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 miodification 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 wildtype (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 (similar to7- and similar to3-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 (Cys(303), cys(318), and Cys(347) 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.