Fluorescence Investigations of the Rate‐Limiting Step in the HCN Ion Channel Deactivation Pathway

Hyperpolarization‐ and cyclic nucleotide‐activated (HCN) ion channels support electrical oscillations of neuronal networks and help prevent absence seizures. The S4 helix of the voltage‐sensor (VS) region in HCN channels moves inwards upon hyperpolarization and this movement is coupled to pore opening. An open HCN channel can further be stabilized by cAMP binding to its C‐terminal region. Modifying the speed of the rate‐limiting step for HCN channel gating might help treat neuropathies, but the steps in the gating pathway are unclear. Specifically, it has been proposed that the depolarization‐dependent deactivation pathway of HCN channels includes a voltage‐independent pore closure step that becomes rate‐limiting at strong depolarizations. To clarify which step is rate‐limiting at different depolarizations, we performed voltage clamp fluorometry on mouse HCN2 channel derivatives expressed in Xenopus oocytes using a fluorophore attached to the N‐terminal region of the S4 helix. These results represent the first direct measurement of VS movements of an HCN2 channel. The fluorophore tracked depolarization‐dependent VS movements during deactivation that preceded pore closure significantly at strong depolarizations; the current decay rate was approximately 4‐fold slower than the fluorescence decay rate at +20 mV. We call this ratio of decay rates the “transmembrane coupling quotient”. At weaker depolarizations, VS movements still preceded pore closure, but less dramatically (transmembrane coupling quotient approximately 1.5‐fold at ‐60 mV). This voltage dependence of the transmembrane coupling quotient was cAMP‐independent, i.e., it remained similar even when cAMP binding was prevented by a mutation in the C‐terminal region. The voltage dependence of the transmembrane coupling quotient is consistent with a model of mammalian HCN channel deactivation where the VS undergoes movements that have a greater voltage dependence compared to pore closure movements. VS movements may serve as the rate‐limiting step for deactivation at weak depolarizations, but another step – perhaps pore closure – is the rate‐limiting step at strong depolarizations. The temporal association between VS movements and pore movements would thus be loose at strong depolarizations, and tight at weak depolarizations. Support or Funding Information Natural Sciences and Engineering Research Council (NSERC) Postgraduate Scholarship to K.E.A.M; Natural Sciences and Engineering Research Council (NSERC) Discovery Grant to E.C.Y