Effects of thimerosal on the transient kinetics of inositol 1,4,5-trisphosphate-induced Ca2+ release from cerebellar...

Thimerosal, a thiol-reactive reagent, has been shown to increase the cytosolic Ca2+ concentration in a variety of cells by sensitizing inositol 1,4,5-trisphosphate (InsP3) receptors. Thimerosal can have both sensitizing (at concentrations of <2 microM) and inhibitory (at concentrations of >2 microM) effects on InsP3-induced Ca2+ release (IICR) from cerebellar microsomes. Transient kinetic studies were performed by employing a fluorimetric stopped-flow approach using fluo-3. IICR was found to be a bi-exponential process with a fast and a slow component. At a maximal InsP3 concentration (20 microM), the fast phase had a rate constant of 0.9 s-1 and the slow phase had a rate constant of 0.4 s-1. The amplitudes of the two phases were 60% and 40% respectively. When the rate constants for the two phases were plotted as Hill plots, the processes were found to be non-co-operative in both cases (Hill coefficient of 1.0), thus arguing for a simple mechanism linking InsP3 binding to channel opening. At a submaximal InsP3 concentration (0.2 microM), where the sensitizing effects of thimerosal are most pronounced, thimerosal increased the rate constants of both phases in a sigmoidal fashion, with a Hill coefficient of 4.0, suggesting that several cysteine residues (up to four) need to be modified in order for maximum sensitization to occur. The rate constants remained elevated even at thimerosal concentrations that inhibited IICR. The amplitude or extent of Ca2+ release was, however, elevated to a much greater extent in the slow phase, suggesting that the two phases respond differently. At maximal InsP3 concentrations, thimerosal has no effect upon the rate constants but inhibits the amplitude of Ca2+ release.