubMed Scopus (19) Google Scholar, 34Peled-Zehavi H. Arkin I.T. Engelman D.M. Shai Y. Biochemistry. 1996; 35: 6828-6838Crossref PubMed Scopus (36) Google Scholar) and NMR spectroscopy (35Opella S.J. Marassi F.M. Gesell J.J. Valente A.P. Kim Y. Oblatt-Montal M. Montal M. Nat. Struct. Biol. 1999; 6: 374-379Crossref PubMed Scopus (302) Google Scholar) have been used to gather information on the structure of ion channels and/or domains. Although NMR spectroscopy has limitations in terms of the size of proteins whose structure can be determined, it has the advantage of permitting the determination of structures in a lipid environment (36Opella S.J. Nat. Struct. Biol. 1997; 4: 845-848PubMed Google Scholar), thereby eliminating the problems associated with crystallization of membrane proteins (31Rosenbusch J.P. J. Struct. Biol. 2001; 136: 144-157Crossref PubMed Scopus (69) Google Scholar). In this study we have used a combination of CD spectropolarimetry, two-dimensional 1H NMR spectroscopy, and electrophysiology to investigate the structure and function of the S5P linker of the HERG K+ channel. Our findings show that the S5P linker contains an amphipathic α-helix. Exogenous application of the S5P peptide fragment or a peptide corresponding to the amphipathic α-helix results in altered ionic selectivity and disruption of inactivation of the HERG K+ channel. These results suggest that the amphipathic α-helix in S5P is critical for inactivation of HERG K+ channels. Peptide Preparation—The peptides were synthesized on a 0.50-mmol scale using O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate activation of Boc-amino acids with in situ neutralization chemistry, as previously described (37Schnölzer M. Alewood P. Jones A. Alewood D. Kent S.B.H. Int. J. Pep. Prot. Res. 1992; 40: 180-193Crossref PubMed Scopus (945) Google Scholar). The syntheses were performed on Boc-Tyr(2BrZ)-OCH2-Pam resin using standard amino acid side chain protection, except that methionine residues at positions 5 and 10 were replaced by the isosteric norleucine residue to prevent adventitious oxidation of the peptide. This step is necessary to stabilize the synthetic peptide and is not expected to affect the peptide conformation (37Schnölzer M. Alewood P. Jones A. Alewood D. Kent S.B.H. Int. J. Pep. Prot. Res. 1992; 40: 180-193Crossref PubMed Scopus (945) Google Scholar). Each residue was reacted for 10 min, and coupling efficiencies were determined by the quantitative ninhydrin reaction. Prior to a standard HF cleavage (10 ml of p-cresol:HF 1:9, 0 °C, 60 min) and workup, the N-terminal Boc protecting group was removed (100% trifluoroacetic acid), followed by formyl group removal (1.5 ml of ethanolamine in 25 ml of N,N-dimethylformamide and 5% water, twice for 30 min). Three peptides were synthesized in this study: a 42-residue peptide corresponding to the S5P linker of HERG (residues Ala570–Tyr611), to which we refer as the S5P peptide; a 42-residue peptide in which the putative amphipathic helix corresponding to residues Gly584–Lys595 (23Pardo-Lopez L. Zhang M. Liu J. Jiang M. Possani L.D. Tseng G.N. J. Biol. Chem. 2002; 277: 16403-16411Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar) was replaced with a GGGSGGGSGGGS linker, to which we refer as the del-helix peptide; and finally a 19-residue peptide corresponding to the putative amphipathic helix and four residues at each end (i.e. Ser581–Ser599 of wild type HERG), to which we refer as the helix peptide. The NMR spectroscopy sample was prepared by dissolving 2.6 mg of the S5P peptide in ∼400 μl of 90% H2O, 10% D2O (v/v) containing 12 mg of SDS-d25 (∼100 mm; the critical micellar concentration for SDS is 8 mm) in a 5-mm outer diameter susceptibility-matched microcell (Shigemi, Tokyo, Japan). This resulted in a peptide concentration of 1.4 mm and pH 3.3. NMR Spectroscopy—NMR spectroscopy experiments were performed on a Bruker Avance-600 DRX spectrometer with a 5-mm 1H inverse probe with operating temperatures of 20, 30, and 37 °C. The homo-nuclear two-dimensional experiments that were performed included double quantum-filtered correlation spectroscopy (38Derome A.E. Williamson M.P. J. Mag. Res. 1990; 88: 177-185Google Scholar) with a phase cycle modified for fast recycle times (38Derome A.E. Williamson M.P. J. Mag. Res. 1990; 88: 177-185Google Scholar), total correlation spectroscopy (39Bax A. Davis D.G. J. Mag. Res. 1985; 65: 355-360Google Scholar) with MLEV spin-lock periods of 35 and 90 ms, and nuclear Overhauser enhancement spectroscopy (NOESY) (40Kumar A. Ernst R.R. Wuthrich K. Biochem. Biophys. Res. Commun. 