Figure 4. SthK channel voltage dependence.

(A) SthK voltage sensor domain (grey) with the 4 positively-charged residues in S4 in blue and L54 in S2 in green. The boxes zoom into the coordination between R111 and countercharges in S3 and S2 (orange) and between K114 and countercharges in S3 and S2 (green). Experimental density shown in light grey at 6 σ (top) and 4 σ (bottom). Dot representation displays water accessible cavities in the voltage sensor calculated with the program HOLE (red: pore radius <1.15 Å; green: pore radius 1.15–2.30 Å; blue: pore radius >2.30 Å). (B) Representative single-channel recording traces from SthK at different voltages (indicated, top of traces) displaying different open probabilities (indicated, right). The dashed line indicates the closed channel level. The traces were filtered online at 1 kHz and additionally filtered offline at 500 Hz. The ligand concentration was 1 mM cAMP. (C) Open probability as a function of voltage for the SthK channel for which selected traces are displayed in (B). Arrow indicates the open probability value at 0 mV, which is the voltage the channels experience on the cryo-EM grid. This experiment was performed for at least 7 different bilayers containing single SthK channels in saturating cAMP concentrations, and a plot showing an average Po vs. voltage curve is shown in ref. Schmidpeter et al. (2018).

Figure 4—figure supplement 1. Voltage sensor domains in SthK and related channels.

Voltage sensor domains of SthK, HCN1, TAX-4 and KvAP (left to right) highlighting distribution of the charged residues (light blue) along S4 and selected countercharges (pink) in S2 and S3, and the charge transfer center (green) in S2. The protein is grey. PDB codes: apo SthK (6CJQ), cAMP HCN1 (5U6P), TAX-4 (5H3O), KvAP (1ORS).

Figure 4—figure supplement 2. SthK single-channel analysis.

All-amplitude histograms of representative current traces from SthK in 1 mM cAMP and −100 mV (A) and +100 mV (B). Inset in A shows the peak at 16 pA on a different scale, for better visualization. Dwell-time histograms of open (C, D) and closed (E, F) time intervals for the same traces analyzed in A and B. Voltage is −100 mV for C and E, and +100 mV for D and F. The open dwell times were fit with one exponential component (red) and the closed dwell times were fit with 3 exponential components (blue). The dead time was 180 µs and only intervals longer than two dead times were included in the analysis. The few intervals longer than 500 ms were removed before the exponential fitting of the closed dwell times histogram. (G) Open dwell time histograms for single-SthK recordings for increasing concentrations of cAMP. From left to right: 1, 5, 10, 20, and 1000 µM cAMP. All histograms were fit with one exponential component whose time constant increases overall with increasing concentrations of cAMP (from left to right τ = 0.9, 0.7, 4, 7, 9 ms, respectively). The open probabilities corresponding to the recordings from which these dwell-time distributions were extracted are, from left to right, 0.002, 0.01, 0.16, 0.32, 0.36. Voltage was +100 mV and traces were filtered at 1 kHz.