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Article: State-Dependent accessibility of the P-S6 linker of pacemaker (HCN) channels supports a dynamic pore-to-gate coupling model

TitleState-Dependent accessibility of the P-S6 linker of pacemaker (HCN) channels supports a dynamic pore-to-gate coupling model
Authors
KeywordsHCN
Outer pore
Pacemaker channels
Issue Date2009
PublisherSpringer New York LLC. The Journal's web site is located at http://link.springer.de/link/service/journals/00232/
Citation
Journal Of Membrane Biology, 2009, v. 230 n. 1, p. 35-47 How to Cite?
AbstractThe hyperpolarization-activated cyclic nucleotide-modulated channel gene family (HCN1-4) encodes the membrane depolarizing current that underlies pacemaking. Although the topology of HCN resembles Kv channels, much less is known about their structure-function correlation. Previously, we identified several pore residues in the S5-P linker and P-loop that are externally accessible and/or influence HCN gating, and proposed an evolutionarily conserved pore-to-gate mechanism. Here we sought dynamic evidence by assessing the functional consequences of Cys-scanning substitutions in the unexplored P-S6 linker (residues 352-359), the HCN1-R background (that is, resistant to sulfhydryl-reactive agents). None of A352C, Q353C, A354C, P355C, V356C, S357C, M358C, or S359C produced functional currents; the loss-of-function of Q353C, A354C, S357C, and M358C could be rescued by the reducing agent dithiothreitol. Q353C, A354C, and S357C, but not M358C and HCN1-R, were sensitive to Cd2+ blockade (IC50 = 3-12 μM vs. >1 mM). External application of the positively charged covalent sulfhydryl modifier MTSET irreversibly reduced I -140mV of Q353C and A354C to 27.9 ± 3.4% and 58.2 ± 13.1% of the control, respectively, and caused significant steady-state activation shifts (δV 1/2 = -21.1 ± 1.6 for Q353C and -10.0 ± 2.9 mV for A354C). Interestingly, MTSET reactivity was also state dependent. MTSET, however, affected neither S357C nor M358C, indicating site specificity. Collectively, we have identified novel P-S6 residues whose extracellular accessibility was sterically and state dependent and have provided the first functional evidence consistent with a dynamic HCN pore-to-gate model.
Persistent Identifierhttp://hdl.handle.net/10722/144951
ISSN
2015 Impact Factor: 1.991
2015 SCImago Journal Rankings: 0.738
PubMed Central ID
ISI Accession Number ID
Funding AgencyGrant Number
National Institutes of HealthR01 HL72857
Croucher Foundation Fellowship
University of Hong Kong Seed Funding Programme for Basic ResearchHKU 200808159001
Hong Kong Research Grant Council General Research Fund7747/08
CC Wong Stem Cell Fund
Funding Information:

This work was supported by grants from the National Institutes of Health (R01 HL72857 to R.A.L.), Croucher Foundation Fellowship (C.W.S), the University of Hong Kong Seed Funding Programme for Basic Research (HKU 200808159001 to C.W.S), the Hong Kong Research Grant Council General Research Fund (HKU 7747/08 M to C.W.S., H.F.T., and R.A.L.), and the CC Wong Stem Cell Fund (to H.F.T. and R.A.L.).

References

 

