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. Author manuscript; available in PMC: 2022 Jan 1.
Published in final edited form as: Nature. 2021 Jun 30;595(7868):606–610. doi: 10.1038/s41586-021-03686-x

Fig. 2: fcAMP binds non-cooperatively to both HCN isoforms.

Fig. 2:

Normalized state occupancy distributions for HCN1SM (a) and HCN2SM (b) at various fcAMP concentrations with total number of molecules (n), data points (m) and binomial success rate for four subunits (P) indicated (see also Supplementary Table 2 and Extended Data Fig. 5). (c) Sequential ligand binding model (Model 1 in Supplementary Table 4). White squares indicate unbound (U), black squares indicate bound (B), and L represents ligand. Optimized transition rates (mean ± s.e.m.) for sequential binding (Supplementary Table 5) for HCN1SM (d) and HCN2SM (e) with a linear fit (black solid). Dashed lines indicate expected rates from constrained and sequential non-cooperative binding model (Model 2 in Supplementary Table 4). (f) Equilibrium association constants (KA) for each ligand binding step (mean ± s.e.m). Dashed lines indicate fits from Model 2. Previously reported KA values for fcAMP binding to HCN2 using PCF of open (−130 mV)2 and closed channels (−30 mV)3 shown for comparison (grey). For d, e, f, parameters were fit to all events across all fcAMP concentrations (HCN1SM: 739 molecules, 180,480 events; HCN2SM: 444 molecules, 82,030 events).