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Article: Novel structural determinants of μ-conotoxin (GIIIB) block in rat skeletal muscle (μ1) Na+ channels

TitleNovel structural determinants of μ-conotoxin (GIIIB) block in rat skeletal muscle (μ1) Na+ channels
Authors
KeywordsSpecies Index: Animalia
Issue Date2000
PublisherAmerican Society for Biochemistry and Molecular Biology, Inc. The Journal's web site is located at http://www.jbc.org/
Citation
Journal Of Biological Chemistry, 2000, v. 275 n. 36, p. 27551-27558 How to Cite?
Abstractμ-Conotoxin (μ-CTX) specifically occludes the pore of voltage-dependent Na+ channels. In the rat skeletal muscle Na+ channel (μ1), we examined the contribution of charged residues between the P loops and S6 in all four domains to μ-CTX block. Conversion of the negatively charged domain II (DII) residues Asp-762 and Glu-765 to cysteine increased the IC50 for μ-CTX block by -100-fold (wild-type = 22.3 ± 7.0 nM; D762C = 2558 ± 250 nM; E765C = 2020 ± 379 nM). Restoration or reversal of charge by external modification of the cysteine-substituted channels with methanethiosulfonate reagents (methanethiosulfonate ethylsulfonate (MTSES) and methanethiosulfonate ethylammonium (MTSEA)) did not affect μ-CTX block (D762C: IC(50, MTSEA+) = 2165.1 ± 250 nM; IC(50, MTSES-) = 2753.5 ± 456.9 nM; E765C: IC(50, MTSEA+) = 2200.1 ± 550.3 nM; IC(50, MTSES-) = 3248.1 ± 2011.9 nM) compared with their unmodified counterparts. In contrast, the charge-conserving mutations D762E (IC50 = 21.9 ± 4.3 nM) and E765D (IC50 = 22.0 ± 7.0 nM) preserved wild-type blocking behavior, whereas the charge reversal mutants D762K (IC50 = 4139.9 ± 687.9 nM) and E765K (IC50 = 4202.7 ± 1088.0 nM) destabilized μ-CTX block even further, suggesting a prominent electrostatic component of the interactions between these DII residues and μ-CTX. Kinetic analysis of μ-CTX block reveals that the changes in toxin sensitivity are largely due to accelerated toxin dissociation (k(off)) rates with little changes in association (k(on)) rates. We conclude that the acidic residues at positions 762 and 765 are key determinants of μ-CTX block, primarily by virtue of their negative charge. The inability of the bulky MTSES or MTSEA side chain to modify μ-CTX sensitivity places steric constraints on the sites of toxin interaction.
Persistent Identifierhttp://hdl.handle.net/10722/91665
ISSN
2020 Impact Factor: 5.157
2023 SCImago Journal Rankings: 1.766
ISI Accession Number ID
References

 

