Abstract
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 Glu(235), an acidic S3-S4 linker residue conserved in all hyperpolarization-activated channels, by Ala substitution produced a depolarizing activation shift (V(12) = -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 Mg(2+) mimicked the progressive rightward shifts of E235A and E235R by gradually shifting activation (V(12) = 1 < 3 < 10 < 30 mm); Delta V(12) induced by 30 mm Mg(2+) 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 Delta V(12) resulting from various Glu(235) substitutions (i.e. Asp, Ala, Pro, His, Lys, and Arg) displayed a strong correlation with their charges (Delta Delta G = -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 Glu(235) influences activation gating, probably by acting as a surface charge.
Highlights
Background) was not externally accessible when probed with methanethiosulfonate ethylammonium (MTSEA)
Unlike act, the deactivation time constant (deact) became faster with increasing depolarization. Plotting these time constants together against the test potential revealed that the voltage dependence of act and deact had a bell-shaped form (Fig. 2D); ␣0 and 0 for WT HCN1 channels derived from this curve were (3.6 Ϯ 0.5) ϫ 10Ϫ1 and (2.3 Ϯ 0.2) ϫ 10 sϪ1, respectively
The S3-S4 Linker Residue Glu235 Acts as an External Surface Charge—Previous studies have demonstrated that the extracellular S3-S4 linker is a determinant of activation in various Kϩ and Ca2ϩ channels [12,13,14,15, 19]
Summary
Background) was not externally accessible when probed with methanethiosulfonate ethylammonium (MTSEA). Neutralizing Glu235, an acidic S3-S4 linker residue conserved in all hyperpolarization-activated channels, by Ala substitution produced a depolarizing activation shift (V1⁄2 ؍؊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).
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