Orai channel-dependent activation of phospholipase C-δ: a novel mechanism for the effects of calcium entry on calcium oscillations

The frequency of oscillatory Ca2+ signals is a major determinant in the selective activation of discrete downstream responses in non-excitable cells. An important modulator of this oscillation frequency is known to be the rate of agonist-activated Ca2+ entry. However precisely how this is achieved and the respective roles of store-operated versus store-independent Ca2+ entry pathways in achieving this are unclear. Here, we examine the possibility that a direct stimulation of a phospholipase C (PLC) by the entering Ca2+ can induce a modulation of Ca2+ oscillation frequency, and examine the roles of the endogenous store-operated and store-independent Orai channels (CRAC and ARC channels, respectively) in such a mechanism. Using the decline in the magnitude of currents through expressed PIP2-dependent Kir2.1 channels as a sensitive assay for PLC activity, we show that simple global increases in Ca2+ concentrations over the physiological range do not significantly affect PLC activity. Similarly, maximal activation of endogenous CRAC channels also fails to affect PLC activity. In contrast, equivalent activation of endogenous ARC channels resulted in a 10-fold increase in the measured rate of PIP2 depletion. Further experiments show that this effect is strictly dependent on the Ca2+ entering via these channels, rather than the gating of the channels or the arachidonic acid used to activate them, and that it reflects the activation of a PLC delta by local Ca2+ concentrations immediately adjacent to the active channels. Finally, based on the effects of expression of either a dominant-negative mutant Orai3 that is an essential component of the ARC channel, or a catalytically compromised mutant PLC delta, it was shown that this specific action of the store-independent ARC channel-mediated Ca2+ entry on PLC delta has a significant impact on the oscillation frequency of the Ca2+ signals activated by low concentrations of agonist.