Evidence that Ca(2+) cycling by the plasma membrane Ca(2+)-ATPase increases the 'excitability' of the extracellular Ca(2+)-sensing receptor.

The extracellular Ca(2+)-sensing receptor (CaR) is a widely expressed G-protein-coupled receptor that translates information about [Ca(2+)] in the extracellular milieu to the interior of the cell, usually via intracellular Ca(2+) signaling pathways. Using fura-2 imaging of cytoplasmic [Ca(2+)], we observed that HEK293 cells expressing CaR oscillated readily under conditions permissive for CaR activation. Spiking was also triggered in the absence of external Ca(2+) by the CaR agonist spermine (1 mM). Oscillating cells were typically located in clusters of closely apposed cells, but Ca(2+) spiking was insensitive to the gap junction inhibitor 18alpha-glycyrrhetinic acid. We hypothesized that Ca(2+) signals might be amplified, in part, through a positive feedback loop in which Ca(2+) extrusion via the plasma membrane Ca(2+)-ATPase (PMCA) activates CaRs on the same cell or adjacent cells through local increases in [Ca(2+)](out). In support of this idea, addition of exogenous Ca(2+) buffers (keeping free [Ca(2+)](out) constant) attenuated or eliminated Ca(2+) signals (manifested as oscillations), as did PMCA inhibitors (HgCl(2), orthovanadate and Caloxin 2A1). Measurement of extracellular [Ca(2+)] using the near membrane probe fura-C(18) revealed that external [Ca(2+)] rose following receptor activation, sometimes displaying an oscillatory pattern. Our data suggest that PMCA-mediated cycling of Ca(2+) across the plasma membrane leads to localized increases in [Ca(2+)](out) that increase the excitability of CaR.