A Pore-blocking Hydrophobic Motif at the Cytoplasmic Aperture of the Closed-state Nav1.7 Channel Is Disrupted by the Erythromelalgia-associated F1449V Mutation
2008; Elsevier BV; Volume: 283; Issue: 35 Linguagem: Inglês
10.1074/jbc.m802900200
ISSN1083-351X
AutoresAngelika Lampert, Andrias O. O’Reilly, Sulayman D. Dib‐Hajj, Lynda Tyrrell, B.A. Wallace, Stephen G. Waxman,
Tópico(s)Cardiac electrophysiology and arrhythmias
ResumoSodium channel Nav1.7 has recently elicited considerable interest as a key contributor to human pain. Gain-of-function mutations of Nav1.7 produce painful disorders, whereas loss-of-function Nav1.7 mutations produce insensitivity to pain. The inherited erythromelalgia Nav1.7/F1449V mutation, within the C terminus of domain III/transmembrane helix S6, shifts channel activation by -7.2 mV and accelerates time to peak, leading to nociceptor hyperexcitability. We constructed a homology model of Nav1.7, based on the KcsA potassium channel crystal structure, which identifies four phylogenetically conserved aromatic residues that correspond to DIII/F1449 at the C-terminal end of each of the four S6 helices. The model predicted that changes in side-chain size of residue 1449 alter the pore's cytoplasmic aperture diameter and reshape inter-domain contact surfaces that contribute to closed state stabilization. To test this hypothesis, we compared activation of wild-type and mutant Nav1.7 channels F1449V/L/Y/W by whole cell patch clamp analysis. All but the F1449V mutation conserve the voltage dependence of activation. Compared with wild type, time to peak was shorter in F1449V, similar in F1449L, but longer for F1449Y and F1449W, suggesting that a bulky, hydrophobic residue is necessary for normal activation. We also substituted the corresponding aromatic residue of S6 in each domain individually with valine, to mimic the naturally occurring Nav1.7 mutation. We show that DII/F960V and DIII/F1449V, but not DI/Y405V or DIV/F1752V, regulate Nav1.7 activation, consistent with well established conformational changes in DII and DIII. We propose that the four aromatic residues contribute to the gate at the cytoplasmic pore aperture, and that their ring side chains form a hydrophobic plug which stabilizes the closed state of Nav1.7. Sodium channel Nav1.7 has recently elicited considerable interest as a key contributor to human pain. Gain-of-function mutations of Nav1.7 produce painful disorders, whereas loss-of-function Nav1.7 mutations produce insensitivity to pain. The inherited erythromelalgia Nav1.7/F1449V mutation, within the C terminus of domain III/transmembrane helix S6, shifts channel activation by -7.2 mV and accelerates time to peak, leading to nociceptor hyperexcitability. We constructed a homology model of Nav1.7, based on the KcsA potassium channel crystal structure, which identifies four phylogenetically conserved aromatic residues that correspond to DIII/F1449 at the C-terminal end of each of the four S6 helices. The model predicted that changes in side-chain size of residue 1449 alter the pore's cytoplasmic aperture diameter and reshape inter-domain contact surfaces that contribute to closed state stabilization. To test this hypothesis, we compared activation of wild-type and mutant Nav1.7 channels F1449V/L/Y/W by whole cell patch clamp analysis. All but the F1449V mutation conserve the voltage dependence of activation. Compared with wild type, time to peak was shorter in F1449V, similar in F1449L, but longer for F1449Y and F1449W, suggesting that a bulky, hydrophobic residue is necessary