Opening and Closing of the HCN2 Channel Pore is Voltage-Independent

Abstract

Recent studies have suggested that the opening and closing of Hyperpolarization-activated Cyclic Nucleotide-gated 'HCN' channels involve a step that is voltage-independent, which depends upon a region that resides within the S4 and S6 transmembrane domains of the channels. In the current study, we use two electrode voltage clamp of the HCN2 channel expressed in Xenopus oocytes to show that mutation of a residue site in the S6 segment, in region that likely corresponds to the pore, dramatically slows channel closing, without large effects on the rate of channel opening, and decreases the dependence of channel closing on voltage. A 6-state,but not a 4-state, cyclic allosteric model incorporating voltage-dependent transitions moving the channels between resting and active states and voltage-independent transitions between closed and open states was best able to describe the complex gating behaviour of both the wild type and pore mutant channels in response to changes in voltage. Modification of the voltage-independent closing transition recapitulates both the very slow and voltage-independent closing of mutant channel. In summary, our data supports a cyclic allosteric model of HCN2 channel gating in which the opening and closing of the pore is voltage-independent.