title: Sensitivity of HCN channel deactivation to cAMP is amplified by an S4 mutation combined with activation mode shift.
abstract: Hyperpolarisation-activation of HCN ion channels relies on the movement of a charged S4 transmembrane helix, preferentially stabilising the open conformation of the ion pore gate. The open state is additionally stabilised, (a) when cyclic AMP (cAMP) is bound to a cytoplasmic C-terminal domain or (b) when the "mode I" open state formed initially by gate opening undergoes a "mode shift" into a "mode II" open state with a new S4 conformation. We isolated a mutation (lysine 381 to glutamate) in S4 of mouse HCN4; patch-clamp of homomeric channels in excised inside-out membranes revealed a conditional phenotype. When cAMP-liganded K381E channels are previously activated by hyperpolarisation, tens of seconds are required for complete deactivation at a weakly depolarised potential; this "ultra-sustained activation" is not observed without cAMP. Whilst cAMP slows deactivation of wild-type channels, the K381E mutation amplifies this effect to enable extraordinary kinetic stabilisation of the open state. K381E channels retain S4-gate coupling, with strong voltage dependence of the rate-limiting step for deactivation of mode II channels near -40 mV. At these voltages, the mode I deactivation pathway shows a different rate-limiting step, lacking strong voltage or cAMP dependence. Ultra-sustained activation thus reflects stabilisation of the mode II open state by the K381E mutation in synergistic combination with cAMP binding. Thus, the voltage-sensing domain is subject to strong functional coupling not only to the pore domain but also to the cytoplasmic cAMP-sensing domain in a manner specific to the voltage sensor conformation.
authors: Department of Molecular Biology and Biochemistry, Simon Fraser University, 8888 University Drive, Burnaby, BC, Canada, V5A 1S6.
authors: Nadine L
authors: NL
authors: Wicks
authors: Kerry S C
authors: KS
authors: Chan
authors: Zarina
authors: Z
authors: Madden
authors: Bina
authors: B
authors: Santoro
authors: Edgar C
authors: EC
authors: Young
mesh_terms: D019943:Amino Acid Substitution / Q000502:physiology*; D000818:Animals; D000242:Cyclic AMP / Q000378:metabolism* / Q000494:pharmacology; D054815:Cyclic Nucleotide-Gated Cation Channels / Q000235:genetics / Q000502:physiology*; D055724:Electrophysiological Phenomena / Q000502:physiology; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology*; D007473:Ion Channels / Q000235:genetics; D007700:Kinetics; D008564:Membrane Potentials / Q000502:physiology; D051379:Mice; D008954:Models, Biological; D009865:Oocytes / Q000378:metabolism; D018075:RNA, Complementary / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000502:physiology; D014982:Xenopus laevis
chemical_list: D054815:Cyclic Nucleotide-Gated Cation Channels; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D007473:Ion Channels; D018075:RNA, Complementary; D011993:Recombinant Fusion Proteins; D000242:Cyclic AMP