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Article: Pore residues critical for μ-CTX binding to rat skeletal muscle Na+ channels revealed by cysteine mutagenesis

TitlePore residues critical for μ-CTX binding to rat skeletal muscle Na+ channels revealed by cysteine mutagenesis
Authors
KeywordsSpecies Index: Animalia
Trixis
Xenopus Laevis
Issue Date1997
PublisherCell Press. The Journal's web site is located at http://www.cell.com/biophysj/
Citation
Biophysical Journal, 1997, v. 73 n. 4, p. 1874-1884 How to Cite?
AbstractWe have studied μ-conotoxin (μ-CTX) block of rat skeletal muscle sodium channel (rSkM1) currents in which single amine acids within the pore (P-loop) were substituted with cysteine. Among 17 cysteine mutants expressed in Xenopus oocytes, 7 showed significant alterations in sensitivity to μ- CTX compared to wild-type rSkM 1 channel (IC50- = 17.5 ± 2.8 nM). E758C and D1241C were less sensitive to μ-CTX block (IC50 = 220 ± 39 nM and 112 ± 24 nM, respectively), whereas the tryptophan mutants W402C, W1239C, and W1531C showed enhanced μ-CTX sensitivity (IC50 = 1.9 ± 0.1, 4.9 ± 0.9, and 5.5 ± 0.4 nM, respectively). D400C and Y401C also showed statistically significant yet modest (approximately twofold) changes in sensitivity to μ- CTX block compared to WT (p < 0.05). Application of the negatively charged, sulfhydryl-reactive compound methanethiosulfonate-ethylsulfonate (MTSES) enhanced the toxin sensitivity of D1241C (IC50 = 46.3 ± 12 nM) while having little effect on E758C mutant channels (IC50 = 199.8 ± 21.8 nM). On the other hand, the positively charged methanethiosulfonate-ethylammonium (MTSEA) completely abolished the μ-CTX sensitivity of E758C (IC50 > 1 μM) and increased the IC50 of D1241C by about threefold. Applications of MTSEA, MTSES, and the neutral MTSBN (benzyl methanethiosulfonate) to the tryptophan- to-cysteine mutants partially or fully restored the wild-type μ-CTX sensitivity, suggesting that the bulkiness of the tryptophan's indole group is a determinant of toxin binding. In support of this suggestion, the blocking IC50 of W1531A (7.5 ± 1.3 nM) was similar to W1531C, whereas W1531Y showed reduced toxin sensitivity (14.6 ± 3.5 nM) similar to that of the wild-type channel. Our results demonstrate that charge at positions 758 and 1241 are important for μ-CTX toxin binding and further suggest that the tryptophan residues within the pore in domains I, III, and IV negatively influence toxin-channel interaction.
Persistent Identifierhttp://hdl.handle.net/10722/91448
ISSN
2015 Impact Factor: 3.632
2015 SCImago Journal Rankings: 2.188
ISI Accession Number ID
References

 

