Research Article SARAF modulates TRPC1, but not TRPC6, channel function in a STIM1-independent manner Letizia Albarrán, José J. López, Luis J. Gómez, Ginés M. Salido and Juan A. Rosado Department of Physiology (Cellular Physiology Research Group), University of Extremadura, Cáceres 10003, Spain Correspondence: Juan A. Rosado (jarosado@unex.es) 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-inde- pendent behaviour. The STIM1 inhibitor SARAF (for store-operated Ca 2+ entry (SOCE)- associated regulatory factor) modulates SOCE by interaction with the STIM1 region responsible for Orai1 activation (SOAR). Furthermore, SARAF modulates Ca 2+ entry through the arachidonate-regulated Ca 2+ (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 Ca 2+ 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 Ca 2+ entry evoked by agonists in TRPC6 overexpressing cells, as well as in Ca 2+ 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 Ca 2+ homeostasis. Introduction Since the characterization of the Drosophila trp gen [1] and subsequent identification of the first mam- malian homologue of the transient receptor potential (TRP) channels [2,3], the role of TRPC1 in agonist-induced Ca 2+ entry has been extensively investigated. TRPC1 forms cation channels activated by the occupation of receptors coupled to phosphatidylinositol 4,5-bisphosphate hydrolysis and subse- quent discharge of the intracellular Ca 2+ stores, mostly the endoplasmic reticulum (ER) [4,5]. TRPC1 channels have been reported to be involved in store-independent and store-operated Ca 2+ entry (SOCE), although this channel, as well as the other TRPC members, is unable to generate a current that resembles the Ca 2+ selective, store-operated, I CRAC . One of the critical pieces of evidence support- ing a role of TRPC1 in SOCE comes from studies, demonstrating the interaction and regulation of TRPC1 channels by the ER-Ca 2+ sensor, STIM1, both in native and transfected cell systems [6–10]. Current evidence has identified two types of Ca 2+ currents operated by Ca 2+ store depletion: I CRAC , mediated by the activation of Orai1 subunits by STIM1, and I SOC , involving Orai1, TRPC1 and STIM1 [11,12]. Accepted Manuscript online: 9 August 2016 Version of Record published: 11 October 2016 Received: 15 April 2016 Revised: 5 August 2016 Accepted: 9 August 2016 © 2016 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society 3581 Biochemical Journal (2016) 473 3581–3595 DOI: 10.1042/BCJ20160348 Downloaded from https://portlandpress.com/biochemj/article-pdf/473/20/3581/689598/bcj-2016-0348.pdf by guest on 16 June 2020