Regulation of Ca2+-release-activated Ca2+ current (I-crac) by ryanodine receptors in inositol 1,4,5-trisphosphate-receptor-deficient DT40 cells

Persistence of capacitative Ca2+ influx in inositol 1,4,5-trisphosphate (IP 3) receptor (IP3R)-deficient DT40 cells (DT40(IP3R-/-)) raises the question of whether gating of Ca2+ release activated Ca2+ current (I-crae) by confo rmational coupling to Ca2+-release channels is a general mechanism of gatin g of these channels. In the present work we examined the properties and mec hanism of activation of Ca2+ current in wild-type and DT40(IP3R-/-) cells. In both cell types passive depletion of internal Ca2+ stores by infusion of EGTA activated a Ca2+ current with similar characteristics and time course . The current was highly Ca2+-selective and showed strong inward rectificat ion, all typical of The activator of I-crae ryanodine receptor (RyR), cADP- ribose (cADPR), facilitated activation of I-crae, and the inhibitors of the RyRs, 8-N-cADPR, ryanodine and Ruthenium Red, all inhibited I-crae, activa tion in DT40(IP3R-/-) cells, even after complete depletion of intracellular Ca2+ stores by ionomycin. Wild-type and DT40(IP3R-/-) cells express RyR is oforms I and 3. RyR levels were adapted in DT40(IP3R-/-) cells to a lower R yR3/RyR1 ratio than in wild-type cells. These results suggest that IP(3)Rs and RyRs can efficiently gate I-crae in DT40 cells and explain the persiste nce of I-crae gating by internal stores in the absence of IP(3)Rs.