A number of thiol-reactive agents induce repetitive Ca spiking in cells by a mechanism thought to involve sensitization of the inositol 1,4,5-trisphosphate receptor (IPR). To further define the basis of this interaction, we have studied the effect of several thiol-reactive agents on [H]IP binding, IP-gated channel activity, and conformation of the IPR in membranes from hepatocytes, cultured WB rat liver epithelial cells, and cerebellum microsomes. At 4 °C, the organomercurial thiol-reactive agent mersalyl markedly stimulates (3-4-fold) [H]IP binding to permeabilized hepatocytes. The closely related molecule, thimerosal, has only a small stimulatory effect under these conditions, and GSSG or N-ethylmaleimide are without effect. The stimulatory effect of mersalyl was associated with a decrease in K of the IPR with no change in B. Mersalyl was without effect on detergent-solubilized hepatocyte binding sites or on the [H]IP binding activity of cerebellum microsomes. In contrast to thimerosal, which potentiates IP-mediated Ca release, mersalyl blocked IP-gated Ca channels. Mersalyl pretreatment of WB membranes altered the pattern of immunoreactive receptor fragments generated upon subsequent cleavage of the receptor with proteinase K. This effect was not reproduced by thimerosal and was also not observed in experiments on cerebellum microsomes. We conclude that the WB cell and brain IP receptors are differently regulated by modification of thiol groups. Reaction of the WB cell IP receptor with mersalyl alters its conformation and modifies the accessibility of sites on the protein that are cleaved by proteinase K. In the presence of mersalyl, the receptor has high affinity for IP but is inactive as a Ca channel. This contrasts with the high affinity receptor/active Ca channel induced by thimerosal, suggesting that even closely related thiol agents may interact at different thiol groups.

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