for normal activation. We also substituted the corresponding aromatic residue of S6 in each domain individually with valine, to mimic the naturally occurring Nav1.7 mutation. We show that DII/F960V and DIII/F1449V, but not DI/Y405V or DIV/F1752V, regulate Nav1.7 activation, consistent with well established conformational changes in DII and DIII. We propose that the four aromatic residues contribute to the gate at the cytoplasmic pore aperture, and that their ring side chains form a hydrophobic plug which stabilizes the closed state of Nav1.7. Neuropathic pain is a common disorder, often refractory to existing treatments, linked to hyperexcitability of dorsal root ganglion (DRG) 5The abbreviations used are:DRGdorsal root ganglionIEMinherited erythromelalgiaDI-IIIdomains I-IIIWTwild typeTTXtetrodotoxin. neurons (1Wall P.D. Gutnick M. Nature. 1974; 248: 740-743Crossref PubMed Scopus (313) Google Scholar, 2Devor M. Wall P.D. J. Neurophysiol. 1990; 64: 1733-1746Crossref PubMed Scopus (234) Google Scholar, 3Wall P.D. Devor M. Pain. 1983; 17: 321-339Abstract Full Text PDF PubMed Scopus (532) Google Scholar). Dysregulated expression of sodium channels contributes to neuropathic pain (4Waxman S.G. Nat. Rev. Neurosci. 2001; 2: 652-659Crossref PubMed Scopus (113) Google Scholar), and some nonspecific sodium channel blockers provide relief, albeit usually partial, for neuropathic pain (5Rice A.S. Hill R.G. Annu. Rev. Med. 2006; 57: 535-551Crossref PubMed Scopus (58) Google Scholar). A direct link of specific sodium channel isoform and neuropathic pain has recently been established in humans by the discovery of mutations in SCN9A, the gene that encodes Nav1.7, a sodium channel preferentially expressed in DRG neurons (6Dib-Hajj S.D. Cummins T.R. Black J.A. Waxman S.G. Trends Neurosci. 2007; 30: 555-563Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar). Gain-of-function mutations in Nav1.7 have been identified in patients with inherited erythromelalgia (IEM, also called erythermalgia), a disorder characterized by severe burning pain in the distal extremities in response to warmth (6Dib-Hajj S.D. Cummins T.R. Black J.A. Waxman S.G. Trends Neurosci. 2007; 30: 555-563Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar), and paroxysmal extreme pain disorder, in which patients experience perirectal and periorbital pain (7Fertleman C.R. Baker M.D. Parker K.A. Moffatt S. Elmslie F.V. Abrahamsen B. Ostman J. Klugbauer N. Wood J.N. 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Meijer I.A. Meury L. Mills T. Moody A. Morinville A. Morten J. O'Donnell D. Raynoschek C. Salter H. Rouleau G.A. Krupp J.J. Hum. Mol. Genet. 2007; 16: 2114-2121Crossref PubMed Scopus (156) Google Scholar). All of the IEM mutations studied to date lower the threshold for Nav1.7 activation, whereas less than half show impaired steady-state fast inactivation (11Choi J.S. Dib-Hajj S.D. Waxman S.G. Neurology. 2006; 67: 1563-1567Crossref PubMed Scopus (77) Google Scholar, 12Cummins T.R. Dib-Hajj S.D. Waxman S.G. J. Neurosci. 2004; 24: 8232-8236Crossref PubMed Scopus (309) Google Scholar, 13Dib-Hajj S.D. Rush A.M. Cummins T.R. Hisama F.M. Novella S. Tyrrell L. Marshall L. Waxman S.G. Brain. 2005; 128: 1847-1854Crossref PubMed Scopus (375) Google Scholar, 14Han C. Rush A.M. Dib-Hajj S.D. Li S. Xu Z. Wang Y. Tyrrell L. Wang X. Yang Y. Waxman S.G. Ann. Neurol. 2006; 59: 553-558Crossref PubMed Scopus (149) Google Scholar, 15Harty T.P. Dib-Hajj S.D. Tyrrell L. Blackman R. Hisama F.M. Rose J.B. Waxman S.G. J. Neurosci. 