1980; 95: 1-6Crossref PubMed Scopus (2051) Google Scholar) with mixing times of 200 and 300 ms. All two-dimensional spectra were acquired using time proportional phase detection (41Marion D. Wuthrich K. Biochem. Biophys. Res. Commun. 1983; 113: 967-974Crossref PubMed Scopus (3540) Google Scholar). In double quantum-filtered correlation spectroscopy and NOESY experiments, water signal suppression was achieved by low power irradiation at the water resonance during the relaxation delay (1.3 s) and during the mixing period in NOESY experiments. In total correlation spectroscopy experiments, the water signal was suppressed using the WATERGATE gradient module (42Piotto M. Saudek V. Sklenar V. J. Biomol. NMR. 1992; 2: 661-665Crossref PubMed Scopus (3563) Google Scholar). All of the spectra were processed using XWIN-NMR software (Bruker, Karlsrühe, Germany). Structure Calculations—Analyses of two-dimensional spectra were carried out using the XEASY program (43Bartels C. Xia T.H. Billeter M. Guntert P. Wuthrich K. J. Biomol. NMR. 1995; 6: 1-10Crossref PubMed Scopus (1614) Google Scholar). Distance constraints were obtained from cross-peak volumes in the NOESY spectra recorded at 30 °C with a mixing time of 200 ms. This yielded 416 nonredundant upper distance constraints. An additional 14 distant constraints for hydrogen bonding were obtained from a hydrogen-deuterium exchange experiment. No ϕ dihedral angle constraints were used in the structure calculations because backbone amide peaks were too broad, probably because of conformational averaging of the peptide structure when in the presence of SDS micelles. The simulated annealing protocol in the torsion angle dynamics program DYANA (44Guntert P. Mumenthaler C. Wuthrich K. J. Mol. Biol. 1997; 273: 283-298Crossref PubMed Scopus (2569) Google Scholar) was used to obtain preliminary three-dimensional structures prior to refinement. Of the 1600 structures generated in DYANA, 40 of the "best" structures, with the lowest NOE violations, were chosen for refinement using the standard simulated annealing script in CNS (45Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (17024) Google Scholar). In this refinement process, the high temperature dynamics and cooling cycle were performed in Cartesian space. Analysis of the ensemble of S5P structures was also carried out using PROMOTIF, a program that identifies structural motifs in proteins (46Hutchinson E.G. Thornton J.M. Protein Sci. 1996; 5: 212-220Crossref PubMed Scopus (1007) Google Scholar). The figures were generated using MOLMOL (47Koradi R. Billeter M. Wuthrich K. J. Mol. Graphics. 1996; 14: 51-55Crossref PubMed Scopus (6519) Google Scholar). Circular Dichroism Spectropolarimetry—CD spectropolarimetry spectra were recorded on a Jasco J-720 spectropolarimeter equipped with a Neslab RTE-111 temperature controller. CD spectropolarimetry data were collected using a 1-mm cuvette over the wavelength range 190–250 nm and with a resolution of 0.5 nm, a bandwidth of 1 nm, and a response time of 1 s. Final spectra were the sums of three scans accumulated at a speed of 20 nm/min and were base line-corrected. Peptide concentration was 0.45 mg/ml for the S5P peptide or 0.15 mg/ml for the del-helix and helix peptides, in 10 mm sodium phosphate, pH 3.0 and 7.0, with or without the addition of 100 mm SDS. The data are presented as molar ellipticity [θ], where [θ] = θ/(10 × c × l) and θ is ellipticity (mdeg), c is the molar concentration of the sample (mol/liter), and l is the pathlength in cm. Electrophysiology—Chinese hamster ovary (CHO) cells stably transfected with HERG K+ channels were maintained in culture using Dulbecco's modified Eagle's medium/Ham's F-12 medium (Invitrogen) with 5% fetal bovine serum, as previously described (48Walker B.D. Valenzuela S.M. Singleton C.B. Tie H. Bursill J.A. Wyse K.R. Qiu M.R. Breit S.N. Campbell T.J. Br. J. Pharmacol. 