The functionally diverse cyclic nucleotide binding domain (CNBD) superfamily of cation channels contains both depolarization-gated (e.g., metazoan EAG family K+ channels) and hyperpolarization-gated channels (e.g., metazoan HCN pacemaker cation channels and the plant K+ channel KAT1). In both types of CNBD channels, the S4 transmembrane helix of the voltage sensor domain (VSD) moves outward in response to depolarization. This movement opens depolarization-gated channels and closes hyperpolarization-gated channels. External divalent cations and protons prevent or slow movement of S4 by binding to a cluster of acidic charges on the S2 and S3 transmembrane domains of the VSD and therefore inhibit activation of EAG family channels. However, a similar divalent ion/proton binding pocket has not been described for hyperpolarization-gated CNBD family channels. We examined the effects of external Cd2+ and protons on Arabidopsisthaliana KAT1 expressed in Xenopus oocytes and found that these ions strongly potentiate voltage activation. Cd2+ at 300 µM depolarizes the V50 of KAT1 by 150 mV, while acidification from pH 7.0 to 4.0 depolarizes the V50 by 49 mV. Regulation of KAT1 by Cd2+ is state dependent and consistent with Cd2+ binding to an S4-down state of the VSD. Neutralization of a conserved acidic charge in the S2 helix in KAT1 (D95N) eliminates Cd2+ and pH sensitivity. Conversely, introduction of acidic residues into KAT1 at additional S2 and S3 cluster positions that are charged in EAG family channels (N99D and Q149E in KAT1) decreases Cd2+ sensitivity and increases proton potentiation. These results suggest that KAT1, and presumably other hyperpolarization-gated plant CNBD channels, can open from an S4-down VSD conformation homologous to the divalent/proton-inhibited conformation of EAG family K+ channels.

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