THE HIGH-AFFINITY STATE OF INOSITOL 1,4,5-TRISPHOSPHATE RECEPTOR IS AFUNCTIONAL-STATE

Inositol 1,4,5-trisphosphate (InsP3) is a second messenger responsible for the rapid and discontinuous release of Ca2+ from intracellular st ores. In this study, the effects of the sulfhydryl reagent thimerosal were investigated on Ca2+ mobilization and on InsP3 binding. Thimerosa l was shown to release Ca2+, in a dose-dependent manner, with an EC50 of 135.8 +/- 5.2 muM, from bovine adrenal cortex microsomes. Thimerosa l-induced Ca2+ release was not prevented by heparin (250 mug/ml), ruli ng out a participation of InsP3 receptor in that effect. The slow rate of thimerosal-induced Ca2+ release rather suggested an inhibition of microsomal Ca2+ ATPase. At submaximal concentration, thimerosal (100 m uM) was also shown to potentiate the release of Ca2+ induced by InsP3. Dose-response experiments revealed that thimerosal enhanced the appar ent affinity of InsP3 by a factor 2.21 +/- 0.28, without modifying the maximal amount of Ca2+ released by InsP3. Thimerosal also enhanced, i n a dose-dependent manner, [H-3]InsP3 binding to adrenal cortex micros omes (EC50 = 43.3 +/- 7.6 muM). A similar effect was also observed on [H-3]InsP3 binding to solubilized receptors, suggesting a direct modif ication of the receptor protein by thimerosal. The effects of thimeros al on Ca2+ release and [H-3]InsP3 binding were abolished in the presen ce of the reducing agent dithiothreitol (1 mM), suggesting a modificat ion by thimerosal of specific thiol groups on these microsomal protein s. Scatchard analysis revealed that thimerosal (100 muM) increased Ins P3 receptor affinity by 1.87 +/- 0.26-fold. Kinetic analysis indicated that this increased affinity was due to an enhancement of InsP3 assoc iation rate constant. The concomitant increases of binding affinity an d Ca2+ releasing potency suggest that the high affinity state of InsP3 receptor is a functional state.