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. 2001 Feb;80(2):698–706. doi: 10.1016/S0006-3495(01)76049-3

Specific neosaxitoxin interactions with the Na+ channel outer vestibule determined by mutant cycle analysis.

J L Penzotti 1, G Lipkind 1, H A Fozzard 1, S C Dudley Jr 1
PMCID: PMC1301268  PMID: 11159437

Abstract

The voltage-gated Na+ channel alpha-subunit consists of four homologous domains arranged circumferentially to form the pore. Several neurotoxins, including saxitoxin (STX), block the pore by binding to the outer vestibule of this permeation pathway, which is composed of four pore-forming loops (P-loops), one from each domain. Neosaxitoxin (neoSTX) is a variant of STX that differs only by having an additional hydroxyl group at the N1 position of the 1,2,3 guanidinium (N1-OH). We used this structural variant in mutant cycle experiments to determine interactions of the N1-OH and its guanidinium with the outer vestibule. NeoSTX had a higher affinity for the adult rat skeletal muscle Na+ channel (muI or Scn4a) than for STX (DeltaG approximately = 1.3 kcal/mol). Mutant cycle analysis identified groups that potentially interacted with each other. The N1 toxin site interacted most strongly with muI Asp-400 and Tyr-401. The interaction between the N1-OH of neoSTX and Tyr-401 was attractive (DeltaDeltaG = -1.3 +/- 0.1 kcal/mol), probably with formation of a hydrogen bond. A second possible attractive interaction to Asp-1532 was identified. There was repulsion between Asp-400 and the N1-OH (DeltaDeltaG = 1.4 +/- 0.1 kcal/mol), and kinetic analysis further suggested that the N1-OH was interacting negatively with Asp-400 at the transition state. Changes in pH altered the affinity of neoSTX, as would be expected if the N1-OH site were partially deprotonated. These interactions offer an explanation for most of the difference in blocking efficacy between neoSTX and STX and for the sensitivity of neoSTX to pH. Kinetic analysis suggested significant differences in coupling energies between the transition and the equilibrium, bound states. This is the first report to identify points of interaction between a channel and a non-peptide toxin. This interaction pattern was consistent with previous proposals describing the interactions of STX with the outer vestibule (Lipkind, G. M., and H. A. Fozzard. 1994. Biophys. J. 66:1-13; Penzotti, J. L., G. Lipkind, H. A. Fozzard, and S. C. Dudley, Jr. 1998. Biophys. J. 75:2647-2657).

