Differential Sensitivity of TRPC- and ORAI-Mediated Calcium Entries to 1-[2-(4-Methoxyphenyl)-2-[3-(4-methoxyphenyl)propoxy]ethyl]imidazole Chloride (SKF-96365)

Abstract

Background and Purpose: SKF-96365 is widely used as a broad-spectrum inhibitor of calcium entry. It was initially identified as a Receptor-Operated Ca2+ Entry (ROCE) blocker and was later shown to inhibit ORAI1−STIM1-mediated Store- Operated Ca2+ Entry (SOCE). However, its selectivity for TRPC versus the ORAI channels remains unclear. Experimental Approach: To examine the selectivity of SKF-96365, we evaluated its effects on SOCE and ROCE in wild-type and TRPC hepta-KO mouse embryonic fibroblasts (MEFs), as well as on TRPCmediated OAG-induced calcium entry in HEK293 cells overexpressing TRPC3, TRPC6, or TRPC7. Additional assays were conducted on HEK293 cells expressing the muscarinic M5 receptor (M5R). Half-maximal inhibitory concentration (IC50) values were determined under all conditions. Key Results: SKF-96365 suppressed thapsigargin (Tg)-induced SOCE similarly in wild-type and TRPC hepta-KO MEFs, with IC50 values around 4−5 μM. Comparable inhibition was observed for carbachol (CCh)-activated ROCE in TRPC-deficient cells. In contrast, OAG-activated Ca2+ entry by TRPC3/6/7 was only weakly inhibited, with IC50 values exceeding 100 μM. Notably, TRPC-mediated Ca2+ entry was unaffected by CRAC channel blockers or ORAI coexpression, confirming its independence of SOCE mechanisms Conclusions and Implications: Our findings demonstrate that ORAI-mediated SOCE is approximately 25-fold more sensitive to SKF-96365 than TRPC-mediated calcium entry. GSK-7975A further confirmed the ORAI selectivity by blocking SOCE without affecting TRPC channels. These results clarify the pharmacological profile of SKF- 96365, confirming its primary action on ORAI channels and highlight the need for concentration-aware interpretation in calcium signaling studies, particularly in the context of CRAC channel-related disorders.