Structural elements of instantaneous and slow gating in hyperpolarization-activated cyclic nucleotide-gated channels

Hyperpolarization-activated cyclic nucleotide-gated (HCN) subunits produce a slowly activating current in response to hyperpolarization (If) and an instantaneous voltage-independent current (Iinst) when expressed in Chinese hamster ovary (CHO) cells. Here we found that a mutation in the S4-S5 linker of HCN2 (Y331D) produced an additional mixed cationic instantaneous current. However, this current was inhibited by external Cs+ like If and unlike Iinst. Together with a concomitant reduction in If, the data suggest that the Y331D mutation disrupted channel closing placing the channel in a "If-like," and not an "Iinst-like," state. The "If-like" instantaneous current represented approximately 70% of total If over voltages ranging from +20 to -150 mV in high K+ solutions. If activated at more depolarized potentials and the activation curve was less steep, whereas deactivation was significantly slowed, consistent with the idea that the mutation inhibited channel closing. The data suggest that the mutation produced allosteric effects on the activation gate (S6 segment) and/or on voltage-sensing elements. We also found that decreases in the ratio of external K+/Na+ further disrupted channel closing in the mutant channel. Finally, our data suggest that the structures involved in producing Iinst are similar between the HCN1 and HCN2 isoforms and that excess HCN protein on the plasma membrane of CHO cells relative to native cells is not responsible for Iinst. The data are consistent with Iinst flowing through a "leaky" closed state but do not rule out flow through a second configuration of recombinant HCN channels or up-regulated endogenous channels/subunits.