2006; 26: 12566-12575Crossref PubMed Scopus (128) Google Scholar, 16Lampert A. Dib-Hajj S.D. Tyrrell L. Waxman S.G. J. Biol. Chem. 2006; 281: 36029-36035Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 17Sheets P.L. Jackson Ii J.O. Waxman S.G. Dib-Hajj S. Cummins T.R. J. Physiol. (Lond.). 2007; 581: 1019-1031Crossref Scopus (147) Google Scholar). The effect on electrogenesis has been examined in transfected DRG neurons for three of these mutations (L858H, A863P, and F1449V), which have been shown to lower the threshold for single action potentials and increase the frequency of action potential firing in DRG neurons in response to graded stimuli (13Dib-Hajj S.D. Rush A.M. Cummins T.R. Hisama F.M. Novella S. Tyrrell L. Marshall L. Waxman S.G. Brain. 2005; 128: 1847-1854Crossref PubMed Scopus (375) Google Scholar, 15Harty T.P. Dib-Hajj S.D. Tyrrell L. Blackman R. Hisama F.M. Rose J.B. Waxman S.G. J. Neurosci. 2006; 26: 12566-12575Crossref PubMed Scopus (128) Google Scholar, 18Rush A.M. Dib-Hajj S.D. Liu S. Cummins T.R. Black J.A. Waxman S.G. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 8245-8250Crossref PubMed Scopus (323) Google Scholar). dorsal root ganglion inherited erythromelalgia domains I-III wild type tetrodotoxin. Most IEM mutations in Nav1.7 are clustered in transmembrane segments (S1, S4, S5, and S6) and linkers joining S4 and S5 of domains I (DI) and DII (6Dib-Hajj S.D. Cummins T.R. Black J.A. Waxman S.G. Trends Neurosci. 2007; 30: 555-563Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar, 19Waxman S.G. Dib-Hajj S. Trends Mol. Med. 2005; 11: 555-562Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). However, the F1449V mutation, which causes inherited erythromelalgia in the largest family studied to date, is located more distally at the cytoplasmic end of the S6 segment of DIII and the juncture with L3, the cytosolic loop which joins DIII and DIV (13Dib-Hajj S.D. Rush A.M. Cummins T.R. Hisama F.M. Novella S. Tyrrell L. Marshall L. Waxman S.G. Brain. 2005; 128: 1847-1854Crossref PubMed Scopus (375) Google Scholar). Because L3 carries the peptide motif IFM, which functions as the fast inactivation gate of sodium channels (20Catterall W.A. Neuron. 2000; 26: 13-25Abstract Full Text Full Text PDF PubMed Scopus (1705) Google Scholar), it is not surprising that the F1449V Nav1.7 mutation affects steady state fast inactivation (13Dib-Hajj S.D. Rush A.M. Cummins T.R. Hisama F.M. Novella S. Tyrrell L. Marshall L. Waxman S.G. Brain. 2005; 128: 1847-1854Crossref PubMed Scopus (375) Google Scholar). In contrast, the basis of the F1449V effect on channel activation is not obvious. We therefore constructed a homology model of Nav1.7, to test possible structural features that might underlie the altered channel activation. Here we show that phylogenetically conserved aromatic residues, located at equivalent positions in each S6 helix relative to the conserved "gating-hinge" glycine (or serine in DIV/S6) residue, contribute to the putative cytoplasmic activation gate of Nav1.7, possibly by forming a hydrophobic plug, which stabilizes the closed state of the channel. Our results also show that substitution of DIII/F1449 with bulky hydrophobic residues does not alter the voltage dependence of activation. Compared with wild-type Nav1.7, however, time to peak was shorter in F1449V, similar in F1449L, but longer for F1449Y and F1449W. The DIII/F1449V and DII/F960V mutations destabilize the closed state of the channel, consistent with channel activation being initiated by conformational changes in DII and DIII. These findings