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 StoreOperated 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 SKF96365, 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.