Calcium is a ubiquitous signaling molecule involved in various cellular processes, including fertilization, muscle contraction or synaptic transmission. There are two major sources for Ca2+: the extracellular media and intracellular Ca2+ stores, particularly the endoplasmic reticulum (ER). When intracellular stores are depleted, a signal is transmitted from a Ca2+ sensor protein that localizes to the ER membrane (STIM) to the plasma membrane to trigger the opening of the Ca2+ channel Orai. This mechanism, known as store-operated calcium entry (SOCE) activates multiple physiological processes and provides Ca2+ to the SERCA pump to refill the ER stores. To avoid unspecific Ca2+ diffusion in the cytosol, SOCE is a highly localized signal, spatially restricted to dense domains (clusters) created by protein-protein interactions between STIM and Orai. Ca2+-sensitive effectors that respond rapidly to Ca2+ influx through SOCE are therefore in the immediate vicinity of the cluster. We here describe a novel mechanism that expands this Ca2+ signaling microdomain through functional coupling of SOCE, SERCA and the IP3 receptor, to allow efficient activation of Ca2+-activated Chloride channels (CACC) localized away from SOCE clusters. When intracellular Ca2+ stores were emptied using ionomycin, a small CACC activation by SOCE was observed. Conversely, when the stores were depleted following injection of IP3 there was a significantly larger CACC activation (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 similar levels of Ca2+ influx lead to different CACC activation. The same mechanism could also be induced when IP3 production was stimulated by lysophosphatidic acid (LPA) and we did show that the cell membrane depolarization induced by LPA required SOCE activation, indicating a physiological function of the process in regulating membrane potential. Ca2+ injection experiments ruled out any increase in the CACC sensitivity to Ca2+, while imaging intracellular Ca2+ levels reflected the amplitude of the Cl- currents Analysis of the localization of the Xenopus CACC (TMEM16a) and of the ER calcium sensor STIM1 indicated that they did not co-localize but rather have a tendency to exclude each other following store depletion. Simultaneous expression of STIM1, Orai1 and SERCA2b tagged with fluorescent markers indicated that the three proteins co-localized in clusters at the plasma membrane following store depletion, creating a very restricted cytoplasmic volume where Ca2+ is increased prior to its pumping into the ER lumen. Finally, GFP-tagged IP3 receptors were not found to be enriched in the clusters. Our results show functional coupling between SOCE, SERCA and IP3 receptors to effectively activate CACCs in response to agonist induced store depletion. Here, Ca2+ 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 CACC. This mechanism extends the function of SOCE to the activation of channels that or not located in the immediate vicinity of the extracellular Ca2+ source without requiring large intracellular calcium events to target distant Ca2+ sensitive elements.


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