Role of Amino Acid Residues in Transmembrane Segments IS6 and IIS6 of the Na+ Channel α Subunit in Voltage-dependent Gating and Drug Block
2002; Elsevier BV; Volume: 277; Issue: 38 Linguagem: Inglês
10.1074/jbc.m206126200
ISSN1083-351X
AutoresVladimir Yarov‐Yarovoy, Jancy C. McPhee, Diane Idsvoog, Caroline Pate, Todd Scheuer, William A. Catterall,
Tópico(s)Cardiac electrophysiology and arrhythmias
ResumoAlanine-scanning mutagenesis of transmembrane segments IS6 and IIS6 of the rat brain Nav1.2 channel α subunit identified mutations N418A in IS6 and L975A in IIS6 as causing strong positive shifts in the voltage dependence of activation. In contrast, mutations V424A in IS6 and L983A in IIS6 caused strong negative shifts. Most IS6 mutations opposed inactivation from closed states, but most IIS6 mutations favored such inactivation. Mutations L421C and L983A near the intracellular ends of IS6 and IIS6, respectively, exhibited significant sustained Na+ currents at the end of 30-ms depolarizations, indicating a role for these residues in Na+ channel fast inactivation. These residues, in combination with residues at the intracellular end of IVS6, are well situated to form an inactivation gate receptor. Mutation I409A in IS6 reduced the affinity of the local anesthetic etidocaine for the inactivated state by 6-fold, and mutations I409A and N418A reduced use-dependent block by etidocaine. No IS6 or IIS6 mutations studied affected inactivated-state affinity or use-dependent block by the neuroprotective drug sipatrigine (compound 619C89). These results suggest that the local anesthetic receptor site is formed primarily by residues in segments IIIS6 and IVS6 with the contribution of a single amino acid in segment IS6. Alanine-scanning mutagenesis of transmembrane segments IS6 and IIS6 of the rat brain Nav1.2 channel α subunit identified mutations N418A in IS6 and L975A in IIS6 as causing strong positive shifts in the voltage dependence of activation. In contrast, mutations V424A in IS6 and L983A in IIS6 caused strong negative shifts. Most IS6 mutations opposed inactivation from closed states, but most IIS6 mutations favored such inactivation. Mutations L421C and L983A near the intracellular ends of IS6 and IIS6, respectively, exhibited significant sustained Na+ currents at the end of 30-ms depolarizations, indicating a role for these residues in Na+ channel fast inactivation. These residues, in combination with residues at the intracellular end of IVS6, are well situated to form an inactivation gate receptor. Mutation I409A in IS6 reduced the affinity of the local anesthetic etidocaine for the inactivated state by 6-fold, and mutations I409A and N418A reduced use-dependent block by etidocaine. No IS6 or IIS6 mutations studied affected inactivated-state affinity or use-dependent block by the neuroprotective drug sipatrigine (compound 619C89). These results suggest that the local anesthetic receptor site is formed primarily by residues in segments IIIS6 and IVS6 with the contribution of a single amino acid in segment IS6. Voltage-gated Na+ channels are integral membrane proteins that are responsible for the initiation and propagation of action potentials in nerve and muscle cells (1Hille B. Ionic Channels of Excitable Membranes. 3rd Ed. Sinauer Associates, Inc., Sunderland, MA2001Google Scholar, 2Catterall W.A. Neuron. 2000; 26: 13-25Abstract Full Text Full Text PDF PubMed Scopus (1686) Google Scholar, 3Cohen S.A. Barchi R.L. Int. Rev. Cytol. 1993; 137C: 55-103PubMed Google Scholar, 4Fozzard H.A. Hanck D.A. Physiol. Rev. 1996; 76: 887-926Crossref PubMed Scopus (233) Google Scholar, 5Marban E. Yamagishi T. Tomaselli G. J. Physiol. (Lond.). 1998; 508: 647-657Crossref Scopus (256) Google Scholar). The rat brain Na+ channel as isolated biochemically consists of α (260 kDa), β1 (36 kDa), and β2 (33 kDa) subunits (2Catterall W.A. Neuron. 