DC FieldValueLanguage
dc.contributor.authorLi, RAen_HK
dc.contributor.authorTsushima, RGen_HK
dc.contributor.authorKallen, RGen_HK
dc.contributor.authorBackx, PHen_HK
dc.date.accessioned2010-09-17T10:19:34Z-
dc.date.available2010-09-17T10:19:34Z-
dc.date.issued1997en_HK
dc.identifier.citationBiophysical Journal, 1997, v. 73 n. 4, p. 1874-1884en_HK
dc.identifier.issn0006-3495en_HK
dc.identifier.urihttp://hdl.handle.net/10722/91448-
dc.description.abstractWe have studied μ-conotoxin (μ-CTX) block of rat skeletal muscle sodium channel (rSkM1) currents in which single amine acids within the pore (P-loop) were substituted with cysteine. Among 17 cysteine mutants expressed in Xenopus oocytes, 7 showed significant alterations in sensitivity to μ- CTX compared to wild-type rSkM 1 channel (IC50- = 17.5 ± 2.8 nM). E758C and D1241C were less sensitive to μ-CTX block (IC50 = 220 ± 39 nM and 112 ± 24 nM, respectively), whereas the tryptophan mutants W402C, W1239C, and W1531C showed enhanced μ-CTX sensitivity (IC50 = 1.9 ± 0.1, 4.9 ± 0.9, and 5.5 ± 0.4 nM, respectively). D400C and Y401C also showed statistically significant yet modest (approximately twofold) changes in sensitivity to μ- CTX block compared to WT (p < 0.05). Application of the negatively charged, sulfhydryl-reactive compound methanethiosulfonate-ethylsulfonate (MTSES) enhanced the toxin sensitivity of D1241C (IC50 = 46.3 ± 12 nM) while having little effect on E758C mutant channels (IC50 = 199.8 ± 21.8 nM). On the other hand, the positively charged methanethiosulfonate-ethylammonium (MTSEA) completely abolished the μ-CTX sensitivity of E758C (IC50 > 1 μM) and increased the IC50 of D1241C by about threefold. Applications of MTSEA, MTSES, and the neutral MTSBN (benzyl methanethiosulfonate) to the tryptophan- to-cysteine mutants partially or fully restored the wild-type μ-CTX sensitivity, suggesting that the bulkiness of the tryptophan's indole group is a determinant of toxin binding. In support of this suggestion, the blocking IC50 of W1531A (7.5 ± 1.3 nM) was similar to W1531C, whereas W1531Y showed reduced toxin sensitivity (14.6 ± 3.5 nM) similar to that of the wild-type channel. Our results demonstrate that charge at positions 758 and 1241 are important for μ-CTX toxin binding and further suggest that the tryptophan residues within the pore in domains I, III, and IV negatively influence toxin-channel interaction.en_HK
dc.languageengen_HK
dc.publisherCell Press. The Journal's web site is located at http://www.cell.com/biophysj/ en_HK
dc.relation.ispartofBiophysical Journalen_HK
dc.subjectSpecies Index: Animaliaen_HK
dc.subjectTrixisen_HK
dc.subjectXenopus Laevisen_HK
dc.subject.meshAnimalsen_HK
dc.subject.meshBinding Sites - geneticsen_HK
dc.subject.meshBinding, Competitiveen_HK
dc.subject.meshBiophysical Phenomenaen_HK
dc.subject.meshBiophysicsen_HK
dc.subject.meshCadmium - metabolismen_HK
dc.subject.meshCystine - chemistry - geneticsen_HK
dc.subject.meshFemaleen_HK
dc.subject.meshMembrane Potentialsen_HK
dc.subject.meshMollusk Venoms - metabolism - pharmacologyen_HK
dc.subject.meshMuscle, Skeletal - metabolismen_HK
dc.subject.meshMutagenesis, Site-Directeden_HK
dc.subject.meshOligopeptides - metabolism - pharmacologyen_HK
dc.subject.meshOocytes - metabolismen_HK
dc.subject.meshRatsen_HK
dc.subject.meshRecombinant Proteins - chemistry - genetics - metabolismen_HK
dc.subject.meshSodium Channel Blockersen_HK
dc.subject.meshSodium Channels - genetics - metabolismen_HK
dc.subject.meshStatic Electricityen_HK
dc.subject.meshXenopus laevisen_HK
dc.titlePore residues critical for μ-CTX binding to rat skeletal muscle Na+ channels revealed by cysteine mutagenesisen_HK
dc.typeArticleen_HK
dc.identifier.emailLi, RA:ronaldli@hkucc.hku.hken_HK
dc.identifier.authorityLi, RA=rp01352en_HK
dc.description.naturelink_to_subscribed_fulltext-
dc.identifier.pmid9336183-
dc.identifier.scopuseid_2-s2.0-0030770381en_HK
dc.relation.referenceshttp://www.scopus.com/mlt/select.url?eid=2-s2.0-0030770381&selection=ref&src=s&origin=recordpageen_HK
dc.identifier.volume73en_HK
dc.identifier.issue4en_HK
dc.identifier.spage1874en_HK
dc.identifier.epage1884en_HK
dc.identifier.isiWOS:A1997XY95500017-
dc.publisher.placeUnited Statesen_HK
dc.identifier.scopusauthoridLi, RA=7404724466en_HK
dc.identifier.scopusauthoridTsushima, RG=7006183117en_HK
dc.identifier.scopusauthoridKallen, RG=7005985453en_HK
dc.identifier.scopusauthoridBackx, PH=7006796226en_HK

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