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.

可激发细胞中的离子通道在大分子复合物中起作用，其中辅助蛋白质调节成孔亚基的生物物理特性。超极化激活的环状核苷酸敏感的HCN4通道是全身细胞（包括丘脑皮层神经元和心脏起搏器细胞）中膜兴奋性的关键决定因素。我们先前表明，HCN4通道的特性在不同的细胞类型中有显着差异，这可能是由于辅助蛋白的内源性表达所致。在这里，我们报告发现的内质网（ER）跨膜蛋白家族与HCN4关联并对其进行调节。淋巴限制膜蛋白（LRMP，Jaw1）和肌醇三磷酸受体相关的鸟苷酸激酶底物（IRAG，Mrvi1，和Jaw1L）是具有小的ER内腔结构域和大的胞质结构域的同源蛋白。尽管它们具有同源性，但LRMP和IRAG对HCN4具有明显的作用。LRMP是一种功能丧失的调制器，可响应cAMP的结合抑制HCN4电压依赖性的典型去极化移位。相反，IRAG通过在不存在cAMP的情况下使基础电压依赖性去极化来引起HCN4功能的获得。LRMP和IRAG的作用机制与运输和cAMP结合无关，并且对HCN4亚型具有特异性。我们还发现IRAG在小鼠窦房结中高表达，在那里计算机建模预测其存在会增加HCN4电流。我们的结果表明LRMP和IRAG在调节细胞兴奋性中起重要作用，