identify a conserved hydrophobic motif at the cytoplasmic aperture of Nav1.7 and provide a mechanistic basis for the decreased threshold for activation by the IEM mutation DIII/F1449V. Computational Modeling of Nav1.7—A homology model of the closed-state pore domain of Nav1.7 was generated using the crystal structure of the KcsA potassium channel (PDB code 1BL8) (21Doyle 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) as the structural template. KcsA was chosen over the structure of Kv2.1, because the latter channel was crystallized in the open state (22Long S.B. Campbell E.B. MacKinnon R. Science. 2005; 309: 897-903Crossref PubMed Scopus (1848) Google Scholar). KcsA was also chosen over the closed-state KirBac1.1 (23Kuo 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) and KirBac3.1 channel structures, because more extensive experimental studies have been carried with KcsA to investigate the structural basis of pore gating. Moreover, although differences in prokaryotic and eukaryotic bilayer thickness and the presence of the S1-S4 voltage sensor may alter pore structure, numerous studies have used an homology model generated from the open-state MthK channel (e.g. Cronin et al. (24Cronin N.B. O'Reilly A. Duclohier H. Wallace B.A. J. Biol. Chem. 2003; 278: 10675-10682Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar)), and KcsA is the more common template for homology modeling studies of closed state eukaryotic sodium (25Lipkind G.M. Fozzard H.A. Biochemistry. 2000; 39: 8161-8170Crossref PubMed Scopus (165) Google Scholar, 26Scheib H. McLay I. Guex N. Clare J.J. Blaney F.E. Dale T.J. Tate S.N. Robertson G.M. J. Mol. Model (Online). 2006; 12: 813-822Crossref PubMed Scopus (38) Google Scholar), potassium (27Ranatunga K.M. Law R.J. Smith G.R. Sansom M.S.P Eur. Biophys. J. 2001; 30: 295-303Crossref PubMed Scopus (22) Google Scholar, 28Stansfeld P.J. Gedeck P. Gosling M. Cox B. Mitcheson J.S. Sutcliffe M.J. Proteins. 2007; 68: 568-580Crossref PubMed Scopus (99) Google Scholar), and calcium (29Cosconati S. Marinelli L. Lavecchia A. Novellino E. J. Med. Chem. 2007; 50: 1504-1513Crossref PubMed Scopus (89) Google Scholar, 30Tikhonov D.B. Zhorov B.S. J. Biol. Chem. 2008; 283: 17594-17604Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar) channel pores, demonstrating the suitability of potassium channels as structural templates for homology modeling. The Nav1.7 sequence (Swiss-Prot accession Q15858) was aligned with the KcsA channel sequence (Swiss-Prot accession P0A334) using ClustalW (31Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (55752) Google Scholar). The outer helices (PDB file residues 23-48), pore-pointing helices (residues 63-74), and pore-lining inner helices (residues 88-117) of KcsA provided the coordinates for modeling the equivalent sequences in each of the four domains in the Nav1.7 model: S5 (Swiss-Prot sequence residues DI:239-264, DII:860-885, DIII:1315-1340, and DIV: 1638-1663), pore-pointing helices (residues DI:348-359, DII: 914-925, DIII:1393-1404, and DIV:1685-1696), and pore-lining S6 helices (residues DI:378-407, DII:944-973, DIII:1433-1462, and DIV:1736-1765) (Fig. 1). Aromatic residues (phenylalanine or tyrosine) are present at the C terminus of the four S6 helices at equivalent positions relative to the conserved glycine gating-hinge residues (or the corresponding serine residue in DIV/S6). Residues of the KcsA structure were mutated to the Nav1.7 sequence using the Biopolymer module of SYBYL (version 7.0, Tripos Inc., St. Louis, MO). Additional mutations were introduced to generate the F1449V, F1449L, F1449Y, and F1449W mutant Nav1.7 models. Both the wild-type and mutant models were subjected to 500 rounds of conjugate-gradient minimization in SYBYL using the Tripos force-field (32Clark M. Cramer R. Van Opdenbosch N. J. Computat. Chem. 1989; 10: 982-1012Crossref Scopus (2821) Google Scholar). The pore diameters of the channel models were calculated using the program HOLE (33Smart O.S. Neduvelil J.G. Wang X. Wallace B.A. Sansom M.S.P J. Mol. Graph. 