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. 2002 Jun;82(6):3048–3055. doi: 10.1016/S0006-3495(02)75645-2

The outermost lysine in the S4 of domain III contributes little to the gating charge in sodium channels.

Michael F Sheets 1, Dorothy A Hanck 1
PMCID: PMC1302092  PMID: 12023227

Abstract

We investigated the contribution the four outermost basic residues (K1, R2, R3, R4) in segment 4 of domain III in the human cardiac Na channel (hH1a, Na(V)1.5) to the total gating charge (Q(max)). Each of the four basic residues were mutated individually to a cysteine. In addition, R2 was also mutated to a glutamate. All mutant channels were transiently expressed with the alpha1 subunit in fused tsA201 cells. We used the relative reduction in Q(max) caused by anthopleurin-A (ApA) toxin, a site-3 toxin known to inhibit the movement of gating charge associated with domain IV, to estimate the size of the contribution from each basic residue. Studies of the toxin's ability to inhibit gating charge in mutant channels showed that R2 contributed 19-20% to the Q(max), R3 contributed 10%, and K1 and R4 made almost no contribution. In contrast to the outermost basic residue in the S4 of Shaker K channels and in the S4 of domain IV in hH1a, the outermost charge (K1) in domain III of Na channels is outside the voltage field.

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Selected References

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  1. Aggarwal S. K., MacKinnon R. Contribution of the S4 segment to gating charge in the Shaker K+ channel. Neuron. 1996 Jun;16(6):1169–1177. doi: 10.1016/s0896-6273(00)80143-9. [DOI] [PubMed] [Google Scholar]
  2. Armstrong C. M., Bezanilla F. Currents related to movement of the gating particles of the sodium channels. Nature. 1973 Apr 13;242(5398):459–461. doi: 10.1038/242459a0. [DOI] [PubMed] [Google Scholar]
  3. Armstrong C. M., Bezanilla F. Inactivation of the sodium channel. II. Gating current experiments. J Gen Physiol. 1977 Nov;70(5):567–590. doi: 10.1085/jgp.70.5.567. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Benzinger G. R., Kyle J. W., Blumenthal K. M., Hanck D. A. A specific interaction between the cardiac sodium channel and site-3 toxin anthopleurin B. J Biol Chem. 1998 Jan 2;273(1):80–84. doi: 10.1074/jbc.273.1.80. [DOI] [PubMed] [Google Scholar]
  5. Bezanilla F., Perozo E., Stefani E. Gating of Shaker K+ channels: II. The components of gating currents and a model of channel activation. Biophys J. 1994 Apr;66(4):1011–1021. doi: 10.1016/S0006-3495(94)80882-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bezanilla F. The voltage sensor in voltage-dependent ion channels. Physiol Rev. 2000 Apr;80(2):555–592. doi: 10.1152/physrev.2000.80.2.555. [DOI] [PubMed] [Google Scholar]
  7. Catterall W. A. A 3D view of sodium channels. Nature. 2001 Feb 22;409(6823):988-9, 991. doi: 10.1038/35059188. [DOI] [PubMed] [Google Scholar]
  8. Cha A., Ruben P. C., George A. L., Jr, Fujimoto E., Bezanilla F. Voltage sensors in domains III and IV, but not I and II, are immobilized by Na+ channel fast inactivation. Neuron. 1999 Jan;22(1):73–87. doi: 10.1016/s0896-6273(00)80680-7. [DOI] [PubMed] [Google Scholar]
