Extended Data Fig. 1: Characterization of HCN1SM and HCN2SM.

(a) Representative electrophysiological recordings (top) of HCN1SM (left) and HCN2SM (right) with voltage protocol. Tail currents (arrow) were collected at −130 mV and were used to generate the activation curves. (b) Normalized activation curves of HCN1SM (left) and HCN2SM (right) in the absence or presence of saturating concentrations (500 μM) of internal cAMP with a Boltzmann fit (red). Data points are mean ± s.e.m. (n = 5 patches). V_1/2 values for are HCN1SM −71.2 ± 0.4 mV without cAMP and −69.1 ± 0.5 mV with cAMP. V_1/2 values for are HCN2SM −105.2 ± 0.6 mV without cAMP and −84.6 ± 0.5 mV with cAMP). (c) Size exclusion chromatography (SEC) profiles of HCN1SM (grey) and HCN2SM (orange dashed). Triangles indicate the peak fraction (0.3 mL) used for single-molecule measurements. (d) Example fluorescence vs time trajectory of photobleaching eGFP-tagged HCN2SM tetramers via TIRFM. (e) Distributions of photobleaching steps overlaid with a maximum likelihood estimate of a zero-truncated binomial distribution (red) for a tetrameric complex with a probability (P) of observing eGFP (HCN1: P = 0.65, 95% CI [0.63, 0.67], n = 752; HCN1: P = 0.67, 95% CI [0.65, 0.69], n = 588).