The proximal N terminus of IRAG is required for potentiation of HCN4 channels.

The inositol triphosphate-associated, ER transmembrane proteins inositol triphosphate receptor-associated guanylate kinase substrate (IRAG) and lymphoid restricted membrane protein (LRMP) are isoform-specific regulators of the hyperpolarization-activated cyclic nucleotide-sensitive isoform 4 (HCN4) channel. LRMP prevents cAMP-dependent potentiation of HCN4, while IRAG mimics the effect of cAMP on the channel. We previously showed that regulation by LRMP requires both the N terminus of HCN4 as well as a unique set of residues in the cAMP transduction center of HCN4, which is comprised of the N-terminal HCN domain (HCND), the C-linker, and the S4-S5 linker. However, it remains unknown if the homologous IRAG requires similar structural features to mimic cAMP-dependent potentiation, or if the site and mechanism of action are different between the two regulators. Using patch-clamp electrophysiology, we determined that the initial 43 amino acids of IRAG are necessary and sufficient to confer regulation of HCN4. Similar to LRMP, IRAG also requires a portion of the N terminus of HCN4 to confer its regulatory effects. Also like LRMP, two point mutations in the C-linker region, near where the C-linker of one subunit contacts its neighboring subunit, were able to eliminate the effect of IRAG. Taken together, these findings suggest a model whereby IRAG and LRMP make complex interactions with a number of intracellular domains of HCN4. For both proteins, regulation of HCN4 requires a portion of the N terminus and at least some portions of the structural domains required to convert the binding of cAMP to the cyclic nucleotide-binding domain (CNBD) to a change in channel gating.