  9. Chen L. Q., Santarelli V., Horn R., Kallen R. G. A unique role for the S4 segment of domain 4 in the inactivation of sodium channels. J Gen Physiol. 1996 Dec;108(6):549–556. doi: 10.1085/jgp.108.6.549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gellens M. E., George A. L., Jr, Chen L. Q., Chahine M., Horn R., Barchi R. L., Kallen R. G. Primary structure and functional expression of the human cardiac tetrodotoxin-insensitive voltage-dependent sodium channel. Proc Natl Acad Sci U S A. 1992 Jan 15;89(2):554–558. doi: 10.1073/pnas.89.2.554. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. HODGKIN A. L., HUXLEY A. F. A quantitative description of membrane current and its application to conduction and excitation in nerve. J Physiol. 1952 Aug;117(4):500–544. doi: 10.1113/jphysiol.1952.sp004764. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hanck D. A., Sheets M. F., Fozzard H. A. Gating currents associated with Na channels in canine cardiac Purkinje cells. J Gen Physiol. 1990 Mar;95(3):439–457. doi: 10.1085/jgp.95.3.439. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hanck D. A., Sheets M. F. Modification of inactivation in cardiac sodium channels: ionic current studies with Anthopleurin-A toxin. J Gen Physiol. 1995 Oct;106(4):601–616. doi: 10.1085/jgp.106.4.601. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hanck D. A., Sheets M. F. Time-dependent changes in kinetics of Na+ current in single canine cardiac Purkinje cells. Am J Physiol. 1992 Apr;262(4 Pt 2):H1197–H1207. doi: 10.1152/ajpheart.1992.262.4.H1197. [DOI] [PubMed] [Google Scholar]
  15. Hartmann H. A., Tiedeman A. A., Chen S. F., Brown A. M., Kirsch G. E. Effects of III-IV linker mutations on human heart Na+ channel inactivation gating. Circ Res. 1994 Jul;75(1):114–122. doi: 10.1161/01.res.75.1.114. [DOI] [PubMed] [Google Scholar]
  16. Hirschberg B., Rovner A., Lieberman M., Patlak J. Transfer of twelve charges is needed to open skeletal muscle Na+ channels. J Gen Physiol. 1995 Dec;106(6):1053–1068. doi: 10.1085/jgp.106.6.1053. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Horn R. Conversation between voltage sensors and gates of ion channels. Biochemistry. 2000 Dec 26;39(51):15653–15658. doi: 10.1021/bi0020473. [DOI] [PubMed] [Google Scholar]
  18. Khera P. K., Benzinger G. R., Lipkind G., Drum C. L., Hanck D. A., Blumenthal K. M. Multiple cationic residues of anthopleurin B that determine high affinity and channel isoform discrimination. Biochemistry. 1995 Jul 11;34(27):8533–8541. doi: 10.1021/bi00027a003. [DOI] [PubMed] [Google Scholar]
  19. Kontis K. J., Goldin A. L. Sodium channel inactivation is altered by substitution of voltage sensor positive charges. J Gen Physiol. 1997 Oct;110(4):403–413. doi: 10.1085/jgp.110.4.403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kontis K. J., Rounaghi A., Goldin A. L. Sodium channel activation gating is affected by substitutions of voltage sensor positive charges in all four domains. J Gen Physiol. 1997 Oct;110(4):391–401. doi: 10.1085/jgp.110.4.391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kühn F. J., Greeff N. G. Movement of voltage sensor S4 in domain 4 is tightly coupled to sodium channel fast inactivation and gating charge immobilization. J Gen Physiol. 1999 Aug;114(2):167–183. doi: 10.1085/jgp.114.2.167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Noceti F., Baldelli P., Wei X., Qin N., Toro L., Birnbaumer L., Stefani E. Effective gating charges per channel in voltage-dependent K+ and Ca2+ channels. J Gen Physiol. 1996 Sep;108(3):143–155. doi: 10.1085/jgp.108.3.143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Satin J., Kyle J. W., Fan Z., Rogart R., Fozzard H. A., Makielski J. C. Post-repolarization block of cloned sodium channels by saxitoxin: the contribution of pore-region amino acids. Biophys J. 1994 May;66(5):1353–1363. doi: 10.1016/S0006-3495(94)80926-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Satin J., Limberis J. T., Kyle J. W., Rogart R. B., Fozzard H. A. The saxitoxin/tetrodotoxin binding site on cloned rat brain IIa Na channels is in the transmembrane electric field. Biophys J. 1994 Sep;67(3):1007–1014. doi: 10.1016/S0006-3495(94)80566-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Sato C., Ueno Y., Asai K., Takahashi K., Sato M., Engel A., Fujiyoshi Y. The voltage-sensitive sodium channel is a bell-shaped molecule with several cavities. Nature. 2001 Feb 22;409(6823):1047–1051. doi: 10.1038/35059098. [DOI] [PubMed] [Google Scholar]