1996; 14 (376): 354-360Crossref PubMed Scopus (1094) Google Scholar). The figures were produced using the PyMOL molecular graphics system (DeLano Scientific, San Carlos, CA). Plasmids and HEK 293 Cell Transfections—The plasmid carrying the TTX-resistant (TTX-R) version of human Nav1.7 cDNA (hNav1.7R) (34Herzog R.I. Cummins T.R. Ghassemi F. Dib-Hajj S.D. Waxman S.G. J. Physiol. (Lond.). 2003; 551: 741-750Crossref Scopus (270) Google Scholar) and the DIII/F1449V mutant derivative (13Dib-Hajj S.D. Rush A.M. Cummins T.R. Hisama F.M. Novella S. Tyrrell L. Marshall L. Waxman S.G. Brain. 2005; 128: 1847-1854Crossref PubMed Scopus (375) Google Scholar) were previously described. The DIII/F1449L, DIII/F1449Y, DIII/F1449W, DI/Y405V, DII/F960V, and DIV/F1752V mutants and the double mutant F960V/F1449V were introduced into hNav1.7R using the QuikChange XL site-directed mutagenesis reagents (Stratagene, La Jolla, CA). Human embryonic kidney cells (HEK 293) were transfected with each individual mutant channel construct using Lipofectamine 2000 (Invitrogen) according to the procedures recommended by the manufacturer. Transfected HEK 293 cells were grown under standard culture conditions (5% CO2, 37 °C) in 50% Dulbecco's modified Eagle's medium 50% F-12 supplemented with 10% fetal bovine serum. We have previously shown that gating properties of Nav1.7R in transfected HEK 293 cells are similar to those of Nav1.7R in native DRG neurons (34Herzog R.I. Cummins T.R. Ghassemi F. Dib-Hajj S.D. Waxman S.G. J. Physiol. (Lond.). 2003; 551: 741-750Crossref Scopus (270) Google Scholar). Electrophysiology—Whole cell voltage clamp recordings (35Hamill O.P. Neher M.A. Sakmann B. Sigworth F.J. Pflügers Arch. 1981; 391: 85-100Crossref PubMed Scopus (15145) Google Scholar) of HEK 293 cells transiently transfected with the sodium channels Nav1.7R, DI/Y405V, DII/F960V, DIII/F1449V, DIII/F1449L, DIII/F1449Y, DIII/F1449W, DIV/F1752V, or F960V/F1449V double mutant derivatives were performed with an EPC-9 amplifier (HEKA Electronics, Lambrecht/Pfalz, Germany) using fire-polished 0.5- to 1.5-MΩ electrodes (World Precision Instruments, Inc., Sarasota, FL). The pipette solution contained (in mm): 140 CsF, 10 NaCl, 1 EGTA, and 10 HEPES; 302 mosmol (pH 7.4, adjusted with CsOH), and the extracellular bath contained (in mm): 140 NaCl, 3 KCl, 10 glucose, 10 HEPES, 1 MgCl2, 1 CaCl2, 0.0003 TTX; 310 mosmol (pH 7.4, adjusted with NaOH). TTX was added to the bath solution to block all endogenous voltage-gated sodium currents that might be present in HEK 293 cells (36Cummins T.R. Zhou J. Sigworth F.J. Ukomadu C. Stephan M. Ptacek L.J. Agnew W.S. Neuron. 