Abstract
Ion channels in the cyclic nucleotide-binding domain (CNBD) family, including hyperpolarization-activated cyclic nucleotide-gated (HCN) channels and human ether-à-go-go-related gene (hERG) channels, play pivotal roles in regulating cardiac action potentials. HCN channels are uniquely activated by hyperpolarization, rather than depolarization, a critical mechanism for controlling the involuntary pacemaker activity of the heart. In contrast, hERG channels are depolarization-activated and mediate K(+) currents essential for action potential repolarization. Notably, certain hERG mutations, including those associated with long-QT syndrome, can induce biphasic activation by both hyperpolarization and depolarization. Despite the diverse voltage-dependent gating behaviors observed in CNBD channels, a unified mechanistic framework remains lacking. Here, we propose an allosteric model for their electromechanical coupling, featuring a single voltage-sensor transition coupled to two distinct conformational coupling modes between voltage-sensing and pore domains. With only three or four free parameters, this model recapitulates the biphasic U-shaped and bell-shaped conductance-voltage relationships commonly seen in CNBD channels. Fluorescence anisotropy-based homo-FRET experiments employing site-specifically incorporated noncanonical amino acids provide further support for the hypothesis, suggesting that the S5 helix movement plays a key role in hyperpolarization-dependent activation, while S4-S6 helix interactions are required for depolarization-dependent gating.