REGULATION OF INOSITOL TRISPHOSPHATE RECEPTORS BY LUMINAL CA2+ CONTRIBUTES TO QUANTAL CA2+ MOBILIZATION

The quantal behaviour of inositol trisphosphate (InsP(3)) receptors al lows rapid graded release of Ca2+ from intracellular stores, but the m echanisms are unknown, In Ca2+-depleted stores loaded with Fura 2, Ins P(3) caused concentration dependent increases in the rates of fluoresc ence quench by Mn2+ that were unaffected by prior incubation with InsP (3), indicating that InsP(3) binding did not cause desensitization. Wh en Fura 2 was used to report the luminal free [Ca2+] after inhibition of further Ca2+ uptake, submaximal concentrations of InsP(3) caused ra pid, partial decreases in fluorescence ratios, Subsequent addition of a maximal InsP(3) concentration caused the fluorescence to fall to wit hin 5% of that recorded after ionomycin. Addition of all but the lowes t concentrations of InsP(3) to stores loaded with the lower affinity i ndicator, Calcium Green-5N, caused almost complete emptying of the sto res at rates that increased with InsP(3) concentration, The lowest con centration of InsP(3) (10 nM) slowly emptied similar to 80% of the sto res, but within 3 min the rate of Ca2+ release slowed leaving similar to 7 mu M Ca2+ within the stores, which was then rapidly released by a maximal InsP(3) concentration, In stores co-loaded with both indicato rs, InsP(3)-evoked Ca2+ release appeared quantal with Fura 2 and large ly non-quantal with Calcium Green-5N; the discrepancy is not, therefor e, a direct effect of the indicators, The fall in luminal [Ca2+] after activation of InsP(3) receptors may, therefore, cause their inactivat ion, but only after the Ca2+ content of the stores has fallen by simil ar to 95% to less than or equal to 10 mu M.