DC FieldValueLanguage
dc.contributor.authorSiu, CWen_HK
dc.contributor.authorAzene, EMen_HK
dc.contributor.authorAu, KWen_HK
dc.contributor.authorLau, CPen_HK
dc.contributor.authorTse, HFen_HK
dc.contributor.authorLi, RAen_HK
dc.date.accessioned2012-02-21T05:44:54Z-
dc.date.available2012-02-21T05:44:54Z-
dc.date.issued2009en_HK
dc.identifier.citationJournal Of Membrane Biology, 2009, v. 230 n. 1, p. 35-47en_HK
dc.identifier.issn0022-2631en_HK
dc.identifier.urihttp://hdl.handle.net/10722/144951-
dc.description.abstractThe hyperpolarization-activated cyclic nucleotide-modulated channel gene family (HCN1-4) encodes the membrane depolarizing current that underlies pacemaking. Although the topology of HCN resembles Kv channels, much less is known about their structure-function correlation. Previously, we identified several pore residues in the S5-P linker and P-loop that are externally accessible and/or influence HCN gating, and proposed an evolutionarily conserved pore-to-gate mechanism. Here we sought dynamic evidence by assessing the functional consequences of Cys-scanning substitutions in the unexplored P-S6 linker (residues 352-359), the HCN1-R background (that is, resistant to sulfhydryl-reactive agents). None of A352C, Q353C, A354C, P355C, V356C, S357C, M358C, or S359C produced functional currents; the loss-of-function of Q353C, A354C, S357C, and M358C could be rescued by the reducing agent dithiothreitol. Q353C, A354C, and S357C, but not M358C and HCN1-R, were sensitive to Cd2+ blockade (IC50 = 3-12 μM vs. >1 mM). External application of the positively charged covalent sulfhydryl modifier MTSET irreversibly reduced I -140mV of Q353C and A354C to 27.9 ± 3.4% and 58.2 ± 13.1% of the control, respectively, and caused significant steady-state activation shifts (δV 1/2 = -21.1 ± 1.6 for Q353C and -10.0 ± 2.9 mV for A354C). Interestingly, MTSET reactivity was also state dependent. MTSET, however, affected neither S357C nor M358C, indicating site specificity. Collectively, we have identified novel P-S6 residues whose extracellular accessibility was sterically and state dependent and have provided the first functional evidence consistent with a dynamic HCN pore-to-gate model.en_HK
dc.languageengen_US
dc.publisherSpringer New York LLC. The Journal's web site is located at http://link.springer.de/link/service/journals/00232/en_HK
dc.relation.ispartofJournal of Membrane Biologyen_HK
dc.rightsThe Author(s)en_US
dc.rightsCreative Commons: Attribution 3.0 Hong Kong Licenseen_US
dc.subjectHCNen_HK
dc.subjectOuter poreen_HK
dc.subjectPacemaker channelsen_HK
dc.subject.meshCadmium - pharmacology-
dc.subject.meshCyclic Nucleotide-Gated Cation Channels - chemistry - drug effects - genetics - metabolism-
dc.subject.meshCysteine - chemistry - genetics-
dc.subject.meshDithiothreitol - pharmacology-
dc.subject.meshElectrophysiology-
dc.titleState-Dependent accessibility of the P-S6 linker of pacemaker (HCN) channels supports a dynamic pore-to-gate coupling modelen_HK
dc.typeArticleen_HK
dc.identifier.openurlhttp://library.hku.hk:4551/resserv?sid=springerlink&genre=article&atitle=State-Dependent Accessibility of the P-S6 Linker of Pacemaker (HCN) Channels Supports a Dynamic Pore-to-Gate Coupling Model&title=Journal of Membrane Biology&issn=00222631&date=2009-07-01&volume=230&issue=1& spage=35&authors=Chung Wah Siu, Ezana M. Azene, Ka Wing Au, <i>et al.</i>en_US
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.naturepublished_or_final_versionen_US
dc.identifier.doi10.1007/s00232-009-9184-2en_HK
dc.identifier.pmid19609824-
dc.identifier.pmcidPMC2718208-
dc.identifier.scopuseid_2-s2.0-68649126236en_HK
dc.identifier.hkuros158537-
dc.identifier.hkuros182835-
dc.identifier.hkuros182836-
dc.identifier.hkuros239714-
dc.relation.referenceshttp://www.scopus.com/mlt/select.url?eid=2-s2.0-68649126236&selection=ref&src=s&origin=recordpageen_HK
dc.identifier.volume230en_HK
dc.identifier.issue1en_HK
dc.identifier.spage35en_HK
dc.identifier.epage47en_HK
dc.identifier.eissn1432-1424en_US
dc.identifier.isiWOS:000268513100004-
dc.publisher.placeUnited Statesen_HK
dc.description.otherSpringer Open Choice, 21 Feb 2012en_US
dc.identifier.scopusauthoridSiu, CW=7006550690en_HK
dc.identifier.scopusauthoridAzene, EM=6602472909en_HK
dc.identifier.scopusauthoridAu, KW=9738204200en_HK
dc.identifier.scopusauthoridLau, CP=7401968501en_HK
dc.identifier.scopusauthoridTse, HF=7006070805en_HK
dc.identifier.scopusauthoridLi, RA=7404724466en_HK
dc.identifier.citeulike5282949-

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