Separable gating mechanisms in a mammalian pacemaker channel

Abstract

Despite permeability to both K+ and Na+, hyperpolarization-activated cyclic nucleotide-gated (HCN) pacemaker channels contain the K+ channel signature sequence, GYG, within the selectivity filter of the pore. Here, we show that this region is involved in regulating gating in a mouse isoform of the pacemaker channel (mHCN2). A mutation in the GYG sequence of the selectivity filter (G404S) had different effects on the two components of the wild-type current; it eliminated the slowly activating current (If) but, surprisingly, did not affect the instantaneous current (Iinst). Confocal imaging and immunocytochemistry showed G404S protein on the periphery of the cells, consistent with the presence of channels on the plasma membrane. Experiments with the wild-type channel showed that the rate of If deactivation and If amplitude had a parallel dependence on the ratio of K+/Na+ driving forces. In addition, the amplitude of fully activated If, unlike Iinst, was not well predicted by equal and independent flow of K+ and Na+. The data are consistent with two separable gating mechanisms associated with pacemaker channels: one (If) that is sensitive to voltage, to a mutation in the selectivity filter, and to driving forces for permeating cations and another (Iinst) that is insensitive to these influences.