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 (IMTSEA/IControl = 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 (V1/2(MTSEA) = -125.8 ± 9.0 mV versus V1/2(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 (IMTSEA/IControl = 101 ± 2%, V1/2(MTSEA) = -78.4 ± 1.1 mV, and V1/2(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+ (IMTSES/IControl = 22 ± 1.6% and V1/2(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.
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