Abstract 2577: The Cytoplasmic cAMP-Sensing Domain of Pacemaker HCN Channels Uses Two Structurally Distinct Mechanisms to Regulate Voltage-Gating
Hyperpolarization-activated cation (HCN) channels in the sinoatrial node produce Ih current, providing a “depolarization reserve” in diastole to prevent excessive hyperpolarizations and to accelerate rhythmic firing. The cytoplasmic cAMP-sensing domain (CSD) of HCN channels inhibits the intrinsic voltage-gating when unliganded; cAMP binding relieves this autoinhibition, depolarizing the V1/2. Cyclic AMP also slows deactivation kinetics, but does this enhanced kinetic trapping of the open state correlate with the thermodynamic effect of autoinhibition relief? We previously tested homomers of HCN4, the predominant cardiac HCN subtype, using inside-out patch-clamp; we showed that a mutation in the S4 helix (K381E) leaves voltage-sensing unchanged, but magnifies cAMP-dependent slowing of deactivation (τdeact at −40 mV increases 10-fold with cAMP). These gating properties were preserved when cytoplasmic regions of HCN4 K381E were replaced with HCN2 sequence. In this new study we tried to mimic cAMP binding in our K381E channel by deleting the CSD. This abolished autoinhibition (depolarizing V1/2 by 25 mV), but paradoxically disrupted kinetic trapping of the open state, with τdeact 3-fold faster than in unliganded intact channels. We also tested intact K381E channels with a mutation in the CSD (K472E, HCN2 numbering), located in the predicted interface between two neighbouring CSD’s. This CSD mutation preserved relief of autoinhibition, but disrupted open state trapping of cAMP-liganded channels, and reduced cAMP-dependence of deactivation (τdeact increasing <2-fold with cAMP). We conclude that adding cAMP to intact HCN channels enhances kinetic open state trapping by a novel mechanism that is not simply the removal of autoinhibitory structures. Rather, specific structures in the cAMP-liganded CSD contribute actively to delaying Ih deactivation.
This research has received full or partial funding support from the American Heart Association, National Center.