Steady-State Regulation of Secretory Cargo Export by Inositol Trisphosphate Receptors and Penta EF Hand Proteins

Abstract

ABSTRACTCa2+ release by inositol trisphosphate receptors (IP3Rs) regulates diverse physiological processes in many cell types. Notably, agonist-stimulated Ca2+ signaling involving IP3Rs can modulate ER export rates through activation of the Ca2+-dependent adaptor ALG-2. It is unknown, however, whether IP3Rs and ALG-2 regulate ER export rates at steady state. Here we report that partial depletion of IP3Rs from normal rat kidney (NRK) epithelial cells caused a marked increase of ER export of the transmembrane cargo VSV-G. Depletion of IP3R-3, the major IP3R isoform in NRK cells, had a larger impact on secretion than the other extant isoform, IP3R-l. Under standard growth conditions, spontaneous cytosolic Ca2+ oscillations usually occurred simultaneously in successive groups of contiguous cells, generating intercellular Ca2+ waves (ICWs) that moved across the monolayer. Unexpectedly, IP3R-3-depleted cells exhibited increased cell participation in this spontaneous signaling. IP3R-3-depleted cells also exhibited increased agonist-dependent Ca2+ signaling, suggesting a general potentiation of the remaining IP3R channels. Increased spontaneous signaling was accompanied by increased ALG-2 and decreased peflin at ER exit sites (ERES), resulting in increased targeting of COPII coat to ERES. Furthermore, the increased secretion rate associated with IP3R depletion required ALG-2. Unexpectedly, IP3R depletion also resulted in partial depletion of ER luminal Ca2+ stores, though this phenomenon was not involved in the secretion change. We conclude that in NRK cells, IP3Rs regulate spontaneous Ca2+ signaling that helps determine the basal secretion rate by modulating COPII targeting to ERES. Furthermore, the IP3R channel density and/or isoform composition significantly impacts this phenomenon.