The important discrepancy between the apparent affinity observed in Ca 2+ mobilization studies and the K d measured in binding studies is a consequence of the quantal process by which inositol 1,4,5-trisphosphate releases Ca 2+ from bovine adrenal cortex microsomes

Abstract

Inositol 1,4,5-trisphosphate (InsP 3) is a second meassenger responsible for Ca 2+ release from a non-mitochondrial intracellular store. An important discrepancy has been observed between the affinity measured in binding studies (K d) and the apparent affinity obtained in Ca 2+ mobilization studies (EC 50). It has been proposed that this discrepancy could be due to different experimental conditions used for Ca 2+ mobilization studies and for InsP 3 binding studies. With the fluorescent indicator Fura-2, we studied InsP 3-induced Ca 2+ release activity at 7°C and at 37°C, in bovine adrenal cortex microsomes. Under both conditions, the Ca 2+ releasing effect of InsP 3 (1 μM) was completed within about 2 s, as a result of the quantal process of InsP 3 receptor action. The apparent affinity (EC 50) observed for InsP 3-induced Ca 2+ release at 7°C and at 37°C were 0.64 ± 0.2 μM and 0.9 ± 0.2 μM respectively. InsP 3 degradation studies, at 37°C, indicated that less than 10% of [ 3H]-InsP 3 was degraded within the first 10 s of incubation. InsP 3 association rates were evaluated, at low temperature, with increasing concentrations of [ 3H]-InsP 3. These kinetic studies revealed a direct relationship between the initial rate of association (V i) and InsP 3 concentration. From this relationship, we evaluated that the concentration of InsP 3 needed to occupy half of the binding sites within the first second of incubation was 271 nM. We conclude that the discrepancy between K d and EC 50 is related to a kinetic constraint dictated by the quantal process by which InsP 3 releases Ca 2+.