GSK-7975-A

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.

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 TRPC-mediated 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.

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.

Urethral smooth muscle cells (USMC) contract to occlude the internal urethral sphincter during bladder filling. Interstitial cells also exist in urethral smooth muscles and are hypothesized to influence USMC behaviours and neural responses. These cells are similar to Kit⁺ interstitial cells of Cajal (ICC), which are gastrointestinal pacemakers and neuroeffectors. Isolated urethral ICC-like cells (ICC-LC) exhibit spontaneous intracellular Ca²⁺ signalling behaviours that suggest these cells may serve as pacemakers or neuromodulators similar to ICC in the gut, although observation and direct stimulation of ICC-LC within intact urethral tissues is lacking. We used mice with cell-specific expression of the Ca²⁺ indicator, GCaMP6f, driven off the endogenous promoter for Kit (Kit-GCaMP6f mice) to identify ICC-LC in situ within urethra muscles and to characterize spontaneous and nerve-evoked Ca²⁺ signalling. ICC-LC generated Ca²⁺ waves spontaneously that propagated on average 40.1 ± 0.7 μm, with varying amplitudes, durations, and spatial spread. These events originated from multiple firing sites in cells and the activity between sites was not coordinated. ICC-LC in urethra formed clusters but not interconnected networks. No evidence for entrainment of Ca²⁺ signalling between ICC-LC was obtained. Ca²⁺ events in ICC-LC were unaffected by nifedipine but were abolished by cyclopiazonic acid and decreased by an antagonist of Orai Ca²⁺ channels (GSK-7975A). Phenylephrine increased Ca²⁺ event frequency but a nitric oxide donor (DEA-NONOate) had no effect. Electrical field stimulation (EFS, 10 Hz) of intrinsic nerves, which evoked contractions of urethral rings and increased Ca²⁺ event firing in USMC, failed to evoke responses in ICC-LC. Our data suggest that urethral ICC-LC are spontaneously active but are not regulated by autonomic neurons.