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Selected References

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  1. Braiman M. S., Dioumaev A. K., Lewis J. R. A large photolysis-induced pKa increase of the chromophore counterion in bacteriorhodopsin: implications for ion transport mechanisms of retinal proteins. Biophys J. 1996 Feb;70(2):939–947. doi: 10.1016/S0006-3495(96)79637-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Chang N. S., French R. J., Lipkind G. M., Fozzard H. A., Dudley S., Jr Predominant interactions between mu-conotoxin Arg-13 and the skeletal muscle Na+ channel localized by mutant cycle analysis. Biochemistry. 1998 Mar 31;37(13):4407–4419. doi: 10.1021/bi9724927. [DOI] [PubMed] [Google Scholar]
  3. Chen S., Hartmann H. A., Kirsch G. E. Cysteine mapping in the ion selectivity and toxin binding region of the cardiac Na+ channel pore. J Membr Biol. 1997 Jan 1;155(1):11–25. doi: 10.1007/s002329900154. [DOI] [PubMed] [Google Scholar]
  4. Chen X. H., Tsien R. W. Aspartate substitutions establish the concerted action of P-region glutamates in repeats I and III in forming the protonation site of L-type Ca2+ channels. J Biol Chem. 1997 Nov 28;272(48):30002–30008. doi: 10.1074/jbc.272.48.30002. [DOI] [PubMed] [Google Scholar]
  5. Chiamvimonvat N., Pérez-García M. T., Ranjan R., Marban E., Tomaselli G. F. Depth asymmetries of the pore-lining segments of the Na+ channel revealed by cysteine mutagenesis. Neuron. 1996 May;16(5):1037–1047. doi: 10.1016/s0896-6273(00)80127-0. [DOI] [PubMed] [Google Scholar]
  6. Davoodi J., Wakarchuk W. W., Campbell R. L., Carey P. R., Surewicz W. K. Abnormally high pKa of an active-site glutamic acid residue in Bacillus circulans xylanase. The role of electrostatic interactions. Eur J Biochem. 1995 Sep 15;232(3):839–843. [PubMed] [Google Scholar]
  7. Favre I., Moczydlowski E., Schild L. On the structural basis for ionic selectivity among Na+, K+, and Ca2+ in the voltage-gated sodium channel. Biophys J. 1996 Dec;71(6):3110–3125. doi: 10.1016/S0006-3495(96)79505-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Favre I., Moczydlowski E., Schild L. Specificity for block by saxitoxin and divalent cations at a residue which determines sensitivity of sodium channel subtypes to guanidinium toxins. J Gen Physiol. 1995 Aug;106(2):203–229. doi: 10.1085/jgp.106.2.203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fersht A. R., Matouschek A., Serrano L. The folding of an enzyme. I. Theory of protein engineering analysis of stability and pathway of protein folding. J Mol Biol. 1992 Apr 5;224(3):771–782. doi: 10.1016/0022-2836(92)90561-w. [DOI] [PubMed] [Google Scholar]
  10. Guo X. T., Uehara A., Ravindran A., Bryant S. H., Hall S., Moczydlowski E. Kinetic basis for insensitivity to tetrodotoxin and saxitoxin in sodium channels of canine heart and denervated rat skeletal muscle. Biochemistry. 1987 Dec 1;26(24):7546–7556. doi: 10.1021/bi00398a003. [DOI] [PubMed] [Google Scholar]
  11. Hidalgo P., MacKinnon R. Revealing the architecture of a K+ channel pore through mutant cycles with a peptide inhibitor. Science. 1995 Apr 14;268(5208):307–310. doi: 10.1126/science.7716527. [DOI] [PubMed] [Google Scholar]
  12. Hille B. The receptor for tetrodotoxin and saxitoxin. A structural hypothesis. Biophys J. 1975 Jun;15(6):615–619. doi: 10.1016/S0006-3495(75)85842-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hu S. L., Kao C. Y. Interactions of neosaxitoxin with the sodium channel of the frog skeletal muscle fiber. J Gen Physiol. 1991 Mar;97(3):561–578. doi: 10.1085/jgp.97.3.561. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kao C. Y. Structure-activity relations of tetrodotoxin, saxitoxin, and analogues. Ann N Y Acad Sci. 1986;479:52–67. doi: 10.1111/j.1749-6632.1986.tb15561.x. [DOI] [PubMed] [Google Scholar]
  15. Kao C. Y., Walker S. E. Active groups of saxitoxin and tetrodotoxin as deduced from actions of saxitoxin analogues on frog muscle and squid axon. J Physiol. 1982 Feb;323:619–637. doi: 10.1113/jphysiol.1982.sp014095. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kao P. N., James-Kracke M. R., Kao C. Y. The active guanidinium group of saxitoxin and neosaxitoxin identified by the effects of pH on their activities on squid axon. Pflugers Arch. 1983 Aug;398(3):199–203. doi: 10.1007/BF00657151. [DOI] [PubMed] [Google Scholar]
  17. Kirsch G. E., Alam M., Hartmann H. A. Differential effects of sulfhydryl reagents on saxitoxin and tetrodotoxin block of voltage-dependent Na channels. Biophys J. 1994 Dec;67(6):2305–2315. doi: 10.1016/S0006-3495(94)80716-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kontis K. J., Goldin A. L. Site-directed mutagenesis of the putative pore region of the rat IIA sodium channel. Mol Pharmacol. 1993 Apr;43(4):635–644. [PubMed] [Google Scholar]
  19. Langsetmo K., Fuchs J. A., Woodward C. The conserved, buried aspartic acid in oxidized Escherichia coli thioredoxin has a pKa of 7.5. Its titration produces a related shift in global stability. Biochemistry. 1991 Jul 30;30(30):7603–7609. doi: 10.1021/bi00244a032. [DOI] [PubMed] [Google Scholar]
