THIOL REAGENTS INCREASE THE AFFINITY OF THE INOSITOL 1,4,5-TRISPHOSPHATE RECEPTOR

Thiol reagents have been shown to increase cytosolic Ca2+ in several c ell types. In non-muscle cells, these agents induce Ca2+ spikes by inc reasing the sensitivity of the intracellular Ca2+ stores to D-myo-inos itol-1,4,5-trisphosphate (InsP3). We have investigated the effects of thimerosal and oxidized glutathione on the binding properties of the I nsP3 receptor in permeabilized hepatocytes and liver and cerebellar me mbranes. Thimerosal, at the maximal concentration of 100 muM, decrease d the K(D) for the InsP3 binding to permeabilized hepatocytes and cere bellar membranes from 16 to 3 nM and from 25 to 8 nM, respectively, wi thout affecting the maximal binding capacities. On liver membranes, bo th thimerosal and high Ca2+ concentrations increased the affinity for InsP3 binding. The Ca2+ and the thimerosal effects were differentiated by kinetic experiments. In low Ca2+ media, two kinetic components wer e identified and thimerosal decreased the rate of dissociation from bo th these components without affecting the rate of association. In the high Ca2+ medium, a single kinetic component was found with a very slo w rate of dissociation. These data suggest that the InsP3 receptor exi sts in different states. The high-affinity inactive state induced by h igh Ca2+ concentrations displays slow rates of association and dissoci ation. The binding properties of the receptor in its active state can be regulated by thiol reagents which increase the affinity by decreasi ng the dissociation rate constants. At a resting concentration of 100- 200 nM, Ca2+ has two effects: it increases the affinity of the active state of the receptor as thiol reagents do and transforms part of the receptors into the inactive high-affinity state.