DC FieldValueLanguage
dc.contributor.authorLi, RAen_HK
dc.contributor.authorEnnis, ILen_HK
dc.contributor.authorVélez, Pen_HK
dc.contributor.authorTomaselli, GFen_HK
dc.contributor.authorMarbán, Een_HK
dc.date.accessioned2010-09-17T10:23:01Z-
dc.date.available2010-09-17T10:23:01Z-
dc.date.issued2000en_HK
dc.identifier.citationJournal Of Biological Chemistry, 2000, v. 275 n. 36, p. 27551-27558en_HK
dc.identifier.issn0021-9258en_HK
dc.identifier.urihttp://hdl.handle.net/10722/91665-
dc.description.abstractμ-Conotoxin (μ-CTX) specifically occludes the pore of voltage-dependent Na+ channels. In the rat skeletal muscle Na+ channel (μ1), we examined the contribution of charged residues between the P loops and S6 in all four domains to μ-CTX block. Conversion of the negatively charged domain II (DII) residues Asp-762 and Glu-765 to cysteine increased the IC50 for μ-CTX block by -100-fold (wild-type = 22.3 ± 7.0 nM; D762C = 2558 ± 250 nM; E765C = 2020 ± 379 nM). Restoration or reversal of charge by external modification of the cysteine-substituted channels with methanethiosulfonate reagents (methanethiosulfonate ethylsulfonate (MTSES) and methanethiosulfonate ethylammonium (MTSEA)) did not affect μ-CTX block (D762C: IC(50, MTSEA+) = 2165.1 ± 250 nM; IC(50, MTSES-) = 2753.5 ± 456.9 nM; E765C: IC(50, MTSEA+) = 2200.1 ± 550.3 nM; IC(50, MTSES-) = 3248.1 ± 2011.9 nM) compared with their unmodified counterparts. In contrast, the charge-conserving mutations D762E (IC50 = 21.9 ± 4.3 nM) and E765D (IC50 = 22.0 ± 7.0 nM) preserved wild-type blocking behavior, whereas the charge reversal mutants D762K (IC50 = 4139.9 ± 687.9 nM) and E765K (IC50 = 4202.7 ± 1088.0 nM) destabilized μ-CTX block even further, suggesting a prominent electrostatic component of the interactions between these DII residues and μ-CTX. Kinetic analysis of μ-CTX block reveals that the changes in toxin sensitivity are largely due to accelerated toxin dissociation (k(off)) rates with little changes in association (k(on)) rates. We conclude that the acidic residues at positions 762 and 765 are key determinants of μ-CTX block, primarily by virtue of their negative charge. The inability of the bulky MTSES or MTSEA side chain to modify μ-CTX sensitivity places steric constraints on the sites of toxin interaction.en_HK
dc.languageengen_HK
dc.publisherAmerican Society for Biochemistry and Molecular Biology, Inc. The Journal's web site is located at http://www.jbc.org/en_HK
dc.relation.ispartofJournal of Biological Chemistryen_HK
dc.subjectSpecies Index: Animaliaen_HK
dc.subject.meshAmino Acid Substitutionen_HK
dc.subject.meshAnimalsen_HK
dc.subject.meshAspartic Aciden_HK
dc.subject.meshBinding Sitesen_HK
dc.subject.meshCell Lineen_HK
dc.subject.meshConotoxins - pharmacologyen_HK
dc.subject.meshEthyl Methanesulfonate - analogs & derivatives - pharmacologyen_HK
dc.subject.meshGlutamic Aciden_HK
dc.subject.meshHumansen_HK
dc.subject.meshKineticsen_HK
dc.subject.meshMesylates - pharmacologyen_HK
dc.subject.meshModels, Molecularen_HK
dc.subject.meshMuscle, Skeletal - physiologyen_HK
dc.subject.meshMutagenesis, Site-Directeden_HK
dc.subject.meshProtein Structure, Secondaryen_HK
dc.subject.meshRatsen_HK
dc.subject.meshSodium Channel Blockersen_HK
dc.subject.meshSodium Channels - chemistry - physiologyen_HK
dc.subject.meshTransfectionen_HK
dc.titleNovel structural determinants of μ-conotoxin (GIIIB) block in rat skeletal muscle (μ1) Na+ channelsen_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.doi10.1074/jbc.M909719199en_HK
dc.identifier.pmid10859326-
dc.identifier.scopuseid_2-s2.0-0034623047en_HK
dc.relation.referenceshttp://www.scopus.com/mlt/select.url?eid=2-s2.0-0034623047&selection=ref&src=s&origin=recordpageen_HK
dc.identifier.volume275en_HK
dc.identifier.issue36en_HK
dc.identifier.spage27551en_HK
dc.identifier.epage27558en_HK
dc.identifier.isiWOS:000089197100007-
dc.publisher.placeUnited Statesen_HK
dc.identifier.scopusauthoridLi, RA=7404724466en_HK
dc.identifier.scopusauthoridEnnis, IL=6604033332en_HK
dc.identifier.scopusauthoridVélez, P=6701863236en_HK
dc.identifier.scopusauthoridTomaselli, GF=7005223451en_HK
dc.identifier.scopusauthoridMarbán, E=8075977300en_HK
dc.identifier.issnl0021-9258-

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