DIFFERENTIAL REGULATION OF CA2-ACTIVATED CA2+ INFLUX BY HETEROTRIMERIC G-PROTEINS( RELEASE)

The least understood aspect of the agonist-induced Ca2+ signal is the activation and regulation of the Ca2+ release-activated Ca2+ influx (C RAC) across the plasma membrane. To explore the possible role of heter otrimeric G proteins in the various regulatory mechanisms of CRAC, con tinuous renal epithelial cell lines stably expressing alpha(13) and th e constitutively active alpha(qQ209L) were isolated and used to measur e CRAC activity by the Mn2+ quench technique. Release of intracellular Ca2+ by agonist stimulation or thapsigargin was required for activati on of CRAC in all cells. Although the size of the internal stores was similar in all cells, CRAC was 2-3-fold higher in alpha 13- and alpha( qQ209L)-expressing cells. However, the channel was differentially regu lated in the two cell types. Incubation at low [Ca2+](i), inhibition o f the NOS pathway, or inhibition of tyrosine kinases inhibited CRAC ac tivity in alpha(13) but not alpha(qQ209L) cells. Treatment with okadai c acid prevented inhibition of the channel by low [Ca2+], and the prot ein kinase inhibitors in alpha(13) cells. These results suggest that e xpression of alpha(qQ209L) dominantly activates CRAC by stabilizing a phosphorylated state, whereas expression of alpha(13) makes CRAC activ ation completely dependent on phosphomylation by several kinases. G pr oteins may also modulate CRAC activity independently of the phosphoryl ation/dephosphorylation state of the pathway to increase maximal CRAC activity. Furthermore, our results suggest a general mechanism for reg ulation of CRAC that depends on coupling of receptors to specific G pr oteins.