Ion channels in excitable cells function in macromolecular complexes in which auxiliary proteins modulate the biophysical properties of the pore-forming subunits. Hyperpolarization-activated, cyclic nucleotide-sensitive HCN4 channels are critical determinants of membrane excitability in cells throughout the body, including thalamocortical 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 proteins. Here, we report the discovery of a family of endoplasmic reticulum (ER) transmembrane proteins that associate with and modulate HCN4. Lymphoid-restricted membrane protein (LRMP, Jaw1) and inositol trisphosphate receptor-associated guanylate kinase substrate (IRAG, Mrvi1, and Jaw1L) are homologous proteins with small ER luminal domains and large cytoplasmic domains. Despite their homology, LRMP and IRAG have distinct effects on HCN4. LRMP is a loss-of-function modulator that inhibits the canonical depolarizing 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 trafficking 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 mechanistic understanding of HCN4 channel function, and as possible scaffolds for coordination of signaling pathways.

Caveolin proteins are involved in establishing membrane microstructure, lipid raft organization, and cell signaling. In the heart, caveolin-3 (Cav3) predominates. Inherited or disease-induced Cav3 loss increases risk of sudden cardiac death (SCD). We aimed to explore connections between Cav3 loss and arrhythmogenic changes in the ventricular action potential (AP) by investigating the Cav3 dependence of ionic currents. Drugs commonly used to disrupt or remove Cav3 in cultured cells exclude any compensatory process likely to occur in vivo. This motivated us to engineer a novel conditional Cav3 knockout (Cav3-/-) mouse that survives to adulthood. We isolated ventricular cells for electrophysiological experimentation. AP duration (APD90) was prolonged from 2454 ms in WT to 9659 ms in Cav3-/-, and several currents were affected. Reduced peak: L-type Ca 2þ current (ICaL), 21%; slow K þ current, 81%; transient outward K þ current, 57%; steady state outward K þ current (Iss), 43%. Late Na þ current was enhanced~10-fold. These changes were partially offsetting -preventing a simple account for the APD90 increase. To relate changes in currents to changes in the AP, we developed a computational representation of Cav3-/-based on the Morotti et al. mouse ventricular cell model and defined by fractional change in currents. Unexpectedly, the relatively small change in relatively small Iss caused 33% of total simulated AP prolongation. Though Iss conductance was reduced, peak Iss actually increased in the dynamic setting of the simulated AP. Early in the AP, lower Iss indirectly enhanced inward currents (importantly late ICaL) by extending the plateau phase, which in turn allowed Iss to more fully activate. This Iss/ ICaL process largely accounted for the pro-arrhythmic APD90 increase following Cav3 loss and is therefore a candidate target for normalizing SCD risk.

The pore-forming subunits of ion channels are regulated by auxiliary interacting proteins.Hyperpolarization-activated cyclic nucleotide-sensitive isoform 4 (HCN4) channels are critical determinants of electrical excitability in many types of cells including neurons and cardiac pacemaker cells. Here we report the discovery of two novel HCN4 regulatory proteins. Despite their homology, the two proteinslymphoid-restricted membrane protein (LRMP) and inositol trisphosphate receptor-associated guanylate kinase substrate (IRAG)have opposing effects on HCN4, causing loss-and gain-of-function, respectively. LRMP and IRAG are expected to play critical roles in regulation of physiological processes ranging from wakefulness to heart rate through their modulation of HCN4 channel function.

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