2000; 26: 13-25Abstract Full Text Full Text PDF PubMed Scopus (1686) Google Scholar). The α subunit is composed of four homologous domains (I–IV), each with six transmembrane segments (S1–S6) and an additional membrane-reentrant pore loop (P-loop) between segments S5 and S6 (2Catterall W.A. Neuron. 2000; 26: 13-25Abstract Full Text Full Text PDF PubMed Scopus (1686) Google Scholar, 4Fozzard H.A. Hanck D.A. Physiol. Rev. 1996; 76: 887-926Crossref PubMed Scopus (233) Google Scholar,5Marban E. Yamagishi T. Tomaselli G. J. Physiol. (Lond.). 1998; 508: 647-657Crossref Scopus (256) Google Scholar). Analogous to the structural topology of pore-forming M2 segments of the K+ channel from Streptomyces lividans (KcsA) (6Doyle D.A. Cabral J.M. Pfuetzner R.A. Kuo A.L. Gulbis J.M. Cohen S.L. Chait B.T. MacKinnon R. Science. 1998; 280: 69-77Crossref PubMed Scopus (5688) Google Scholar), the S6 segments from each domain of the Na+ channel are thought to be arranged in a square array surrounding the inner pore, whereas P-loops from each domain line the outer pore and form the ion selectivity filter (2Catterall W.A. Neuron. 2000; 26: 13-25Abstract Full Text Full Text PDF PubMed Scopus (1686) Google Scholar, 4Fozzard H.A. Hanck D.A. Physiol. Rev. 1996; 76: 887-926Crossref PubMed Scopus (233) Google Scholar, 5Marban E. Yamagishi T. Tomaselli G. J. Physiol. (Lond.). 1998; 508: 647-657Crossref Scopus (256) Google Scholar).Voltage-gated Na+ channel activation is thought to result from a voltage-driven outward movement of gating charges, which initiates a conformational change in the protein that opens the channel (7Hodgkin A.L. Huxley A.F. J. Physiol. (Lond.). 1952; 117: 500-544Crossref Scopus (14050) Google Scholar, 8Armstrong C.M. Physiol. Rev. 1981; 61: 644-683Crossref PubMed Scopus (355) Google Scholar). Analysis of the primary structure of the Na+channel α subunit led to the prediction that S4 transmembrane segments, which contain repeated motifs of positively charged arginine and lysine residues every three amino acids, might serve as the voltage sensor (9Catterall W.A. Trends Neurosci. 1986; 9: 7-10Abstract Full Text PDF Scopus (121) Google Scholar, 10Guy H.R. Seetharamulu P. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 508-512Crossref PubMed Scopus (421) Google Scholar). Depolarization of the membrane was proposed to cause the S4 segments to move outward, inducing a conformational change in the pore of the channel resulting in activation. Site-directed mutagenesis studies have demonstrated that the positively charged residues in all four S4 segments contribute to the voltage-dependent activation of the Na+ channel (11Stühmer W. Conti F. Suzuki H. Wang X.D. Noda M. Yahagi N. Kubo H. Numa S. Nature. 1989; 339: 597-603Crossref PubMed Scopus (943) Google Scholar, 12Kontis K.J. Rounaghi A. Goldin A.L. J. Gen. Physiol. 1997; 110: 391-401Crossref PubMed Scopus (126) Google Scholar). The proposed outward movement of the S4 segments has been directly detected using mutagenesis, covalent modification, and fluorescent imaging experiments (13Yang N. Horn R. Neuron. 1995; 15: 213-218Abstract Full Text PDF PubMed Scopus (350) Google Scholar, 14Cha A. Ruben P.C. George Jr., A.L. Fujimoto E. Bezanilla F. Neuron. 1999; 22: 73-87Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar, 15Yang N. George Jr., A.L. Horn R. Neuron. 1996; 16: 113-122Abstract Full Text Full Text PDF PubMed Scopus (530) Google Scholar). Thus, the S4 segments play a critical role in voltage-dependent activation. Na+ channel fast inactivation occurs within a few milliseconds of channel opening and is mediated by the intracellular loop connecting domains III and IV (16Vassilev P.M. Scheuer T. Catterall W.A. Science. 1988; 241: 1658-1661Crossref PubMed Scopus (313) Google Scholar, 17Vassilev P. Scheuer T. Catterall W.A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8147-8151Crossref PubMed Scopus (171) Google Scholar). Mutagenesis studies of this loop revealed three hydrophobic residues (isoleucine, phenylalanine, and methionine (IFM motif)) that are critical for fast inactivation (18West J.W. Patton D.E. Scheuer T. Wang Y. Goldin A.L. Catterall W.