SARAF modulates TRPC1, but not TRPC6, channel function in a STIM1-independent manner

Canonical transient receptor potential-1 (TRPC1) is an almost ubiquitously expressed channel that plays a relevant role in cell function. As other TRPC members, TRPC1 forms receptor-operated cation channels that exhibit both STIM1-dependent and store-independent behaviour. The STIM1 inhibitor SARAF (for store-operated Ca²⁺ entry (SOCE)-associated regulatory factor) modulates SOCE by interaction with the STIM1 region responsible for Orai1 activation (SOAR). Furthermore, SARAF modulates Ca²⁺ entry through the arachidonate-regulated Ca²⁺ (ARC) channels, consisting of Orai1 and Orai3 heteropentamers and plasma membrane-resident STIM1. While a role for STIM1-Orai1-mediated signals has been demonstrated, the possible role of SARAF in TRPC1 function remains unknown. Here, we provide evidence for the interaction of SARAF with TRPC1, independently of STIM1 both in STIM1-deficient NG115-401L cells and SH-SY5Y cells endogenously expressing STIM1. Silencing of SARAF expression in STIM1-deficient cells demonstrated that SARAF plays a negative regulatory role in TRPC1-mediated Ca²⁺ entry. The interaction of SARAF with TRPC1 in STIM1-deficient cells, as well as with the TRPC1 pool not associated with STIM1 in STIM1-expressing cells was enhanced by stimulation with the physiological agonist ATP. In contrast with TRPC1, we found that the interaction between SARAF and TRPC6 was constitutive rather than inducible by agonist stimulation. Furthermore, we found that SARAF expression silencing was without effect on Ca²⁺ entry evoked by agonists in TRPC6 overexpressing cells, as well as in Ca²⁺ influx evoked by the TRPC6 activator Hyp9. These findings provide evidence for a new regulator of TRPC1 channel function and highlight the relevance of SARAF in intracellular Ca²⁺ homeostasis.