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Article: Structural and functional determinants in the S5-P region of HCN-encoded pacemaker channels revealed by cysteine-scanning substitutions

TitleStructural and functional determinants in the S5-P region of HCN-encoded pacemaker channels revealed by cysteine-scanning substitutions
Authors
KeywordsCysteine mutagenesis
Hyperpolarization-activated cyclic nucleotide
Sulfhydryl modification
Issue Date2008
PublisherAmerican Physiological Society. The Journal's web site is located at http://intl-ajpcell.physiology.org/
Citation
American Journal Of Physiology - Cell Physiology, 2008, v. 294 n. 1, p. C136-C144 How to Cite?
AbstractHyperpolarization-activated cyclic nucleotide-modulated (HCN) channels are responsible for the membrane pacemaker current that underlies the spontaneous generation of bioelectrical rhythms. However, their structure-function relationship is poorly understood. Previously, we identified several pore residues that influence HCN gating properties and proposed a pore-to-gate mechanism. Here, we systematically introduced cysteine-scanning substitutions into the descending portion of the P loop (residues 339-345) of HCN1-R (where R is resistance to sulfhydryl-reactive agents) channels, in which all endogenous cysteines except C303 have been removed or replaced. F339C, K340C, A341C, M342C, S343C, and M345C did not produce functional currents. Interestingly, the loss of function phenotype of F339C could be rescued by the reducing agent dithiothreitol (DTT). H344C but not HCN1-R and DTT-treated F339C channels were sensitive to blockade by divalent Cd2+ (current with 100 μMCd 2+/control current at -140 mV = 67.6 ± 2.9%, 109.3 ± 3.1%, and 103.8 ± 1.7%, respectively). Externally applied methanethiosulfate ethylammonium, a covalent sulfhydryl-reactive compound, irreversibly modified H344C by reducing the current at -140 mV (to 43.7 ± 6.5%), causing a hyperpolarizing steady-state activation shift (change in half-activation voltage: ∼-6 mV) and decelerated gating kinetics (by up to 3-fold). Based on these results, we conclude that pore residues 339-345 are important determinants of the structure-function properties of HCN channels and that the side chain of H344 is externally accessible. Copyright © 2008 the American Physiological Society.
Persistent Identifierhttp://hdl.handle.net/10722/76498
ISSN
2015 Impact Factor: 3.395
2015 SCImago Journal Rankings: 1.893
ISI Accession Number ID
References

 

