Structural determinants of the direct inhibition of GIRK channels by Sigma-1 receptor antagonist

G-protein-gated inward rectifier K+ (GIRK) channels play critical roles in the regulation of the excitability of cardiomyocytes and neurons and include GIRK1, GIRK2, GIRK3 and GIRK4 subfamily members. BD1047 dihydrobromide (BD1047) is one of the representative antagonists of multi-functional Sigma-1 receptor (S1R). In the analysis of the effect of BD1047 on the inhibition of Gi-coupled receptors by S1R using GIRK channel as an effector, we observed that BD1047 directly inhibits GIRK current even in the absence of S1R. Thus, we aimed to clarify the effect of BD1047 on GIRK channels and its structural determinants. By electrophysiological recordings in Xenopus oocytes, we observed that BD1047 directly inhibited the current of GIRK channels, producing a much stronger inhibition of GIRK4 channels compared to GIRK2. It also inhibited the ACh-induced native GIRK current in isolated rat atrial myocytes. Chimeric and mutagenesis studies of GIRK2 and GIRK4 combining with molecular docking analysis, demonstrate the importance of the Leu77 on the proximal N-terminal cytoplasmic region of GIRK4 for the inhibition by BD1047. The activator of GIRK channel, ivermectin, competed with BD1047 at Leu77 on GIRK4. This study provides us with a novel inhibitor of GIRK channels and information for developing pharmacological treatments for GIRK4 associated diseases. Key pointsO_LISigma-1 receptor antagonist, BD1047, directly inhibits the current of GIRK channels. It strongly inhibits GIRK4 channels but only weakly inhibits GIRK2. C_LIO_LIBD1047 inhibits the ACh-induced GIRK current in isolated rat atrial myocyte. C_LIO_LILeu77 on the proximal N-terminal of GIRK4 is essential for the inhibition by BD1047. C_LIO_LIBinding of BD1047 adjacent to Leu77, but not to Leu77Ile, was confirmed by molecular docking analysis. C_LIO_LIThe activator of GIRK channel, ivermectin, competes with BD1047 at Leu77 on GIRK4. C_LI