Pharmacological characterization of inositol 1,4,5-trisphosphate binding sites: relation to Ca2+ release.

Two subcellular fractions, one enriched in plasma membranes and the other in endoplasmic reticulum membranes, were obtained from WRK1 cells using a combination of differential centrifugations and Percoll gradient fractionation. Specific inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) binding sites were detected in these two preparations. Endoplasmic reticulum membranes exhibited a binding capacity which was about 5-fold higher than that of plasma membranes. Dose-dependent Ins(1,4,5)P3 binding was determined. Experimental data obtained with endoplasmic reticulum membranes could be adequately fitted with a two-site model (a high-affinity binding site with Kd and Bmax values of 0.7 +/- 0.15 nM and 12.9 +/- 5 fmol/mg protein and a low-affinity binding site with Kd and Bmax values of 44.2 +/- 14.6 nM and 143 +/- 43 fmol/mg protein). Both the high- and low-affinity binding sites were selective for Ins(1,4,5)P3. Besides Ins(1,4,5)P3, Ins(1,3,4,5)P4 also discriminated between the two populations of sites while heparin interacted with the high- and low-affinity binding sites with the same affinity. Ins(1,4,5)P3-induced calcium release from endoplasmic reticulum vesicles was determined by monitoring the calcium concentration in the extravesicular compartment with fura-2. Under experimental conditions where the degradation of Ins(1,4,5)P3 was reduced (incubation at 0 degrees C), a high-affinity Ins(1,4,5)P3-induced calcium release (apparent Kact around 20 nM) could be demonstrated. These results suggest that in WRK1 cells, the endoplasmic reticulum is a major site for Ins(1,4,5)P3 action and that the high-affinity binding sites located on the endoplasmic reticulum membranes may contribute to the physiological regulation of the cytosolic free calcium concentration.