An external determinant in the S5-P linker of the pacemaker (HCN) channel identified by sulfhydryl modification

Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels underlie spontaneous rhythmic activities in the heart and brain. Sulfhydryl modification of ion channels is a proven approach for studying their structure-function relationships; here we examined the effects of the hydrophilic sulfhydryl-modifying agents methanethiosulfonate ethylammonium(MTSEA⁺). and methanethiosulfonate ethylsulfonate (MTSES^-) on wild-type (WT) and engineered HCN1 channels. External application of MTSEA⁺ to WT channels irreversibly reduced whole-cell currents (I_MTSEA/I_Control = 42 ± 2%), slowed activation and deactivation kinetics (∼7- and ∼3-fold at -140 and -20 mV, respectively), and produced hyperpolarizing shifts of steady-state activation (V_1/2(MTSEA) = -125.8 ± 9.0 mV versus V_1/2(Control) = -76.4 ± 1.6 mV). Sequence inspection revealed the presence of five endogenous cysteines in the transmembrane domains of HCN1: three are putatively close to the extracellular milieu (Cys³⁰³, Cys³¹⁸, and Cys³⁴⁷ in the S5, S5-P, and P segments, respectively), whereas the remaining two are likely to be cytoplasmic or buried. To identify the molecular constituent(s) responsible for the effects of MTSEA⁺, we mutated the three "external" cysteines individually to serine. C303S did not yield measurable currents. Whereas C347S channels remained sensitive to MTSEA⁺, C318S was not modified (I_MTSEA/I_Control = 101 ± 2%, V_1/2(MTSEA) = -78.4 ± 1.1 mV, and V_1/2(Control) = -79.8 ± 2.3 mV). Likewise, WT (but not C318S) channels were sensitive to MTSES^-. Despite their opposite charges, MTSES^- produced changes directionally similar to those effected by MTSEA⁺ (I_MTSES/I_Control = 22 ± 1.6% and V_1/2(MTSES) = -145.9 ± 4.9 mV). We conclude that S5-P Cys³¹⁸ of HCN1 is externally accessible and that the external pore vestibule and activation gating of HCN channels are allosterically coupled.