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

The frequency of oscillatory Ca(2+) 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 Ca(2+) entry. However precisely how this is achieved and the respective roles of store-operated versus store-independent Ca(2+) entry pathways in achieving this are unclear. Here, we examine the possibility that a direct stimulation of a phospholipase C (PLC) by the entering Ca(2+) can induce a modulation of Ca(2+) 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 PIP(2)-dependent Kir2.1 channels as a sensitive assay for PLC activity, we show that simple global increases in Ca(2+) 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 PIP(2) depletion. Further experiments show that this effect is strictly dependent on the Ca(2+) 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 PLCδ by local Ca(2+) 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 PLCδ, it was shown that this specific action of the store-independent ARC channel-mediated Ca(2+) entry on PLCδ has a significant impact on the oscillation frequency of the Ca(2+) signals activated by low concentrations of agonist.