LRMP inhibits the cAMP-dependence of HCN4 channels by disrupting intramolecular signal transduction

Lymphoid restricted membrane protein (LRMP) is a specific regulator of the hyperpolarization-activated cyclic nucleotide-sensitive isoform 4 (HCN4) channel. LRMP prevents cAMP-dependent potentiation in HCN4 but the interaction domains, mechanisms of action, and basis for isoform-specificity remain unknown. Here we identify the domains of LRMP essential for regulation, we show that LRMP acts by disrupting the intramolecular signal transduction between cyclic nucleotide binding and gating, and we demonstrate that non-conserved regions in HCN4 are required for LRMP isoform-specificity. Using patch clamp electrophysiology and Förster resonance energy transfer (FRET), we show that the initial 227 residues of LRMP and the N-terminus of HCN4 are necessary for LRMP to interact with HCN4. We also found that the HCN4 N-terminus and HCN4-specific residues in the C-linker are necessary for regulation of HCN4 by LRMP. Taken together, these results suggest that LRMP modulates HCN4 through an isoform-specific interaction involving the N-terminals of both proteins, and that this interaction inhibits the transduction between cAMP binding and channel activation via an interface involving the N-terminus, C-linker, and S4-S5 linker of HCN4.