Elucidations of mechanisms involved in activation and regulation of store-operated calcium(2+) entry channels

Abstract

Store-operated Ca2+ entry (SOCaE) is involved in a host of processes within cells, including Ca2+ store replenishment, growth, and gene transcription. Although this process has long since been identified, the molecular nature of the store-operated Ca2+ channel (SOC) as well as the activation and regulatory mechanisms underlying its control have remained elusive. Studies began, using a cellular approach integrated with fluorescent techniques to measure intracellular Ca 2+, examining the possible involvement of the actin cytoskeleton in the stimulation or regulation of SOCaE using cultured smooth muscle cells. Manipulation of the cytoskeleton, either its removal or redistribution, demonstrated that the actin cytoskeleton did not actively participate in the activation or control of SOCaE. Compelling evidence that internal membrane association with the plasma membrane is necessary for the activation of SOCaE was provided by loss of SOCaE when cells were induced to form cortical actin subjacent to the plasma membrane. In addition, these interactions were demonstrated to occur in a reversible and labile manner by disruption of this cortical actin, restoring the activation and function of SOC's. This data led to the proposal of a new model for SOCaE, due to its close parallels to secretion. Using a more molecular approach, activation of human transient receptor potential channel 3 (TRP3), a possible model for SOC, was compared to SOCaE. Using HEK-293 cells stably transfected with TRP3, cortical actin formation demonstrated that TRP3 also required internal membrane associations at the plasma membrane for activation, although TRP3 did not prove to be activated by Ca2+ store-depletion. The direct activator of TRP3, diacylglycerol, could activate TRP3 in the presence or absence of cortical actin, proving that a functional channel exists proceeding cortical actin formation. In conjunction with cortical actin formation inhibiting activation of TRP3 by InsP3 , this provides even more evidence for a "secretion-like" activation process. Use of the inositol tri-phosphate receptor (InsP 3-receptor) antagonists 2-amino-diphenylborate (2-APB) and xestaspongin C had identical properties to cortical actin formation in the inhibition of SOCaE and TRP3. From this we concluded that the InsP3-receptor was an integral member of the SOCaE activation pathway. Use of 2-APB in combination with a novel inhibitor of Ca2+ channels, MDL-12,330A, allowed for a pharmacological profile of SOC, TRP3 and Ca2+ channels activated by S-nitrosylation. Only SOC and TRP3 proved to be sensitive to 2-APB, suggesting that Ca2+ channels activated by S-nitrosylation are either not activated by the InsP3-receptor, or that the InsP 3-receptor is not required for their activation in this manner. MDL-12,330A proved to be a useful tool, not only as a reliable, rapidly reversible antagonist, but also as a direct modifier of Ca2+ channels, providing indirect evidence that a conserved region exists within all of the Ca2+ channels studied. This work taken as a whole implies an important role of internal membranes and inositol signaling in the activation and regulation of SOCaE, in addition to providing evidence that SOC's and TRP's may be closely related, but differentially activated.