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10910-10914Crossref PubMed Scopus (656) Google Scholar). Scanning mutagenesis experiments have identified multiple amino acid residues that may form the inactivation gate receptor within and near the intracellular mouth of the pore, including a cluster of three hydrophobic residues at the intracellular end of segment S6 in domain IV (segment IVS6) (19McPhee J.C. Ragsdale D.S. Scheuer T. Catterall W.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12346-12350Crossref PubMed Scopus (110) Google Scholar, 20McPhee J.C. Ragsdale D.S. Scheuer T. Catterall W.A. J. Biol. Chem. 1995; 270: 12025-12034Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar) and residues in intracellular loops S4–S5 in domain III (21Smith M.R. Goldin A.L. Biophys. J. 1997; 73: 1885-1895Abstract Full Text PDF PubMed Scopus (147) Google Scholar) and S4–S5 in domain IV (22Lerche H. Peter W. Fleischhauer R. PikaHartlaub U. Malina T. Mitrovic N. Lehmann-Horn F. J. Physiol. (Lond.). 1997; 505: 345-352Crossref Scopus (69) Google Scholar, 23Filatov G.N. Nguyen T.P. Kraner S.D. Barchi R.L. J. Gen. Physiol. 1998; 111: 703-715Crossref PubMed Scopus (44) Google Scholar, 24McPhee J.C. Ragsdale D.S. Scheuer T. Catterall W.A. J. Biol. Chem. 1998; 273: 1121-1129Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 25Tang L. Chehab N. Wieland S.J. Kallen R.G. J. Gen. Physiol. 1998; 111: 639-652Crossref PubMed Scopus (23) Google Scholar). The voltage dependence of Na+ channel inactivation derives largely from coupling to the activation process (8Armstrong C.M. Physiol. Rev. 1981; 61: 644-683Crossref PubMed Scopus (355) Google Scholar). Mutagenesis studies have provided strong evidence that outward movement of the S4 segments in domains III and IV initiates a conformational change that leads to fast inactivation of the Na+ channel by closure of the intracellular inactivation gate (14Cha A. Ruben P.C. George Jr., A.L. Fujimoto E. Bezanilla F. Neuron. 1999; 22: 73-87Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar, 26Chen L.Q. Santarelli V. Horn R. Kallen R.G. J. Gen. Physiol. 1996; 108: 549-556Crossref PubMed Scopus (166) Google Scholar, 27Rogers J.C., Qu, Y. Tanada T.N. Scheuer T. Catterall W.A. J. Biol. Chem. 1996; 271: 15950-15962Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar, 28Sheets M.F. Kyle J.W. Kallen R.G. Hanck D.A. Biophys. J. 1999; 77: 747-757Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar).Alanine-scanning mutagenesis was previously used to investigate the role of amino acid residues in the S6 transmembrane segments of domains III and IV of the Na+ channel in voltage-dependent gating and block by clinically important drugs (19McPhee J.C. Ragsdale D.S. Scheuer T. Catterall W.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12346-12350Crossref PubMed Scopus (110) Google Scholar, 20McPhee J.C. Ragsdale D.S. Scheuer T. Catterall W.A. J. Biol. Chem. 1995; 270: 12025-12034Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 29Ragsdale D.S. McPhee J.C. Scheuer T. Catterall W.A. Science. 1994; 265: 1724-1728Crossref PubMed Scopus (722) Google Scholar, 30Yarov-Yarovoy V. Brown J. Sharp E.M. Clare J.J. Scheuer T. Catterall W.A. J. Biol. Chem. 2001; 276: 20-27Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 31Liu G. Scheuer T. Catterall W.A. Biophys. J. 1998; 74 (abstr.): A399Google Scholar). A number of mutations in segments IIIS6 and IVS6 of the rat brain type IIA Na+ channel α subunit, designated the Nav1.2 channel according to the nomenclature of Goldin et al. (32Goldin A.L. Barchi R.L. Caldwell J.H. Hofmann F. Howe J.R. Hunter J.C. Kallen R.G. Mandel G. Meisler M.H. Netter Y.B. Noda M. Tamkun M.M. Waxman S.G. Wood J.N. Catterall W.A. Neuron. 