1993; 10: 667-678Abstract Full Text PDF PubMed Scopus (129) Google Scholar) and thereby permitted study of Nav1.7R in isolation. All recordings were conducted at room temperature (∼21 °C). The pipette potential was adjusted to zero before seal formation, and the voltages were not corrected for liquid junction potential. Capacity transients were cancelled, and series resistance was compensated at 10 μsby 65-95%. Leakage current was subtracted digitally online using hyperpolarizing potentials applied after the test pulse (P/4 procedure). Currents were acquired using Pulse software (HEKA electronics, Lambrecht/Pfalz, Germany), filtered at 10 kHz and sampled at a rate of 100 kHz. Voltage protocols were carried out at a predetermined time after establishing cell access. Standard current-voltage (I-V) families were obtained using 40-ms pulses from a holding potential of -120 mV to a range of potentials (-100 to +60 mV) in 5-mV steps with 5 s between pulses. The peak value at each potential was plotted to form I-V curves. Activation curves were obtained by calculating the conductance G at each voltage V,G=IV-VREV(Eq. 1) with Vrev being the reversal potential, determined for each cell individually. Activation curves were fitted with the following Boltzmann distribution equation,GNA=GNA,max1+e-(Vm-V1/2k)(Eq. 2) where GNa is the voltage-dependent sodium conductance, GNa,max is the maximal sodium conductance, V½ is the potential at which activation is half-maximal, Vm is the membrane potential, and k is the slope factor. Protocols for assessing steady-state fast-inactivation consisted of a series of pre-pulses from -130 to -10 mV, each lasting 500 ms from a holding potential of -100 mV, followed by a 40-ms depolarization to -10 mV to measure the non-inactivated transient current. The normalized curves were fitted using a Boltzmann distribution equation,INaINa,max=11+eVm-V1/2k(Eq. 3) where INa,max is the peak sodium current elicited after the most hyperpolarized pre-pulse, Vm is the preconditioning pulse potential, V½ is the half-maximal sodium current, and k is the slope factor. Analysis of variance tests were carried out using Prism version 4.0 for Windows (GraphPad Software, San Diego, CA). Statistical significance (p < 0.05) was tested using a two-sided Dunnett's multiple comparison test as post-hoc analysis. All data are compared with WT and presented as mean ± S.E. Homology Model of Nav1.7 Pore—The crystal structures of potassium channels have been used as structural templates to generate homology models of human sodium channels (26Scheib H. McLay I. Guex N. Clare J.J. Blaney F.E. Dale T.J. Tate S.N. Robertson G.M. J. Mol. Model (Online). 2006; 12: 813-822Crossref PubMed Scopus (38) Google Scholar, 37Cestele S. Yarov-Yarovoy V. Qu Y. Sampieri F. Scheuer T. Catterall W.A. J. Biol. Chem. 2006; 281: 21332-21344Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). In this study the crystal structure of the bacterial potassium channel KcsA, which has been the subject of molecular dynamics simulations (e.g. Holyoake et al. (38Holyoake J. Domene C. Bright J.N. Sansom M.S.P Eur. Biophys. J. 2004; 33: 238-246Crossref PubMed Scopus (31) Google Scholar)) and direct observation (39Shimizu H. Iwamoto M. Konno T. Nihei A. Sasaki Y.C. Oiki S. Cell. 2008; 132: 67-78Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar) to elucidate the structural basis of pore gating, has provided the structural template for homology modeling of the Nav1.7 pore in a closed conformation. Fig. 1 shows the alignment of the S6 transmembrane segments in each of the four domains of Nav1.7 with the corresponding pore-lining helix from KcsA. The assignment of the C-terminal end of S6 of KcsA and the corresponding coordinates in Nav1.7 in our studies include the terminal tripeptide WFV representing the last helical turn of the inner helix of KcsA (21Doyle 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), which was missing in previously reported sodium channel models (25Lipkind G.M. Fozzard H.A. Biochemistry. 