1999; 127: 243-251Crossref PubMed Scopus (53) Google Scholar). CHO cells were plated on 13-mm glass coverslips 48–72 h prior to patch clamp analysis. The coverslips were then placed in a 0.5-ml perfusion chamber mounted on the stage of a Nikon Eclipse TE200 inverted microscope. The cells were superfused with normal Tyrode solution that contained 130 mm NaCl, 4.8 mm KCl, 0.3 mm KH2PO4, 0.35 mm NaH2PO4, 1.8 mm CaCl2, 1.0 mm MgCl2, 10 mm HEPES, pH adjusted to 7.4 with NaOH. In the Na+-free external solution NaCl was replaced with N-methyl-d-glucamine (NMDG)-Cl, and the NaH2PO4 was omitted. The cells were patched using micropipettes fabricated from thin walled borosilicate glass (Vitrex Microhematocrit tubes, Modulohm I/S, Denmark) with a horizontal pipette puller (model P-87; Sutter Instrument Co.). The pipette solution contained 120 mm potassium gluconate, 20 mm KCl, 1.5 mm MgATP, 5 mm EGTA, 10 mm HEPES, pH adjusted to 7.4 with KOH. The permeability ratio of K+/Na+, α, was calculated from the reversal potential measured in standard Tyrode solution using the following constant field equation, Erev=RT/Fln[(α[K+]o+[Na+]o)/(α[K+]i+[Na+]i)(Eq. 1) and assuming that [K+]i = 145 mm and [Na+]i = 5 mm. Conventional whole cell voltage clamp recordings were performed using an Axopatch 200B amplifier interfaced with a Digidata 1200 A/D converter operated using pClamp software (Axon Instruments, Foster City, CA). All of the experiments were performed at room temperature. Whole cell capacitance was determined from capacitative transient decay in current recordings following voltage steps ± 10 mV from the holding potential, and at least 80% series resistance compensation was achieved in all of the reported experiments. The protocols used in the specific experiments are described in the figure legends. In all protocols a 20-ms duration 20 mV step from the holding potential of -80 to -100 mV was applied at the start of each sweep to enable off-line leak correction. We assumed that the leak was linear in the voltage range -120 to +40 mV. Data analysis was performed using the Clampfit module of the PClamp software. The data are expressed as the means ± S.E. for n experiments, and analysis of variance was performed using Microsoft Excel. A p value of <0.05 was considered significant. For electrophysiology experiments the peptides were prepared as 0.5 mm stock solutions in either normal Tyrode or Na+-free external solution, and aliquots were stored at -20 °C. Once thawed aliquots were stored at 4 °C for up to 2 weeks. The aliquots were diluted as required on the day of experiment. The peptides were applied to cells using a picospritzer II (Intracell, Cambridge, UK) to ensure rapid application (typically less that 20 ms) and minimize the amount of peptide used in each experiment. A new coverslip was used for each experiment to ensure no residual contamination of cells with peptides. Analysis of Peptide Binding Data—Apparent on rates, (λ) and off rates (k -1) for peptide binding were obtained by fitting single exponential functions to the data for onset of current block and recovery of current following washout of the peptide. The on rate (k +1) was calculated using the formula, λ=[peptide]·k+1+k-1(Eq. 2) To obtain the time constant for the apparent on rate of peptide binding at negative potentials, it was necessary to correct for channel deactivation (see Fig. 9). We assumed that the rate of binding of the peptide to the channel was independent of the rate of deactivation, and therefore, Iobs=A·exp(-t/τobs)+C=A·exp(-t/τdeact)·exp(-t/τon)+C(Eq. 3) where A and C are constants, τobs is the observed single exponential time constant measured from the rate of change in current following addition of the peptide, τdeact is the time constant of deactivation (estimated from the single exponential fit to the current recorded in the absence of peptide), and τon is the apparent time constant for peptide binding, i.e. τon = 1/λ. Circular Dichroism Spectropolarimetry—Far-UV CD spectropolarimetry spectra of the S5P HERG peptide in 10 mm sodium phosphate buffer with and without 100 mm SDS at 20 °C and pH 3.0 are shown in Fig. 2. The large minimum between 195 and 200 nm and ellipticity close to zero at 222 nm for the S5P peptide in aqueous solution (thin line) indicates that the peptide does not have a well defined secondary structure under these conditions. This outcome prompted our investigation of the structural properties of the peptide under membrane-like conditions. A dramatic change in the CD spectropolarimetry spectral profile was observed upon addition of 100 mm SDS (Fig. 2, thick line); positive ellipticity was observed between 190 and 195 nm, the