oa "Ca2+ funneling": Functional coupling between SOCE, SERCA and IP3 receptors enhances Ca2+ signaling efficiency in activating the Ca2+-activated Cl channels as downstream effectors.

Store-operated calcium entry (SOCE) is a ubiquitous Ca2+ influx pathway leading to a sustained, low amplitude Ca2+ signal that activates multiple physiological process including gene transcription and secretion, and contributes to store refilling. We here describe a novel mechanism, that we call "Ca2+ funneling", which allows efficient activation by SOCE of Calcium-activated chloride channels (CaCl2) in the xenopus oocyte to regulate the cell's membrane potential. When intracellular Ca2+ stores were emptied using ionomycin, a small CaCl2 current is observed. Conversely, when the stores were depleted following injection of IP3 there was a significantly larger CaCl2 activation than with ionomycin (2.5 ± 0.5 nA, n=14 vs 0.08 ± 0.02 nA, n=20). Surprisingly the size of the SOCE current was similar in both conditions, indicating that an equivalent calcium influx was leading to different CaCl2 activation. Ca2+ injection experiments ruled out any change in the CaCl2 sensitivity to Ca2+. The CaCl2 activated by IP3 could be inhibited by La3+, BTP-2, 2-APB and heparin showing its dependence on both store-operated channels as well as IP3 receptor activation. The same mechanism could also be induced when IP3 production was stimulated by lysophosphatidic acid (LPA). Moreover, we could show that the sustained phase of the biphasic depolarization induced by LPA required SOCE activation. Analysis of the cellular localization of the xenopus TMEM16a (CaCl2) and of the ER calcium sensor STIM1 revealed a tendency of the two proteins to exclude each other following store depletion. We therefore propose the existence of a functional coupling between SOCE, SERCA and IP3 receptors to effectively activate CaCl2 in response to an agonist-induced store depletion. We refer to this coupling as "Ca2+ funneling" where calcium entering the cell through SOCE is rapidly taken up into the ER by SERCAs and released through open IP3 receptors close to its target, the Ca2+-activated Cl channels.