2000; 39: 8161-8170Crossref PubMed Scopus (165) Google Scholar, 26Scheib H. McLay I. Guex N. Clare J.J. Blaney F.E. Dale T.J. Tate S.N. Robertson G.M. J. Mol. Model (Online). 2006; 12: 813-822Crossref PubMed Scopus (38) Google Scholar). Sequence alignment of the nine human sodium channels shows that there are gating-hinge glycine residues in S6 in domains I-III (a serine residue is present in DIV/S6) at positions equivalent to those in the bacterial channels and that the terminal residue in each of the aligned S6 helices is an aromatic amino acid, either tyrosine in domain I (DI) or phenylalanine in the other three domains (Fig. 1). The wild-type Nav1.7 homology model shows that the side chain of the aromatic residue at the cytoplasmic end of S6 in each of the four domains (DI/Y405, DII/F960, DIII/F1449, and DIV/F1752) contribute to a potential pore-obstructing hydrophobic gate (Fig. 2A). A similar hydrophobic gate has been suggested for the acetylcholine receptor (40Unwin N. Nature. 1995; 373: 37-43Crossref PubMed Scopus (909) Google Scholar, 41Beckstein O. Sansom M.S.P Phys. Biol. 2006; 3: 147-159Crossref PubMed Scopus (160) Google Scholar, 42Miyazawa A. Fujiyoshi Y. Unwin N. Nature. 2003; 423: 949-955Crossref PubMed Scopus (1081) Google Scholar) and has been identified in the bacterial potassium channel KirBac1.1 (23Kuo 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). The model suggests that the four aromatic residues share a similar environment in Nav1.7 closed state. Thus, we expected that mutation to the small residue valine (Fig. 2, B and C) might destabilize the hydrophobic cluster in the closed-state conformation of the Nav1.7 pore, permitting the channel to open at more hyperpolarized potentials as has been observed in patch clamp studies (13Dib-Hajj S.D. Rush A.M. Cummins T.R. Hisama F.M. Novella S. Tyrrell L. Marshall L. Waxman S.G. Brain. 2005; 128: 1847-1854Crossref PubMed Scopus (375) Google Scholar). We investigated the effect of substitution of F1449 with an aliphatic hydrophobic residue leucine (F1449L), and the aromatic residues tyrosine (F1449Y) and tryptophan (F1449W). Fig. 3 shows Nav1.7 homology models with F1449V, F1449L, F1449Y, and F1449W substitutions. Calculations of the diameter of the pore in the vicinity of the aromatic residues reveal that the calculated pore diameter of WT channel is 0.6 Å, and that of F1449V is almost double at 1.1 Å. The substitution of another hydrophobic residue leucine (F1449L) is 0.76 Å, whereas substitution of F1449 by aromatic residues tyrosine (F1449Y) reduces the pore diameter to 0.5 Å, and tryptophan (F1449W) to 0.45 Å. Therefore changes in side-chain size alter the effective plugging of the pore in addition to reshaping inter-domain contact surfaces that are involved in the putative closed-state stabilization. Effect of Substitutions at F1449 on Channel Opening—The predicted effects of substitutions at F1449 that emerged from computer modeling were tested in whole cell patch clamp studies. Whole cell patch clamp recordings of HEK 293 cells that were co-transfected with wild-type Nav1.7R (WT) or mutant channels and green fluorescent protein were carried out as described under "Experimental Procedures." To avoid heterogeneity caused by transfections of up to four plasmids (α-subunit, β1 and β2 subunits, and green fluorescent protein), only the Nav1.7 channel α-subunit and green fluorescent protein were transfected into HEK 293 cells. As expected, WT and mutant Nav1.7 constructs produced