title: State-dependent accessibility of the P-S6 linker of pacemaker (HCN) channels supports a dynamic pore-to-gate coupling model.
abstract: The hyperpolarization-activated cyclic nucleotide-modulated channel gene family (HCN1-4) encodes the membrane depolarizing current that underlies pacemaking. Although the topology of HCN resembles K(v) 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 Cd(2+) blockade (IC(50) = 3-12 microM 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 (DeltaV(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.
authors.affiliation: Stem Cell Program, University of California, Davis, CA, USA.
authors.forename: Chung Wah
authors.initials: CW
authors.lastname: Siu
authors.forename: Ezana M
authors.initials: EM
authors.lastname: Azene
authors.forename: Ka Wing
authors.initials: KW
authors.lastname: Au
authors.forename: Chu Pak
authors.initials: CP
authors.lastname: Lau
authors.forename: Hung Fat
authors.initials: HF
authors.lastname: Tse
authors.forename: Ronald A
authors.initials: RA
authors.lastname: Li
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002104:Cadmium / Q000494:pharmacology; D054815:Cyclic Nucleotide-Gated Cation Channels / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D004229:Dithiothreitol / Q000494:pharmacology; D004594:Electrophysiology; D051379:Mice; D008954:Models, Biological; D009865:Oocytes; D013329:Structure-Activity Relationship; D014981:Xenopus
chemical_list: D054815:Cyclic Nucleotide-Gated Cation Channels; D002104:Cadmium; D003545:Cysteine; D004229:Dithiothreitol