2000; 28: 365-368Abstract Full Text Full Text PDF PubMed Scopus (649) Google Scholar), produced strong shifts in the voltage dependence of steady-state activation and inactivation, suggesting that the native residues at those positions might play a particularly important role in the voltage-dependent gating of Na+ channels (20McPhee J.C. Ragsdale D.S. Scheuer T. Catterall W.A. J. Biol. Chem. 1995; 270: 12025-12034Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 30Yarov-Yarovoy V. Brown J. Sharp E.M. Clare J.J. Scheuer T. Catterall W.A. J. Biol. Chem. 2001; 276: 20-27Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). An α-helical pattern of the shifts in the voltage dependence of activation and inactivation produced by alanine mutations in the inner two-thirds of segment IIIS6 suggested rotational movement of segment IIIS6 during channel gating (30Yarov-Yarovoy V. Brown J. Sharp E.M. Clare J.J. Scheuer T. Catterall W.A. J. Biol. Chem. 2001; 276: 20-27Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). Specific amino acid residues in segments IIIS6 and IVS6 were identified that form the receptor sites for Na+ channel pore-blocking drugs such as local anesthetics and antiarrhythmic and anticonvulsant drugs (29Ragsdale D.S. McPhee J.C. Scheuer T. Catterall W.A. Science. 1994; 265: 1724-1728Crossref PubMed Scopus (722) Google Scholar, 30Yarov-Yarovoy V. Brown J. Sharp E.M. Clare J.J. Scheuer T. Catterall W.A. J. Biol. Chem. 2001; 276: 20-27Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 31Liu G. Scheuer T. Catterall W.A. Biophys. J. 1998; 74 (abstr.): A399Google Scholar). Mutations F1764A and Y1771A in segment IVS6 of the rat brain Nav1.2 channel reduced the affinity of inactivated Na+ channels for the local anesthetic etidocaine by 130- and 35-fold, respectively (29Ragsdale D.S. McPhee J.C. Scheuer T. Catterall W.A. Science. 1994; 265: 1724-1728Crossref PubMed Scopus (722) Google Scholar). Mutation of Phe1764 and Tyr1771 in the Nav1.2 channel and their homologs in other Na+ channels also substantially reduced block of inactivated Na+ channels by other local anesthetics and antiarrhythmic and anticonvulsant drugs (29Ragsdale D.S. McPhee J.C. Scheuer T. Catterall W.A. Science. 1994; 265: 1724-1728Crossref PubMed Scopus (722) Google Scholar, 33Qu Y. Rogers J. Tanada T. Scheuer T. Catterall W.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11839-11843Crossref PubMed Scopus (107) Google Scholar, 34Ragsdale D.S. McPhee J.C. Scheuer T. Catterall W.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9270-9275Crossref PubMed Scopus (424) Google Scholar, 35Wang G.K. Quan C. Wang S. Pfluegers Arch. Eur. J. Physiol. 1998; 435: 293-302Crossref PubMed Scopus (78) Google Scholar, 36Wright S.N. Wang S.Y. Wang G.K. Mol. Pharmacol. 1998; 54: 733-739PubMed Google Scholar, 37Weiser T., Qu, Y. Catterall W.A. Scheuer T. Mol. Pharmacol. 1999; 56: 1238-1244Crossref PubMed Scopus (49) Google Scholar, 38Li H.L. Galue A. Meadows L. Ragsdale D.S. Mol. Pharmacol. 1999; 55: 134-141Crossref PubMed Scopus (135) Google Scholar, 39Carter A.J. Grauert M. Pschorn U. Bechtel W.D. Bartmann-Lindholm C., Qu, Y. Scheuer T. Catterall W.A. Weiser T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4944-4949Crossref PubMed Scopus (48) Google Scholar). Less dramatic disruptions in inactivated-state block by etidocaine were observed with mutations L1465A, N1466A, and I1469A in segment IIIS6 of the rat brain Nav1.2 channel, resulting in 6-, 8-, and 7-fold reduction in affinity, respectively (30Yarov-Yarovoy V. Brown J. Sharp E.M. Clare J.J. Scheuer T. Catterall W.A. J. Biol. Chem. 2001; 276: 20-27Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). Mutations L1465A and I1469A also reduced the inactivated-state affinity for the anticonvulsant lamotrigine and its congeners (30Yarov-Yarovoy V. Brown J. Sharp E.M. Clare J.J. Scheuer T. Catterall W.A. J. Biol. Chem. 2001; 276: 20-27Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). Lysine mutations of rat skeletal muscle Nav1.4 channel Ser1276 and Leu1280 (homologous to Leu1465 in the rat brain Nav1.2 channel) in segment IIIS6 reduced the inactivated-state affinity for the local anesthetic bupivacaine by 7–17-fold (40Wang S.Y. Nau C. Wang G.K. Biophys. J. 2000; 79: 1379-1387Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Lysine mutations of rat skeletal muscle Nav1.4 channel Asn434 and Leu437 in segment IS6 reduced the inactivated-state affinity for etidocaine by 7- and 3-fold, respectively (41Wang G.K. Quan C. Wang S.Y. Mol. Pharmacol. 