Regulation of the store-regulated calcium entry pathway in rat thymic lymphocytes
Description
A significant body of evidence has accumulated to show that $\rm Ca\sp{2+}$ influx into mitogen stimulated T-lymphocytes is regulated in large part by the $\rm Ca\sp{2+}$ content of the $\rm IP\sb3$ sensitive pool. The divalent cation permeability sequence of this store-regulated $\rm Ca\sp{2+}$ entry pathway was investigated in rat thymic lymphocytes fluorimetrically using bis-oxonol. On the basis of the present results the following relative permeability sequence of divalent cation permeability was obtained for the store mediated $\rm Ca\sp{2+}$ entry pathway: $\rm Ca\sp{2+} > Mn\sp{2+}\gg Ba\sp{2+}$ and $\rm Sr\sp{2+}.$ Radioisotopic flux measurements were conducted to investigate the role of inactivation on the store-mediated $\rm Ca\sp{2+}$ entry pathway. The results of the these studies revealed the presence of complex inactivation mechanisms involving both $\rm Ca\sp{2+}$-dependent and $\rm Ca\sp{2+}$-independent components in the control of this pathway, perhaps indicative of time dependent alterations in the mechanism(s) linking $\rm Ca\sp{2+}$ stores to the activation of $\rm Ca\sp{2+}$ entry Membrane depolarization caused marked inhibition of the store-regulated $\rm Ca\sp{2+}$ entry as measured by fluorimetric and radioisotopic techniques. Experiments were undertaken to define the relationship between membrane potential and store-mediated unidirectional $\rm Ca\sp{2+}$ entry. The data presented in this study suggests that the store-mediated $\rm Ca\sp{2+}$ entry pathway is modulated by membrane potential in an inwardly rectifying direction, in a manner that is inconsistent with simple alterations in the electrochemical gradient for $\rm Ca\sp{2+}.$ Experiments were also performed to determine the requirement for cellular ATP in store-regulated $\rm Ca\sp{2+}$ uptake. This pathway was found to be markedly inhibited in cells depleted of ATP. These data are consistent with a requirement for ATP or a high energy phosphate donor in the activation of store-mediated $\rm Ca\sp{2+}$ entry Finally, experiments were undertaken to investigate the regulation of store-regulated $\rm Ca\sp{2+}$ entry by phorbol ester sensitive protein kinase C and serine/threonine protein phosphatase activity. The results of these studies demonstrate that modification of the phosphorylation state of target protein(s) on serine/threonine amino acid residues by inhibition of phosphatase type 1/2A, inhibits the store-mediated $\rm Ca\sp{2+}$ entry pathway in rat thymic lymphocytes. Importantly, this pathway was not modulated by activation of phorbol ester-sensitive protein kinase C