Isoform-Specific Regulation of HCN4 Channels by a Family of Novel Interacting Proteins

Abstract

The hyperpolarization-activated cyclic-nucleotide sensitive channel, HCN4, is a key contributor to the pacemaker function of the cardiac sinoatrial node (SAN). Sympathetic nervous system stimulation of the SAN produces cAMP, which is an important physiological regulator of HCN4 channels. cAMP binding increases the open probability and slows closing of HCN4, ultimately contributing to the fight-or-flight increase in heart rate. In contrast, the neuronal HCN channel accessory protein, TRIP8b, decreases cAMP sensitivity of activation and deactivation by binding to the cyclic-nucleotide binding domain of HCN channels. Here, we have identified two novel, isoform-specific modulators of HCN4 (HINT1 and HINT2), which have distinct effects on channel function. Similar to TRIP8b, HINT1 decreases the cAMP sensitivity of HCN4 channel activation. Conversely, HINT2 increases HCN4 channel open probability in the absence of cAMP, but has no effect on channel gating in the presence of cAMP. In contrast to TRIP8b which can regulate multiple HCN channel isoforms, HINT1/2 appear to be specific for HCN4, having no effect on HCN2. Furthermore, HINT1/2 affect only channel activation without altering cAMP-dependent slowing of deactivation. These data suggest that HINT1 and HINT2 do not directly compete for the cAMP binding site of HCN4 and that their mechanisms of action differ from those of TRIP8b. In contrast to TRIP8b, which is expressed exclusively in the brain, qPCR and western blotting experiments show that HINT1 and HINT2 transcript as well as at least HINT2 protein are present in the SAN. Our results suggest that HINT1 and HINT2 are novel, isoform-specific regulators of HCN4 channel function that likely play a physiologically relevant role in control of pacemaker function and heart rate.