1998; 54: 389-396Crossref PubMed Scopus (44) Google Scholar).In this work, we have undertaken a systematic analysis of the role of segments IS6 and IIS6 of the rat brain Nav1.2 channel in channel gating and in block by local anesthetics and anticonvulsant drugs using alanine-scanning site-directed mutagenesis. Our results identify individual residues in segments IS6 and IIS6 that are important for Na+ channel activation and inactivation gating and also define novel determinants of the receptor site for the local anesthetics. Voltage-gated Na+ channels are integral membrane proteins that are responsible for the initiation and propagation of action potentials in nerve and muscle cells (1Hille B. Ionic Channels of Excitable Membranes. 3rd Ed. Sinauer Associates, Inc., Sunderland, MA2001Google Scholar, 2Catterall W.A. Neuron. 2000; 26: 13-25Abstract Full Text Full Text PDF PubMed Scopus (1686) Google Scholar, 3Cohen S.A. Barchi R.L. Int. Rev. Cytol. 1993; 137C: 55-103PubMed Google Scholar, 4Fozzard H.A. Hanck D.A. Physiol. Rev. 1996; 76: 887-926Crossref PubMed Scopus (233) Google Scholar, 5Marban E. Yamagishi T. Tomaselli G. J. Physiol. (Lond.). 1998; 508: 647-657Crossref Scopus (256) Google Scholar). The rat brain Na+ channel as isolated biochemically consists of α (260 kDa), β1 (36 kDa), and β2 (33 kDa) subunits (2Catterall W.A. Neuron. 2000; 26: 13-25Abstract Full Text Full Text PDF PubMed Scopus (1686) Google Scholar). The α subunit is composed of four homologous domains (I–IV), each with six transmembrane segments (S1–S6) and an additional membrane-reentrant pore loop (P-loop) between segments S5 and S6 (2Catterall W.A. Neuron. 2000; 26: 13-25Abstract Full Text Full Text PDF PubMed Scopus (1686) Google Scholar, 4Fozzard H.A. Hanck D.A. Physiol. Rev. 1996; 76: 887-926Crossref PubMed Scopus (233) Google Scholar,5Marban E. Yamagishi T. Tomaselli G. J. Physiol. (Lond.). 1998; 508: 647-657Crossref Scopus (256) Google Scholar). Analogous to the structural topology of pore-forming M2 segments of the K+ channel from Streptomyces lividans (KcsA) (6Doyle D.A. Cabral J.M. Pfuetzner R.A. Kuo A.L. Gulbis J.M. Cohen S.L. Chait B.T. MacKinnon R. Science. 1998; 280: 69-77Crossref PubMed Scopus (5688) Google Scholar), the S6 segments from each domain of the Na+ channel are thought to be arranged in a square array surrounding the inner pore, whereas P-loops from each domain line the outer pore and form the ion selectivity filter (2Catterall W.A. Neuron. 2000; 26: 13-25Abstract Full Text Full Text PDF PubMed Scopus (1686) Google Scholar, 4Fozzard H.A. Hanck D.A. Physiol. Rev. 1996; 76: 887-926Crossref PubMed Scopus (233) Google Scholar, 5Marban E. Yamagishi T. Tomaselli G. J. Physiol. (Lond.). 1998; 508: 647-657Crossref Scopus (256) Google Scholar). Voltage-gated Na+ channel activation is thought to result from a voltage-driven outward movement of gating charges, which initiates a conformational change in the protein that opens the channel (7Hodgkin A.L. Huxley A.F. J. Physiol. (Lond.). 1952; 117: 500-544Crossref Scopus (14050) Google Scholar, 8Armstrong C.M. Physiol. Rev. 1981; 61: 644-683Crossref PubMed Scopus (355) Google Scholar). Analysis of the primary structure of the Na+channel α subunit led to the prediction that S4 transmembrane segments, which contain repeated motifs of positively charged arginine and lysine residues every three amino acids, might serve as the voltage sensor (9Catterall W.A. Trends Neurosci. 1986; 9: 7-10Abstract Full Text PDF Scopus (121) Google Scholar, 10Guy H.R. Seetharamulu P. