Dissecting the structural and functional roles of the S3-S4 linker of pacemaker (hyperpolarization-activated cyclic nucleotide-modulated) channels by systematic length alterations

Abstract

I f or I h, a key player in neuronal and cardiac pacing, is encoded by the hyperpolarization-activated cyclic nucleotide-modulated (HCN) channel gene family. We have recently reported that the S3-S4 linker (i.e. residues 229EKGMDSEVY 237 of HCN1) prominently influences the activation phenotypes of HCN channels and that part of the linker may conform a secondary helical structure. Here we further dissected the structural and functional roles of this linker by systematic alterations of its length. In contrast to voltage-gated K + channels, complete deletion of the S3-S4 linker (Δ229-237) did not produce functional channels. Similarly, the deletions Δ229-234, Δ232-234, and Δ232-237 also abolished normal current activity. Interestingly, Δ229-231, Δ233-237, Δ234-237, Δ235-237, Δ229-231/Δ233-237, Δ229-231/Δ234-237, and Δ229-231/Δ235-237 all yielded robust hyperpolarization-activated inward currents, indicating that loss-of-function caused by deletion could be rescued by keeping the single functionally important residue Met 232 alone. Whereas shortening the linker by deletion generally shifted steady-state activation in the depolarizing direction (e.g. ΔV 1/2 of Δ229-231, Δ233-237, Δ235-237 >+10 mV relative to wild type), linker prolongation by duplicating the entire linker (Dup229-237) or by glutamine insertion (InsQ233Q, InsQQ233QQ and InsQQQ233QQQ, or Ins237QQQ) produced length-dependent progressive hyperpolarizing activation shifts (-35 mV < ΔV 1/2 < -4 mV). Based on these results, we conclude that only Met 232 is prerequisite for channels to function, but the length and other constituents of the S3-S4 linker shape the ultimate activation phenotype. Our results also highlight several evolutionary similarities and differences between HCN and voltage-gated K + channels. Manipulations of the S3-S4 linker length may provide a flexible approach to customize HCN gating for engineering electrically active cells (such as stem cell-derived neuronal and cardiac pacemakers) for gene- and cell-based therapies.