position of the minimum shifted to 205 nm, and a marked shoulder was present at 222 nm. This indicated that the S5P peptide contains helical elements in the micellar environment at pH 3.0. Similar CD spectropolarimetry profiles were obtained at pH 7.0 (also see Fig. 10) and over the temperature range 20–30 °C. NMR spectroscopy experiments were therefore performed at a pH level of ∼3 to facilitate data collection.Fig. 10A, far-UV CD spectropolarimetry spectra for S5P peptides. All three peptides display a predominantly random coil conformation in aqueous environment (thin lines) with little evidence for secondary structure. However, in the presence of SDS micelles (thick lines) both the S5P peptide and the helix peptide display elements of helical structure, whereas the del-helix remains unstructured. Calculation of the mean residue molar ellipticity, which takes into account the number of peptide bonds in the peptide, indicates that the helix peptide is almost completely helical in SDS micelles, with very little of the sequence in alternative conformations, whereas the S5P contains significant random coil content in addition to the helical elements. This is consistent with the experimentally determined structure of the S5P peptide and what would be expected from secondary structure prediction algorithms. B, typical examples of HERG currents recorded at -80 mV test potentials under control conditions (thin traces) and following addition of peptides (thick traces). S5P peptide causes a shift to inward current, the del-helix peptide has minimal effect on the current and the helix peptide has a similar effect to the full-length peptide. The dotted line in each trace indicates the zero current level.View Large Image Figure ViewerDownload Hi-res image Download (PPT) NMR Spectroscopy—The two-dimensional NOESY spectrum of the S5P peptide in aqueous solution at 25 °C had only a few very weak cross-peaks, suggesting that the peptide had a flexible, predominantly random coil structure (data not shown) that was consistent with the CD spectropolarimetry data (Fig. 2). The number and intensity of cross-peaks in the two-dimensional NOESY spectrum improved markedly upon the addition of 100 mm SDS. The appearance of amide-amide cross-peaks indicated that the peptide contained some helical structure when in the presence of SDS micelles (Fig. 3), in accordance with the CD spectropolarimetry results. Assignments of proton resonances from the S5P peptide were made using standard methods (49Wuthrich K. NMR of Proteins and Nucleic Acids. John Wiley & Sons, Inc., New York1986Crossref Google Scholar) of analyzing two-dimensional total correlation spectroscopy and NOESY spectra. The cross-peaks were generally broader than those in aqueous solution, presumably because of the increased correlation time of the peptide and possible conformational averaging and slow exchange between conformations. At 20 °C, NOE cross-peaks were too broad to analyze and in several instances overlapped with other cross-peaks, especially in the "fingerprint" region. Increasing the temperature brought about significant narrowing of the cross-peaks; however, some NOE cross-peaks became less intense, and a few disappeared. The NOESY spectrum obtained at 30 °C was seen to be the best for resonance assignment and structural calculations, but where possible, spectra obtained at 20 °C or 37 °C were used to resolve peaks that overlapped at 30 °C (see Table S1 in the supplementary data). Before doing the structure calculations, the chemical shift values obtained for Cα protons were analyzed to provide information on possible secondary structure present in the peptide. For this we used the chemical shift index method of Wishart et al. (50Wishart D.S. Sykes B.D. Richards F.M. Biochemistry. 1992; 31: 1647-1651Crossref PubMed Scopus (2037) Google Scholar). A prominent grouping of chemical shift deviations below -0.1 ppm for most residues between 16 and 22 implied a prominent helix in this part of the molecule (Fig. 4). It is also possible that a short helix is present in the region formed by residues 39–41 because they form a small cluster of three with low chemical shift deviations. The absence of a group of residues with chemical shift deviations greater than +0.1 ppm indicated that no β-sheet structure is expected for the S5P HERG p
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