  26. Schneider M. F., Chandler W. K. Voltage dependent charge movement of skeletal muscle: a possible step in excitation-contraction coupling. Nature. 1973 Mar 23;242(5395):244–246. doi: 10.1038/242244a0. [DOI] [PubMed] [Google Scholar]
  27. Schoppa N. E., McCormack K., Tanouye M. A., Sigworth F. J. The size of gating charge in wild-type and mutant Shaker potassium channels. Science. 1992 Mar 27;255(5052):1712–1715. doi: 10.1126/science.1553560. [DOI] [PubMed] [Google Scholar]
  28. Seoh S. A., Sigg D., Papazian D. M., Bezanilla F. Voltage-sensing residues in the S2 and S4 segments of the Shaker K+ channel. Neuron. 1996 Jun;16(6):1159–1167. doi: 10.1016/s0896-6273(00)80142-7. [DOI] [PubMed] [Google Scholar]
  29. Sheets M. F., Hanck D. A. Gating of skeletal and cardiac muscle sodium channels in mammalian cells. J Physiol. 1999 Jan 15;514(Pt 2):425–436. doi: 10.1111/j.1469-7793.1999.425ae.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Sheets M. F., Hanck D. A. Voltage-dependent open-state inactivation of cardiac sodium channels: gating current studies with Anthopleurin-A toxin. J Gen Physiol. 1995 Oct;106(4):617–640. doi: 10.1085/jgp.106.4.617. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Sheets M. F., Kyle J. W., Hanck D. A. The role of the putative inactivation lid in sodium channel gating current immobilization. J Gen Physiol. 2000 May;115(5):609–620. doi: 10.1085/jgp.115.5.609. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Sheets M. F., Kyle J. W., Kallen R. G., Hanck D. A. The Na channel voltage sensor associated with inactivation is localized to the external charged residues of domain IV, S4. Biophys J. 1999 Aug;77(2):747–757. doi: 10.1016/S0006-3495(99)76929-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Sheets M. F., Kyle J. W., Krueger S., Hanck D. A. Optimization of a mammalian expression system for the measurement of sodium channel gating currents. Am J Physiol. 1996 Sep;271(3 Pt 1):C1001–C1006. doi: 10.1152/ajpcell.1996.271.3.C1001. [DOI] [PubMed] [Google Scholar]
  34. Tejedor F. J., Catterall W. A. Site of covalent attachment of alpha-scorpion toxin derivatives in domain I of the sodium channel alpha subunit. Proc Natl Acad Sci U S A. 1988 Nov;85(22):8742–8746. doi: 10.1073/pnas.85.22.8742. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Thomsen W. J., Catterall W. A. Localization of the receptor site for alpha-scorpion toxins by antibody mapping: implications for sodium channel topology. Proc Natl Acad Sci U S A. 1989 Dec;86(24):10161–10165. doi: 10.1073/pnas.86.24.10161. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Yang N., George A. L., Jr, Horn R. Molecular basis of charge movement in voltage-gated sodium channels. Neuron. 1996 Jan;16(1):113–122. doi: 10.1016/s0896-6273(00)80028-8. [DOI] [PubMed] [Google Scholar]
  37. Zagotta W. N., Hoshi T., Dittman J., Aldrich R. W. Shaker potassium channel gating. II: Transitions in the activation pathway. J Gen Physiol. 1994 Feb;103(2):279–319. doi: 10.1085/jgp.103.2.279. [DOI] [PMC free article] [PubMed] [Google Scholar]

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