SKF96365 is a selective Inhibitor of Ca2+ release-activated Ca2+ channels (CRAC channels) rather than a TRPC channel-selective inhibitor.

SEBASTIÁN SUSPERREGUY; KARINA FORMOSO; JULIETA MANSILLA RICARTTI; LUTZ BIRNBAUMER

Congreso; LXVI Reunión Anual de la Sociedad Argentina de Investigación Clínica (SAIC); 2021

SAIC

Backgroundand puroposeSKF-96365 (SKF) has been broadly used as a tool todiscern TRPC participation in the Store Operated Ca2+ Entry (SOCE)phenomenon, being generally accepted that SKF96365 is a TRPC channel-selectiveinhibitor and is sold as such (Sigma Aldrich cat #S7809, Tocris cat #1147, Alomone cat #S-175). However, SKF was also used as a non-selective blocker of SOCE.Considering that the effect of SKF96365 onthe SOCE mechanism remains controversial and taking advantage ofthe TRPC 7KO MEFs, we hypothesized that SK96365blocks SOCE (or ROCE) by blocking ORAI1-based channels instead of inhibiting TRPC-based channels.Experimental approachTRPC7KO MEF cells, a unique cell model in which the seven TRPC are absent, wereused to elucidate the effect of SKF 96365 on native ORAI1-based channels. Inaddition TRPC 7KO and ORAI1 KO MEFs were transfected with ORAI1 plus STIM1 toconstitute CRAC channels by heterologous expression.. TRPC-based channels weretest for SKF sensitivity in HEK293 in which TRPC6 was stably expressed and Ca2+influx was activated with OAG. SKF effect was always studied by changes inTg-activated Ca2+ influx monitored by FUR2-AM method and theexpression level of TRPC, ORAI and STIM were analyzed by RT-PCR.KeyresultsWe found Tg-evoked SOCE was nearly 50% suppressed by5µM SKF in TRPC 7KO MEF cells. In addition, the increment in Tg-evoked SOCEproduced by the transfection of ORAI1 in ORAI1 KO MEFs and ORAI1 plus STIM1 inTRPC 7KO MEFs was completely prevented by 5µM SKF. OAG-activated Ca2+entry in HEK293 stably transfected with TRPC6 was insensitive at ORAI1overexpression and GSK7975A treatment in contrast to TRPC7KO MEFs in whichTg-activated Ca2+ influx was sensitive to GSK7975A. Finally, we showedTRPC-based Ca2+ influx in HEK293-TRPC6 was barely reduced by 10 µMSKF and 50 µM dose was needed to block OAG-activated Ca2+ influx.Conclusions and implicationsIn conclusion, we report convincing evidence showingthat SKF96365 acts as a blocker of CRACchannels in MEF cells having no TRPC. In addition, SKF  showed higherpotency to block CRAC channels compared to its potency against TRPC channels. This finding suggests we should be in cautious when analyze results whereSKF is used as a pharmacologic agent to asses TRPC channels activity.