title: Probing S4 and S5 segment proximity in mammalian hyperpolarization-activated HCN channels by disulfide bridging and Cd2+ coordination.
abstract: We explored the structural basis of voltage sensing in the HCN1 hyperpolarization-activated cyclic nucleotide-gated cation channel by examining the relative orientation of the voltage sensor and pore domains. The opening of channels engineered to contain single cysteine residues at the extracellular ends of the voltage-sensing S4 (V246C) and pore-forming S5 (C303) domains is inhibited by formation of disulfide or cysteine:Cd(2+) bonds. As Cd(2+) coordination is promoted by depolarization, the S4-S5 interaction occurs preferentially in the closed state. The failure of oxidation to catalyze dimer formation, as assayed by Western blotting, indicates the V246C:C303 interaction occurs within a subunit. Intriguingly, a similar interaction has been observed in depolarization-activated Shaker voltage-dependent potassium (Kv) channels at depolarized potentials but such an intrasubunit interaction is inconsistent with the X-ray crystal structure of Kv1.2, wherein S4 approaches S5 of an adjacent subunit. These findings suggest channels of opposite voltage-sensing polarity adopt a conserved S4-S5 orientation in the depolarized state that is distinct from that trapped upon crystallization.
authors.affiliation: Department of Neuroscience, Columbia University, 722 West 168th Street, New York, NY 10032, USA.
authors.forename: Damian C
authors.initials: DC
authors.lastname: Bell
authors.forename: Harma K
authors.initials: HK
authors.lastname: Turbendian
authors.forename: Matthew T
authors.initials: MT
authors.lastname: Valley
authors.forename: Lei
authors.initials: L
authors.lastname: Zhou
authors.forename: John H
authors.initials: JH
authors.lastname: Riley
authors.forename: Steven A
authors.initials: SA
authors.lastname: Siegelbaum
authors.forename: Gareth R
authors.initials: GR
authors.lastname: Tibbs
mesh_terms: D000818:Animals; D002104:Cadmium / Q000502:physiology*; D054815:Cyclic Nucleotide-Gated Cation Channels / Q000187:drug effects / Q000502:physiology*; D003545:Cysteine / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism*; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D010618:Phenanthrolines / Q000494:pharmacology; D014982:Xenopus laevis
chemical_list: D054815:Cyclic Nucleotide-Gated Cation Channels; D004220:Disulfides; D010618:Phenanthrolines; D002104:Cadmium; D003545:Cysteine; C025205:1,10-phenanthroline