DC FieldValueLanguage
dc.contributor.authorAu, KWen_HK
dc.contributor.authorSiu, CWen_HK
dc.contributor.authorLau, CPen_HK
dc.contributor.authorTse, HFen_HK
dc.contributor.authorLi, RAen_HK
dc.date.accessioned2010-09-06T07:21:53Z-
dc.date.available2010-09-06T07:21:53Z-
dc.date.issued2008en_HK
dc.identifier.citationAmerican Journal Of Physiology - Cell Physiology, 2008, v. 294 n. 1, p. C136-C144en_HK
dc.identifier.issn0363-6143en_HK
dc.identifier.urihttp://hdl.handle.net/10722/76498-
dc.description.abstractHyperpolarization-activated cyclic nucleotide-modulated (HCN) channels are responsible for the membrane pacemaker current that underlies the spontaneous generation of bioelectrical rhythms. However, their structure-function relationship is poorly understood. Previously, we identified several pore residues that influence HCN gating properties and proposed a pore-to-gate mechanism. Here, we systematically introduced cysteine-scanning substitutions into the descending portion of the P loop (residues 339-345) of HCN1-R (where R is resistance to sulfhydryl-reactive agents) channels, in which all endogenous cysteines except C303 have been removed or replaced. F339C, K340C, A341C, M342C, S343C, and M345C did not produce functional currents. Interestingly, the loss of function phenotype of F339C could be rescued by the reducing agent dithiothreitol (DTT). H344C but not HCN1-R and DTT-treated F339C channels were sensitive to blockade by divalent Cd2+ (current with 100 μMCd 2+/control current at -140 mV = 67.6 ± 2.9%, 109.3 ± 3.1%, and 103.8 ± 1.7%, respectively). Externally applied methanethiosulfate ethylammonium, a covalent sulfhydryl-reactive compound, irreversibly modified H344C by reducing the current at -140 mV (to 43.7 ± 6.5%), causing a hyperpolarizing steady-state activation shift (change in half-activation voltage: ∼-6 mV) and decelerated gating kinetics (by up to 3-fold). Based on these results, we conclude that pore residues 339-345 are important determinants of the structure-function properties of HCN channels and that the side chain of H344 is externally accessible. Copyright © 2008 the American Physiological Society.en_HK
dc.languageengen_HK
dc.publisherAmerican Physiological Society. The Journal's web site is located at http://intl-ajpcell.physiology.org/en_HK
dc.relation.ispartofAmerican Journal of Physiology - Cell Physiologyen_HK
dc.rightsAmerican Journal of Physiology: Cell Physiology. Copyright © American Physiological Society.-
dc.subjectCysteine mutagenesisen_HK
dc.subjectHyperpolarization-activated cyclic nucleotideen_HK
dc.subjectSulfhydryl modificationen_HK
dc.subject.meshAmino Acid Sequence-
dc.subject.meshCyclic Nucleotide-Gated Cation Channels - chemistry - drug effects - genetics - metabolism-
dc.subject.meshCysteine - chemistry - genetics-
dc.subject.meshIon Channel Gating - drug effects - genetics-
dc.subject.meshPotassium Channels - chemistry - drug effects - genetics - metabolism-
dc.titleStructural and functional determinants in the S5-P region of HCN-encoded pacemaker channels revealed by cysteine-scanning substitutionsen_HK
dc.typeArticleen_HK
dc.identifier.openurlhttp://library.hku.hk:4550/resserv?sid=HKU:IR&issn=0363-6143&volume=294&issue=1&spage=C136&epage=144&date=2008&atitle=Structural+and+functional+determinants+in+the+S5-P+region+of+HCN-encoded+pacemaker+channels+revealed+by+cysteine-scanning+substitutionsen_HK
dc.identifier.emailSiu, CW:cwdsiu@hkucc.hku.hken_HK
dc.identifier.emailTse, HF:hftse@hkucc.hku.hken_HK
dc.identifier.emailLi, RA:ronaldli@hkucc.hku.hken_HK
dc.identifier.authoritySiu, CW=rp00534en_HK
dc.identifier.authorityTse, HF=rp00428en_HK
dc.identifier.authorityLi, RA=rp01352en_HK
dc.description.naturelink_to_OA_fulltext-
dc.identifier.doi10.1152/ajpcell.00340.2007en_HK
dc.identifier.pmid17989208en_HK
dc.identifier.scopuseid_2-s2.0-38349145565en_HK
dc.identifier.hkuros159757-
dc.identifier.hkuros160265-
dc.identifier.hkuros222565-
dc.relation.referenceshttp://www.scopus.com/mlt/select.url?eid=2-s2.0-38349145565&selection=ref&src=s&origin=recordpageen_HK
dc.identifier.volume294en_HK
dc.identifier.issue1en_HK
dc.identifier.spageC136en_HK
dc.identifier.epageC144en_HK
dc.identifier.isiWOS:000252507600017-
dc.publisher.placeUnited Statesen_HK
dc.identifier.scopusauthoridAu, KW=9738204200en_HK
dc.identifier.scopusauthoridSiu, CW=7006550690en_HK
dc.identifier.scopusauthoridLau, CP=7401968501en_HK
dc.identifier.scopusauthoridTse, HF=7006070805en_HK
dc.identifier.scopusauthoridLi, RA=7404724466en_HK
dc.identifier.citeulike3688616-

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