Steady-state regulation of COPII-dependent secretory cargo sorting by inositol trisphosphate receptors, calcium, and penta EF hand proteins

Recently, we demonstrated that agonist-stimulated Ca²⁺ signaling involving IP3 receptors modulates ER export rates through activation of the penta-EF Hand proteins apoptosis-linked gene-2 (ALG-2) and peflin. It is unknown, however, whether IP3Rs and penta-EF proteins regulate ER export rates at steady state. Here we tested this idea in normal rat kidney epithelial cells by manipulation of IP3R isoform expression. Under standard growth conditions, spontaneous cytosolic Ca²⁺ oscillations occurred simultaneously in successive groups of contiguous cells, generating intercellular Ca²⁺ waves that moved across the monolayer periodically. Depletion of IP3R-3, typically the least promiscuous IP3R isoform, caused increased cell participation in intercellular Ca²⁺ waves in unstimulated cells. The increased spontaneous signaling was sufficient to cause increased ALG-2 and COPII coat subunit Sec31A and decreased peflin localization at ER exit sites, resulting in increased ER-to-Golgi transport of the COPII client cargo VSV-G. The elevated ER-to-Golgi transport caused greater concentration of VSV-G at ER exit sites and had reciprocal effects on transport of VSV-G and a bulk-flow cargo, though both cargos equally required Sec31A. Inactivation of client cargo sorting using 4-phenylbutyrate had opposing reciprocal effects on client and bulk-flow cargo and neutralized any effect of ALG-2 activation on transport. This work extends our knowledge of ALG-2 mechanisms and indicates that in normal rat kidney cells, IP3R isoforms regulate homeostatic Ca²⁺ signaling that helps determine the basal secretion rate and stringency of COPII-dependent cargo sorting.