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 (IP₃) receptor (IP₃R)-deficient DT40 cells (DT40^IP3R-/-) raises the question of whether gating of C²⁺-release activated Ca²⁺ current (I_crac) by conformational coupling to Ca²⁺-release channels is a general mechanism of gating of these channels. In the present work we examined the properties and mechanism of activation of I_crac 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 rectification, all typical of I_crac. The activator of ryanodine receptor (RyR), cADP-ribose (cADPR), facilitated activation of I_crac, and the inhibitors of the RyRs, 8-N-cADPR, ryanodine and Ruthenium Red, all inhibited I_crac activation in DT40^IP3R-/- cells, even after complete depletion of intracellular Ca²⁺ stores by ionomycin. Wild-type and DT40^IP3R-/- cells express RyR isoforms 1 and 3. RyR levels were adapted in DT40^IP3R-/- cells to a lower RyR3/RyR1 ratio than in wild-type cells. These results suggest that IP₃Rs and RyRs can efficiently gate I_crac in DT40 cells and explain the persistence of I_crac gating by internal stores in the absence of IP₃Rs.