Defining the interactions between HCN4 and the regulatory proteins IRAG and LRMP.

Abstract

Hyperpolarization-activated, cyclic nucleotide-sensitive HCN4 channels are the molecular determinants of the funny (If) current, a mixed Na+/K+ current that controls pacemaking in cardiac cells. In the sinoatrial node (SAN), an increase in If in response to cAMP contributes to an increase in action potential (AP) firing rate and consequently heart rate. We recently discovered two related modulators of HCN4 function: lymphoid-restricted membrane protein (LRMP) and inositol trisphosphate receptor-associated guanylate kinase substrate (IRAG). Despite homology between these two proteins, LRMP inhibits the depolarizing shift in the voltage dependence of activation of HCN4 in response to cAMP while IRAG shifts the basal voltage dependence in the depolarizing direction. Even with a clear picture of the functional effects that these two proteins exert on HCN4, the domains responsible for the interactions are completely unknown. Here, we pair a FRET two-hybrid approach with patch clamp electrophysiology to determine which domains of LRMP, IRAG, and HCN4 are involved in complex formation. Both LRMP and IRAG have small ER luminal domains, ER transmembrane segments, and a large cytoplasmic domain that likely interacts with an intracellular portion of HCN4 channels. By co-expressing cerulean-tagged cytosolic fragments of HCN4 with citrine-tagged fragments of LRMP and IRAG, we determine which regions on each protein are required for complex formation using FRET. We then test the functional effect of removing these critical domains on the regulation of the channel by each protein using patch clamp electrophysiology. Our preliminary results suggest that the N-terminus of HCN4 is critical for both LRMP and IRAG regulation of the channel. Moreover, the first 230 amino acids of LRMP is sufficient to bind HCN4. These results shed light on a novel amino terminus-dependent mechanism for regulating the gating of HCN4 channels.