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 508-512Crossref PubMed Scopus (421) Google Scholar). Depolarization of the membrane was proposed to cause the S4 segments to move outward, inducing a conformational change in the pore of the channel resulting in activation. Site-directed mutagenesis studies have demonstrated that the positively charged residues in all four S4 segments contribute to the voltage-dependent activation of the Na+ channel (11Stühmer W. Conti F. Suzuki H. Wang X.D. Noda M. Yahagi N. Kubo H. Numa S. Nature. 1989; 339: 597-603Crossref PubMed Scopus (943) Google Scholar, 12Kontis K.J. Rounaghi A. Goldin A.L. J. Gen. Physiol. 1997; 110: 391-401Crossref PubMed Scopus (126) Google Scholar). The proposed outward movement of the S4 segments has been directly detected using mutagenesis, covalent modification, and fluorescent imaging experiments (13Yang N. Horn R. Neuron. 1995; 15: 213-218Abstract Full Text PDF PubMed Scopus (350) Google Scholar, 14Cha A. Ruben P.C. George Jr., A.L. Fujimoto E. Bezanilla F. Neuron. 1999; 22: 73-87Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar, 15Yang N. George Jr., A.L. Horn R. Neuron. 1996; 16: 113-122Abstract Full Text Full Text PDF PubMed Scopus (530) Google Scholar). Thus, the S4 segments play a critical role in voltage-dependent activation. Na+ channel fast inactivation occurs within a few milliseconds of channel opening and is mediated by the intracellular loop connecting domains III and IV (16Vassilev P.M. Scheuer T. Catterall W.A. Science. 1988; 241: 1658-1661Crossref PubMed Scopus (313) Google Scholar, 17Vassilev P. Scheuer T. Catterall W.A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8147-8151Crossref PubMed Scopus (171) Google Scholar). Mutagenesis studies of this loop revealed three hydrophobic residues (isoleucine, phenylalanine, and methionine (IFM motif)) that are critical for fast inactivation (18West J.W. Patton D.E. Scheuer T. Wang Y. Goldin A.L. Catterall W.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10910-10914Crossref PubMed Scopus (656) Google Scholar). Scanning mutagenesis experiments have identified multiple amino acid residues that may form the inactivation gate receptor within and near the intracellular mouth of the pore, including a cluster of three hydrophobic residues at the intracellular end of segment S6 in domain IV (segment IVS6) (19McPhee J.C. Ragsdale D.S. Scheuer T. Catterall W.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12346-12350Crossref PubMed Scopus (110) Google Scholar, 20McPhee J.C. Ragsdale D.S. Scheuer T. Catterall W.A. J. Biol. Chem. 1995; 270: 12025-12034Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar) and residues in intracellular loops S4–S5 in domain III (21Smith M.R. Goldin A.L. Biophys. J. 1997; 73: 1885-1895Abstract Full Text PDF PubMed Scopus (147) Google Scholar) and S4–S5 in domain IV (22Lerche H. Peter W. Fleischhauer R. PikaHartlaub U. Malina T. Mitrovic N. Lehmann-Horn F. J. Physiol. (Lond.). 1997; 505: 345-352Crossref Scopus (69) Google Scholar, 23Filatov G.N. Nguyen T.P. Kraner S.D. Barchi R.L. J. Gen. Physiol. 1998; 111: 703-715Crossref PubMed Scopus (44) Google Scholar, 24McPhee J.C. Ragsdale D.S. Scheuer T. Catterall W.A. J. Biol. Chem. 1998; 273: 1121-1129Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 25Tang L. Chehab N. Wieland S.J. Kallen R.G. J. Gen. Physiol. 1998; 111: 639-652Crossref PubMed Scopus (23) Google Scholar). The voltage dependence of Na+ channel inactivation derives largely from coupling to the activation process (8Armstrong C.M. Physiol. Rev. 1981; 61: 644-683Crossref PubMed Scopus (355) Google Scholar). Mutagenesis studies have provided strong evidence that outward movement of the S4 segments in domains III and IV initiates a conformational change that leads to fast inactivation of the Na+ channel by closure of the intracellular inactivation gate (14Cha A. Ruben P.C. George Jr., A.L. Fujimoto E. Bezanilla F. Neuron. 1999; 22: 73-87Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar, 26Chen L.Q. Santarelli V. Horn R. Kallen R.G. J. Gen. Physiol. 