title: Alanine scanning of the S6 segment reveals a unique and cAMP-sensitive association between the pore and voltage-dependent opening in HCN channels.
abstract: Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels resemble Shaker K+ channels in structure and function. In both, changes in membrane voltage produce directionally similar movement of positively charged residues in the voltage sensor to alter the pore structure at the intracellular side and gate ion flow. However, HCNs open when hyperpolarized, whereas Shaker opens when depolarized. Thus, electromechanical coupling between the voltage sensor and gate is opposite. A key determinant of this coupling is the intrinsic stability of the pore. In Shaker, an alanine/valine scan of residues across the pore, by single point mutation, showed that most mutations made the channel easier to open and steepened the response of the channel to changes in voltage. Because most mutations likely destabilize protein packing, the Shaker pore is most stable when closed, and the voltage sensor works to open it. In HCN channels, the pore energetics and vector of work by the voltage sensor are unknown. Accordingly, we performed a 22-residue alanine/valine scan of the distal pore of the HCN2 isoform and show that the effects of mutations on channel opening and on the steepness of the response of the channel to voltage are mixed and smaller than those in Shaker. These data imply that the stabilities of the open and closed pore are similar, the voltage sensor must apply force to close the pore, and the interactions between the pore and voltage sensor are weak. Moreover, cAMP binding to the channel heightens the effects of the mutations, indicating stronger interactions between the pore and voltage sensor, and tips the energetic balance toward a more stable open state.
authors.affiliation: Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Macri
authors.forename: Hamed
authors.initials: H
authors.lastname: Nazzari
authors.forename: Evan
authors.initials: E
authors.lastname: McDonald
authors.forename: Eric A
authors.initials: EA
authors.lastname: Accili
mesh_terms: D000409:Alanine; D019943:Amino Acid Substitution; D000818:Animals; D016466:CHO Cells; D002462:Cell Membrane / Q000187:drug effects / Q000502:physiology; D006224:Cricetinae; D003412:Cricetulus; D000242:Cyclic AMP / Q000494:pharmacology / Q000502:physiology*; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D007473:Ion Channels / Q000737:chemistry / Q000187:drug effects / Q000235:genetics* / Q000502:physiology*; D051379:Mice; D018408:Patch-Clamp Techniques; D015221:Potassium Channels; D051637:Shaker Superfamily of Potassium Channels / Q000187:drug effects / Q000502:physiology; D013816:Thermodynamics; D014633:Valine
chemical_list: C578711:Hcn2 protein, mouse; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D007473:Ion Channels; D015221:Potassium Channels; D051637:Shaker Superfamily of Potassium Channels; D000242:Cyclic AMP; D014633:Valine; D000409:Alanine