  20. Levitt M., Perutz M. F. Aromatic rings act as hydrogen bond acceptors. J Mol Biol. 1988 Jun 20;201(4):751–754. doi: 10.1016/0022-2836(88)90471-8. [DOI] [PubMed] [Google Scholar]
  21. Lipkind G. M., Fozzard H. A. A structural model of the tetrodotoxin and saxitoxin binding site of the Na+ channel. Biophys J. 1994 Jan;66(1):1–13. doi: 10.1016/S0006-3495(94)80746-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Matouschek A., Kellis J. T., Jr, Serrano L., Fersht A. R. Mapping the transition state and pathway of protein folding by protein engineering. Nature. 1989 Jul 13;340(6229):122–126. doi: 10.1038/340122a0. [DOI] [PubMed] [Google Scholar]
  23. Moczydlowski E., Hall S., Garber S. S., Strichartz G. S., Miller C. Voltage-dependent blockade of muscle Na+ channels by guanidinium toxins. J Gen Physiol. 1984 Nov;84(5):687–704. doi: 10.1085/jgp.84.5.687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Noda M., Suzuki H., Numa S., Stühmer W. A single point mutation confers tetrodotoxin and saxitoxin insensitivity on the sodium channel II. FEBS Lett. 1989 Dec 18;259(1):213–216. doi: 10.1016/0014-5793(89)81531-5. [DOI] [PubMed] [Google Scholar]
  25. Penzotti J. L., Fozzard H. A., Lipkind G. M., Dudley S. C., Jr Differences in saxitoxin and tetrodotoxin binding revealed by mutagenesis of the Na+ channel outer vestibule. Biophys J. 1998 Dec;75(6):2647–2657. doi: 10.1016/S0006-3495(98)77710-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Pérez-García M. T., Chiamvimonvat N., Marban E., Tomaselli G. F. Structure of the sodium channel pore revealed by serial cysteine mutagenesis. Proc Natl Acad Sci U S A. 1996 Jan 9;93(1):300–304. doi: 10.1073/pnas.93.1.300. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Rousso I., Friedman N., Sheves M., Ottolenghi M. pKa of the protonated Schiff base and aspartic 85 in the bacteriorhodopsin binding site is controlled by a specific geometry between the two residues. Biochemistry. 1995 Sep 19;34(37):12059–12065. doi: 10.1021/bi00037a049. [DOI] [PubMed] [Google Scholar]
  28. Sampogna R. V., Honig B. Environmental effects on the protonation states of active site residues in bacteriorhodopsin. Biophys J. 1994 May;66(5):1341–1352. doi: 10.1016/S0006-3495(94)80925-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Schantz E. J., Ghazarossian V. E., Schnoes H. K., Strong F. M., Springer J. P., Pezzanite J. O., Clardy J. Letter: The structure of saxitoxin. J Am Chem Soc. 1975 Mar 5;97(5):1238–1238. doi: 10.1021/ja00838a045. [DOI] [PubMed] [Google Scholar]
  30. Schreiber G., Fersht A. R. Energetics of protein-protein interactions: analysis of the barnase-barstar interface by single mutations and double mutant cycles. J Mol Biol. 1995 Apr 28;248(2):478–486. doi: 10.1016/s0022-2836(95)80064-6. [DOI] [PubMed] [Google Scholar]
  31. Shimizu Y., Alam M., Oshima Y., Fallon W. E. Presence of four toxins in red tide infested clams and cultured Gonyaulax tamarensis cells. Biochem Biophys Res Commun. 1975 Sep 16;66(2):731–737. doi: 10.1016/0006-291x(75)90571-9. [DOI] [PubMed] [Google Scholar]
  32. Shimizu Y. Chemistry and biochemistry of saxitoxin analogues and tetrodotoxin. Ann N Y Acad Sci. 1986;479:24–31. doi: 10.1111/j.1749-6632.1986.tb15558.x. [DOI] [PubMed] [Google Scholar]
  33. Strichartz G. Structural determinants of the affinity of saxitoxin for neuronal sodium channels. Electrophysiological studies on frog peripheral nerve. J Gen Physiol. 1984 Aug;84(2):281–305. doi: 10.1085/jgp.84.2.281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Sun Y. M., Favre I., Schild L., Moczydlowski E. On the structural basis for size-selective permeation of organic cations through the voltage-gated sodium channel. Effect of alanine mutations at the DEKA locus on selectivity, inhibition by Ca2+ and H+, and molecular sieving. J Gen Physiol. 1997 Dec;110(6):693–715. doi: 10.1085/jgp.110.6.693. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Terlau H., Heinemann S. H., Stühmer W., Pusch M., Conti F., Imoto K., Numa S. Mapping the site of block by tetrodotoxin and saxitoxin of sodium channel II. FEBS Lett. 1991 Nov 18;293(1-2):93–96. doi: 10.1016/0014-5793(91)81159-6. [DOI] [PubMed] [Google Scholar]
  36. Woodhull A. M. Ionic blockage of sodium channels in nerve. J Gen Physiol. 1973 Jun;61(6):687–708. doi: 10.1085/jgp.61.6.687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Yamagishi T., Janecki M., Marban E., Tomaselli G. F. Topology of the P segments in the sodium channel pore revealed by cysteine mutagenesis. Biophys J. 1997 Jul;73(1):195–204. doi: 10.1016/S0006-3495(97)78060-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Yang L., Kao C. Y., Oshima Y. Actions of decarbamoyloxysaxitoxin and decarbamoylneosaxitoxin on the frog skeletal muscle fiber. Toxicon. 1992 May-Jun;30(5-6):645–652. doi: 10.1016/0041-0101(92)90858-3. [DOI] [PubMed] [Google Scholar]
  39. Zhang J. F., Siegelbaum S. A. Effects of external protons on single cardiac sodium channels from guinea pig ventricular myocytes. J Gen Physiol. 1991 Dec;98(6):1065–1083. doi: 10.1085/jgp.98.6.1065. [DOI] [PMC free article] [PubMed] [Google Scholar]

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