1996; 108: 549-556Crossref PubMed Scopus (166) Google Scholar, 27Rogers J.C., Qu, Y. Tanada T.N. Scheuer T. Catterall W.A. J. Biol. Chem. 1996; 271: 15950-15962Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar, 28Sheets M.F. Kyle J.W. Kallen R.G. Hanck D.A. Biophys. J. 1999; 77: 747-757Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). Alanine-scanning mutagenesis was previously used to investigate the role of amino acid residues in the S6 transmembrane segments of domains III and IV of the Na+ channel in voltage-dependent gating and block by clinically important drugs (19McPhee J.C. Ragsdale D.S. Scheuer T. Catterall W.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12346-12350Crossref PubMed Scopus (110) Google Scholar, 20McPhee J.C. Ragsdale D.S. Scheuer T. Catterall W.A. J. Biol. Chem. 1995; 270: 12025-12034Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 29Ragsdale D.S. McPhee J.C. Scheuer T. Catterall W.A. Science. 1994; 265: 1724-1728Crossref PubMed Scopus (722) Google Scholar, 30Yarov-Yarovoy V. Brown J. Sharp E.M. Clare J.J. Scheuer T. Catterall W.A. J. Biol. Chem. 2001; 276: 20-27Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 31Liu G. Scheuer T. Catterall W.A. Biophys. J. 1998; 74 (abstr.): A399Google Scholar). A number of mutations in segments IIIS6 and IVS6 of the rat brain type IIA Na+ channel α subunit, designated the Nav1.2 channel according to the nomenclature of Goldin et al. (32Goldin A.L. Barchi R.L. Caldwell J.H. Hofmann F. Howe J.R. Hunter J.C. Kallen R.G. Mandel G. Meisler M.H. Netter Y.B. Noda M. Tamkun M.M. Waxman S.G. Wood J.N. Catterall W.A. Neuron. 2000; 28: 365-368Abstract Full Text Full Text PDF PubMed Scopus (649) Google Scholar), produced strong shifts in the voltage dependence of steady-state activation and inactivation, suggesting that the native residues at those positions might play a particularly important role in the voltage-dependent gating of Na+ channels (20McPhee J.C. Ragsdale D.S. Scheuer T. Catterall W.A. J. Biol. Chem. 1995; 270: 12025-12034Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 30Yarov-Yarovoy V. Brown J. Sharp E.M. Clare J.J. Scheuer T. Catterall W.A. J. Biol. Chem. 2001; 276: 20-27Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). An α-helical pattern of the shifts in the voltage dependence of activation and inactivation produced by alanine mutations in the inner two-thirds of segment IIIS6 suggested rotational movement of segment IIIS6 during channel gating (30Yarov-Yarovoy V. Brown J. Sharp E.M. Clare J.J. Scheuer T. Catterall W.A. J. Biol. Chem. 2001; 276: 20-27Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). Specific amino acid residues in segments IIIS6 and IVS6 were identified that form the receptor sites for Na+ channel pore-blocking drugs such as local anesthetics and antiarrhythmic and anticonvulsant drugs (29Ragsdale D.S. McPhee J.C. Scheuer T. Catterall W.A. Science. 1994; 265: 1724-1728Crossref PubMed Scopus (722) Google Scholar, 30Yarov-Yarovoy V. Brown J. Sharp E.M. Clare J.J. Scheuer T. Catterall W.A. J. Biol. 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J. 2000; 79: 1379-1387Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Lysine mutations of rat skeletal muscle Nav1.4 channel Asn434 and Leu437 in segment IS6 reduced the inactivated-state affinity for etidocaine by 7- and 3-fold, respectively (41Wang G.K. Quan C. Wang S.Y. Mol. Pharmacol. 1998; 54: 389-396Crossref PubMed Scopus (44) Google Scholar). In this work, we have undertaken a systematic analysis of the role of segments IS6 and IIS6 of the rat brain Nav1.2 channel in channel gating and in block by local anesthetics and anticonvulsant drugs using alanine-scanning site-directed mutagenesis. Our results identify individual residues in segments IS6 and IIS6 that are important for Na+ channel activation and inactivation gating and also define novel determinants of the receptor site for the local anesthetics.
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