Identification of a surface charged residue in the S3-S4 linker of the pacemaker (HCN) channel that influences activation gating

I_f, encoded by the hyperpolarization-activated cyclic nucleotide-modulated (HCN) channel family, is a key player in cardiac and neuronal pacing. Although HCN channels structurally resemble voltage-gated K⁺ (Kv) channels, their structure-function correlation is much less clear. Here we probed the functional importance of the HCN1 S3-S4 linker by multiple substitutions of its residues. Neutralizing Glu²³⁵, an acidic S3-S4 linker residue conserved in all hyperpolarization-activated channels, by Ala substitution produced a depolarizing activation shift (V_1/2 = -65.0 ± 0.7 versus -70.6 ± 0.7 mV for wild-type HCN1); the charge-reversed mutation E235R shifted activation even more positively (-56.2 ± 0.5 mV). Increasing external Mg²⁺ mimicked the progressive rightward shifts of E235A and E235R by gradually shifting activation (V_1/2 = 1 < 3 < 10 < 30 mM); ΔV_1/2, induced by 30 mM Mg²⁺ was significantly attenuated for E235A (+7.9 ± 1.2 versus +11.3 ± 0.9 mV for wild-type HCN1) and E235R (+3.3 ± 1.4 mV) channels, as if surface charges were already shielded. Consistent with an electrostatic role, the energetic changes associated with ΔV_1/2 resulting from various Glu²³⁵ substitutions (i.e. Asp, Ala, Pro, His, Lys, and Arg) displayed a strong correlation with their charges (ΔΔG = -2.1 ± 0.3 kcal/mol/charge; r = 0.94). In contrast, D233E, D233A, D233G, and D233R did not alter activation gating. D233C (in C318S background) was also not externally accessible when probed with methanethiosulfonate ethylammonium (MTSEA). We conclude that the S3-S4 linker residue Glu²³⁵ influences activation gating, probably by acting as a surface charge.