fast inactivating transient sodium current (Fig. 4A, 5A, and Fig. 6A). The capacitance of HEK 293 cells did not significantly change when transfected with WT or mutant Nav1.7R constructs (Table 1). WT and DIII/F1449V, DIII/F1449L, or DIII/F1449Y channels produced comparable current densities while DIII/F1449W channels produced a significantly smaller current density that is about half that of WT channels (Table 1).FIGURE 5Contribution of C terminus residues of DII/S6 and DIII/S6 to channel opening. A, representative traces of current families recorded from HEK 293 cells transiently expressing Nav1.7R WT (black traces), DII/F960V (green traces), DIII/F1449V (cyan traces), or the double mutation F960V/F1449V (magenta traces). B, voltage dependence of activation is shown for Nav1.7R (black squares), DII/F960V (green triangles), DIII/F1449V (cyan circles), or the double mutation F960V/F1449V (magenta triangles). C, voltage dependence of time to peak is shown for step depolarizations from a holding potential of -120 mV to the indicated voltages. The mutations DII/F960V (green triangles), DIII/F1449V (cyan circles), or the double mutation F960V/F1449V (magenta triangles) open faster than Nav1.7R (black squares) at -10 mV. WT and DIII/F1449V data are identical to those of Fig. 4 and are added for comparison.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 6Mutations DI/Y405V and DIV/F1752V do not alter activation kinetics. A, representative current-voltage (I-V) families recorded from HEK293 cells expressing DI/Y405V (blue traces) or DIV/F1752V (gray traces). B, conductance curves of Nav1.7R WT (black squares), DI/Y405V (blue triangles), and DIV/F1752V (gray diamonds), deduced from current-voltage families as described in methods. Lines are Boltzmann fits of mean values. C, time to peak from the onset of stimulation pulse is shown at various potentials for Nav1.7R WT (black squares), DI/Y405 (blue triangles), and DIV/F1752V (gray diamonds). WT data are identical with those of Fig. 4 and are added for comparison.View Large Image Figure ViewerDownload Hi-res image Download (PPT)TABLE 1Activation and steady-state fast-inactivation parameters of Nav 1.7 channels with substitutions at the Phe-1449 positionWTDIII/F1449VDIII/F1449LDIII/F1449YDIII/F1449WCapacitance (pF)15.4 ± 1.1 (n = 57)14.1 ± 1.1 (n = 13)17.8 ± 1.8 (n = 16)20.7 ± 4.6 (n = 15)16.8 ± 3.2 (n = 13)Current density (pA/pF)264.5 ± 28.9 (n = 57)228.6 ± 45.6 (n = 13)165.1 ± 40.1 (n = 16)180.9 ± 46.5 (n = 15)114.1 ± 18.3aSignificantly different compared to WT, p < 0.05. (n = 13)Activation V½ (mV)−10.89 ± 0.78 (n = 53)−18.11 ± 1.9bSignificantly different compared to WT, p < 0.01. (n = 11)−7.73 ± 2.13 (n = 15)−8.99 ± 1.91 (n = 13)−10.83 ± 0.92 (n = 9)Slope factoract9.54 ± 0.26 (n = 53)10.59 ± 0.53 (n = 11)9.81 ± 0.48 (n = 15)10.60 ± 0.74 (n = 13)9.55 ± 0.71 (n = 9)Time to peak at −10 mV (ms)0.54 ± 0.02 (n = 53)0.41 ± 0.02aSignificantly different compared to WT, p < 0.05. (n = 11)0.47 ± 0.03 (n = 13)1.07 ± 0.1bSignificantly different compared to WT, p < 0.01. (n = 13)1.85 ± 0.2bSignificantly different compared to WT, p < 0.01. (n = 9)Fast-inactivation V½ (mV)−78.8 ± 0.7 (n = 56)−74.5 ± 1.6 (n = 10)−77.6 ± 1.3 (n = 10)−57.3 ± 1.5bSignificantly different compared to WT, p < 0.01. (n = 15)−65.0 ± 1.7bSignificantly different compared to WT, p < 0.01. (n = 14)Slope factorinact6.9 ± 0.2 (n = 56)7.2 ± 0.2 (n = 10)8.84 ± 0.6bSignificantly different compared to WT, p < 0.01. (n = 12)8.0 ± 0.9 (n = 15)6.4 ± 0.3 (n
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