Proceedings of The Physiological Society
University of Manchester (2010) Proc Physiol Soc 19, C127
Store-independent ARC channels and the modulation of Ca2+ oscillation frequency by Ca2+ entry
T. Shuttleworth1, J. Thompson1
1. Pharmacology and Physiology, University of Rochester Medical Center, Rochester, New York, United States.
Physiological stimulation of non-excitable cells typically results in oscillatory cytosolic Ca2+ signals, the frequency of which influences the selective activation of downstream responses. Oscillation frequency is modulated by the rate of agonist-activated Ca2+ entry but how this is achieved, and the nature of the entry pathway responsible (store-operated versus store-independent), is unclear. Here, we have explored a potential novel mechanism for this effect involving the direct stimulation of phospholipase C (PLC) by entering Ca2+, and assessed the relative contribution of store-operated and store-independent (via the arachidonic acid regulated "ARC" channel) modes of Ca2+ entry in such a mechanism. PLC activity was monitored by assaying the inhibition of K+ currents through PIP2-dependent Kir2.1 channels (1) in HEK293 cells using whole-cell patch clamp techniques. Maximal activation of endogenous store-operated Ca2+-selective CRAC channels failed to significantly affect the resting inhibition of the Kir2.1 channel currents. In marked contrast, activation of the Ca2+-selective ARC channels with exogenous arachidonic acid (AA) to give a similar whole-cell Ca2+ current density to that of the CRAC channels, resulted in the rapid inhibition of Kir2.1 channel currents, consistent with increased PLC activity. This rate of inhibition was some 10-fold larger than that seen in control cells. This effect was due to the induced entry of Ca2+, rather than any effect of AA itself, as it was eliminated by inhibition of Ca2+ currents through the ARC channel either by external Gd3+ (5 µM), or reducing the driving force (holding at 0 mV). Increasing cytosolic Ca2+ concentrations from 100 nM to1.3 µM failed to significantly effect the rate of Kir2.1 channel current activity, showing that PLC activity was unaffected by global Ca2+ concentrations over the physiological range, and that the ARC channel effect must depend on local high Ca2+ concentrations. Calculations of the mean diffusion distance of free Ca2+ ions at the EGTA concentrations used, indicate that the relevant PLC must be located within ~100 nm of the channel mouth. These data suggest that modulation of the frequency of agonist-induced Ca2+ oscillations by Ca2+ entry results from a local tonic activation of a PLC as a direct, and specific consequence of the activity of the store-independent ARC channels. Such a model is entirely consistent with the previously demonstrated unique activation of these channels at the low agonist concentrations that induce oscillatory Ca2+ signals in various cell types. Moreover, because activation of the ARC channels is independent of store depletion, this phenomenon can also account for the previously unexplained ability of agonist-activated Ca2+ entry to affect signal latency (the delay between agonist addition and the initiation of a Ca2+ response).
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