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Article: An external determinant in the S5-P linker of the pacemaker (HCN) channel identified by sulfhydryl modification

TitleAn external determinant in the S5-P linker of the pacemaker (HCN) channel identified by sulfhydryl modification
Authors
KeywordsSpecies Index: Animalia
Issue Date2002
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, 2002, v. 277 n. 48, p. 46233-46242 How to Cite?
AbstractHyperpolarization-activated, cyclic nucleotide-gated (HCN) channels underlie spontaneous rhythmic activities in the heart and brain. Sulfhydryl modification of ion channels is a proven approach for studying their structure-function relationships; here we examined the effects of the hydrophilic sulfhydryl-modifying agents methanethiosulfonate ethylammonium(MTSEA+). and methanethiosulfonate ethylsulfonate (MTSES-) on wild-type (WT) and engineered HCN1 channels. External application of MTSEA+ to WT channels irreversibly reduced whole-cell currents (IMTSEA/IControl = 42 ± 2%), slowed activation and deactivation kinetics (∼7- and ∼3-fold at -140 and -20 mV, respectively), and produced hyperpolarizing shifts of steady-state activation (V1/2(MTSEA) = -125.8 ± 9.0 mV versus V1/2(Control) = -76.4 ± 1.6 mV). Sequence inspection revealed the presence of five endogenous cysteines in the transmembrane domains of HCN1: three are putatively close to the extracellular milieu (Cys303, Cys318, and Cys347 in the S5, S5-P, and P segments, respectively), whereas the remaining two are likely to be cytoplasmic or buried. To identify the molecular constituent(s) responsible for the effects of MTSEA+, we mutated the three "external" cysteines individually to serine. C303S did not yield measurable currents. Whereas C347S channels remained sensitive to MTSEA+, C318S was not modified (IMTSEA/IControl = 101 ± 2%, V1/2(MTSEA) = -78.4 ± 1.1 mV, and V1/2(Control) = -79.8 ± 2.3 mV). Likewise, WT (but not C318S) channels were sensitive to MTSES-. Despite their opposite charges, MTSES- produced changes directionally similar to those effected by MTSEA+ (IMTSES/IControl = 22 ± 1.6% and V1/2(MTSES) = -145.9 ± 4.9 mV). We conclude that S5-P Cys318 of HCN1 is externally accessible and that the external pore vestibule and activation gating of HCN channels are allosterically coupled.
Persistent Identifierhttp://hdl.handle.net/10722/91605
ISSN
2015 Impact Factor: 4.258
2015 SCImago Journal Rankings: 3.151
ISI Accession Number ID
References

 

DC FieldValueLanguage
dc.contributor.authorXue, Ten_HK
dc.contributor.authorLi, RAen_HK
dc.date.accessioned2010-09-17T10:22:05Z-
dc.date.available2010-09-17T10:22:05Z-
dc.date.issued2002en_HK
dc.identifier.citationJournal Of Biological Chemistry, 2002, v. 277 n. 48, p. 46233-46242en_HK
dc.identifier.issn0021-9258en_HK
dc.identifier.urihttp://hdl.handle.net/10722/91605-
dc.description.abstractHyperpolarization-activated, cyclic nucleotide-gated (HCN) channels underlie spontaneous rhythmic activities in the heart and brain. Sulfhydryl modification of ion channels is a proven approach for studying their structure-function relationships; here we examined the effects of the hydrophilic sulfhydryl-modifying agents methanethiosulfonate ethylammonium(MTSEA+). and methanethiosulfonate ethylsulfonate (MTSES-) on wild-type (WT) and engineered HCN1 channels. External application of MTSEA+ to WT channels irreversibly reduced whole-cell currents (IMTSEA/IControl = 42 ± 2%), slowed activation and deactivation kinetics (∼7- and ∼3-fold at -140 and -20 mV, respectively), and produced hyperpolarizing shifts of steady-state activation (V1/2(MTSEA) = -125.8 ± 9.0 mV versus V1/2(Control) = -76.4 ± 1.6 mV). Sequence inspection revealed the presence of five endogenous cysteines in the transmembrane domains of HCN1: three are putatively close to the extracellular milieu (Cys303, Cys318, and Cys347 in the S5, S5-P, and P segments, respectively), whereas the remaining two are likely to be cytoplasmic or buried. To identify the molecular constituent(s) responsible for the effects of MTSEA+, we mutated the three "external" cysteines individually to serine. C303S did not yield measurable currents. Whereas C347S channels remained sensitive to MTSEA+, C318S was not modified (IMTSEA/IControl = 101 ± 2%, V1/2(MTSEA) = -78.4 ± 1.1 mV, and V1/2(Control) = -79.8 ± 2.3 mV). Likewise, WT (but not C318S) channels were sensitive to MTSES-. Despite their opposite charges, MTSES- produced changes directionally similar to those effected by MTSEA+ (IMTSES/IControl = 22 ± 1.6% and V1/2(MTSES) = -145.9 ± 4.9 mV). We conclude that S5-P Cys318 of HCN1 is externally accessible and that the external pore vestibule and activation gating of HCN channels are allosterically coupled.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.meshAnimalsen_HK
dc.subject.meshCyclic Nucleotide-Gated Cation Channelsen_HK
dc.subject.meshCysteine - geneticsen_HK
dc.subject.meshIon Channels - chemistry - genetics - physiologyen_HK
dc.subject.meshMembrane Potentialsen_HK
dc.subject.meshMiceen_HK
dc.subject.meshMutagenesisen_HK
dc.subject.meshNerve Tissue Proteinsen_HK
dc.subject.meshPotassium Channelsen_HK
dc.subject.meshSerine - geneticsen_HK
dc.subject.meshStructure-Activity Relationshipen_HK
dc.subject.meshSulfhydryl Compounds - chemistryen_HK
dc.titleAn external determinant in the S5-P linker of the pacemaker (HCN) channel identified by sulfhydryl modificationen_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.M204915200en_HK
dc.identifier.pmid12351622-
dc.identifier.scopuseid_2-s2.0-0037195838en_HK
dc.relation.referenceshttp://www.scopus.com/mlt/select.url?eid=2-s2.0-0037195838&selection=ref&src=s&origin=recordpageen_HK
dc.identifier.volume277en_HK
dc.identifier.issue48en_HK
dc.identifier.spage46233en_HK
dc.identifier.epage46242en_HK
dc.identifier.isiWOS:000179529300071-
dc.publisher.placeUnited Statesen_HK
dc.identifier.scopusauthoridXue, T=7005064190en_HK
dc.identifier.scopusauthoridLi, RA=7404724466en_HK

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