A Charged Residue in the HCN Channel C-Linker Stabilizes the Open State

Abstract

Hyperpolarization- and cyclic nucleotide-activated (HCN) channels are regulated by a voltage sensing domain (VSD) as well as a cyclic nucleotide binding domain (CNBD) coupled to the pore by a C-linker. We previously found a mutation in the VSD that slows deactivation kinetics by creating a highly stable open state (Open State Trapping, OST) that is dependent on cAMP binding; deletion of the CNBD and C-linker (ΔC) completely removed OST. I introduced point mutations in the C-linker in an OST mutant, and tested their effects on the stable open conformation. Deactivation kinetics of the mutants, and the ΔC construct, were studied in Xenopus laevis oocytes using two electrode voltage clamp, and fitted to an exponential decay equation. Arginine substitution of a conserved glutamate significantly decreased the deactivation time constant (τ) at +20 mV by ∼3.5 fold when compared to wild type. The same mutation shifted the isochronal deactivation midpoint voltage (Vdeact) by −41 mV. For ΔC, ΔVdeact was −55 mV relative to wild type and τ was ∼8 fold faster than wild type. Therefore, the charge reversal mutation removed a substantial portion of the OST effect on τ and the majority of the OST effect on Vdeact. In comparison, mutating the same residue to glutamine or cysteine partially removed the OST effects, with τ decreased by ∼1.5 fold and 3 fold respectively, and with ΔVdeact of −9 mV and −19 mV respectively. We conclude the native glutamate stabilizes the open state during OST, possibly due to C-linker interactions with other channel regions. Future research will explore how and when this stabilization occurs, for example if it can still occur in the absence of cAMP binding or of the OST mechanism.