Isoform-specific regulation of HCN4 channels by a family of endoplasmic reticulum proteins

Ion channels in excitable cells function in macromolecular com-plexes in which auxiliary proteins modulate the biophysical properties of the pore-forming subunits. Hyperpolarization-activated, cyclic nucleotide-sensitive HCN4 channels are critical determinants of mem-brane excitability in cells throughout the body, including thalamocort-ical neurons and cardiac pacemaker cells. We previously showed that the properties of HCN4 channels differ dramatically in different cell types, possibly due to the endogenous expression of auxiliary pro-teins. Here, we report the discovery of a family of endoplasmic re-ticulum (ER) transmembrane proteins that associate with and modulate HCN4. Lymphoid-restricted membrane protein (LRMP, Jaw1) and inositol trisphosphate receptor-associated guanylate ki-nase substrate (IRAG, Mrvi1, and Jaw1L) are homologous proteins with small ER luminal domains and large cytoplasmic domains. De-spite their homology, LRMP and IRAG have distinct effects on HCN4. LRMP is a loss-of-function modulator that inhibits the canonical depo-larizing shift in the voltage dependence of HCN4 in response to the binding of cAMP. In contrast, IRAG causes a gain of HCN4 function by depolarizing the basal voltage dependence in the absence of cAMP. The mechanisms of action of LRMP and IRAG are independent of traf-ficking and cAMP binding, and they are specific to the HCN4 isoform. We also found that IRAG is highly expressed in the mouse sinoatrial node where computer modeling predicts that its presence increases HCN4 current. Our results suggest important roles for LRMP and IRAG in the regulation of cellular excitability, as tools for advancing mecha-nistic understanding of HCN4 channel function, and as possible scaf-folds for coordination of signaling pathways.