The S4-S5 linker couples voltage sensing and activation of pacemaker channels

Voltage-gated channels are normally opened by depolarization and closed by repolarization of the membrane. Despite sharing significant sequence homolo gy with voltage-gated K+ channels, the gating of hyperpolarization-activate d, cyclic-nucleotide-gated (HCN) pacemaker channels has the opposite depend ence on membrane potential: hyperpolarization opens, whereas depolarization closes, these channels. The mechanism and structural basis of the process that couples voltage sensor movement to HCN channel opening and closing is not understood. On the basis of our previous studies of a mutant HERG (huma n ether-a-go-go-related gene) channel, we hypothesized that the intracellul ar linker that connects the fourth and fifth transmembrane domains (S4-S5 l inker) of HCN channels might be important for channel gating. Here, we used alanine-scanning mutagenesis of the HCN2 S4-S5 linker to identify three re sidues, E324, Y331, and R339, that when mutated disrupted normal channel cl osing. Mutation of a basic residue in the S4 domain (R318Q) prevented chann el opening, presumably by disrupting S4 movement. However, channels with R3 18Q and Y331S mutations were constitutively open, suggesting that these cha nnels can open without a functioning S4 domain. We conclude that the S4-S5 linker mediates coupling between voltage sensing and HCN channel activation . Our findings also suggest that opening of HCN and related channels corres ponds to activation of a gate located near the inner pore, rather than reco very of channels from a C-type inactivated state.