Differential regulation of Ca²⁺ release-activated Ca²⁺ influx by heterotrimeric G proteins

The least understood aspect of the agonist.induced Ca²⁺ signal is the activation and regulation of the Ca²⁺ release-activated Ca²⁺ influx (CRAC) across the plasma membrane. To explore the possible role of heterotrimeric G proteins in the various regulatory mechanisms of CRAC, continuous renal epithelial cell lines stably expressing α₁₃ and the constitutively active α(qQ209L) were isolated and used to measure CRAC activity by the Mn²⁺ quench technique. Release of intracellular Ca²⁺ by agonist stimulation or thapsigargin was required for activation of CRAC in all cells. Although the size of the internal stores was similar in all cells, CRAC was 2-3, fold higher in α₁₃- and α(qQ209L) -expressing cells. However, the channel was differentially regulated in the two cell types. Incubation at low [Ca²⁺](i), inhibition of the NOS pathway, or inhibition of tyrosine kinases inhibited CRAC activity in α₁₃ but not α(qQ2O9L) cells. Treatment with okadaic acid prevented inhibition of the channel by low [Ca²⁺](i) and the protein kinase inhibitors in α₁₃ cells. These results suggest that expression of α(qQ2O9L) dominantly activates CRAC by stabilizing a phosphorylated state, whereas expression of α₁₃ makes CRAC activation completely dependent on phosphorylation by several kinases. G proteins may also modulate CRAC activity independently of the phosphorylation/dephosphorylation state of the pathway to increase maximal CRAC activity. Furthermore, our results suggest a general mechanism for regulation of CRAC that depends on coupling of receptors to specific G proteins.