Journal of Biological Chemistry vol:271 issue:21 pages:12287-93
We developed a unidirectional 45Ca2+ efflux technique in which 60 cumulative doses of inositol 1,4,5-trisphosphate (InsP3), each lasting 6 s, were subsequently added to permeabilized A7r5 cells. This technique allowed an accurate determination of the threshold for InsP3 action, which was around 32 nM InsP3 under control conditions. The InsP3-induced Ca2+ release was characterized by an initial rapid phase, after which the normalized rate progressively decreased. The slowing of the release was associated with a shift of the threshold to higher InsP3 concentrations. Stimulatory concentrations of thimerosal (10 microM) shifted the threshold to 4.5 nM InsP3 and increased both the cooperativity and the maximal normalized rate of Ca2+ release. This low threshold was maintained when the thimerosal concentration was increased to inhibitory levels (100 microM) but then the effects on the cooperativity and on the normalized rate of Ca2+ release disappeared. Oxidized glutathione (5 mM) was much less effective in stimulating the release and did not have an effect on the threshold or on the cooperativity. ATP (5 mM) stimulated the release despite a shift in threshold toward higher InsP3 concentrations. Luminal Ca2+ did not affect the threshold for InsP3 action but stimulated the normalized release at each InsP3 concentration. The inhibitory effect of 10 microM free cytosolic Ca2+ was associated with a shift in threshold to higher InsP3 concentrations and a decreased cooperativity of the release process. We conclude that this novel technique of accurately measuring the threshold for InsP3 action under various experimental conditions has allowed us to refine the analysis of the kinetic parameters involved in the regulation of the InsP3 receptor.