Evidence that Ca2+ cycling by the plasma membrane Ca2+-ATPase increases the 'excitability' of the extracellular Ca2+-sensing receptor

The extracellular Ca2+-sensing receptor (CaR) is a widely expressed G-protein-coupled receptor that translates information about [Ca2+] in the extracellular milieu to the interior of the cell, usually via intracellular Ca2+ signaling pathways. Using fura-2 imaging of cytoplasmic [Ca2+], we observed that HEK293 cells expressing CaR oscillated readily under conditions permissive for CaR activation. Spiking was also triggered in the absence of external Ca2+ by the CaR agonist spermine (1 mM). Oscillating cells were typically located in clusters of closely apposed cells, but Ca2+ spiking was insensitive to the gap junction inhibitor 18alpha-glycyrrhetinic acid. We hypothesized that Ca2+ signals might be amplified, in part, through a positive feedback loop in which Ca2+ extrusion via the plasma membrane Ca2+-ATPase (PMCA) activates CaRs on the same cell or adjacent cells through local increases in [Ca2+](out). In support of this idea, addition of exogenous Ca2+ buffers (keeping free [Ca2+](out) constant) attenuated or eliminated Ca2+ signals (manifested as oscillations), as did PMCA inhibitors (HgCl2, orthovanadate and Caloxin 2A1). Measurement of extracellular [Ca2+] using the near membrane probe fura-C-18 revealed that external [Ca2+] rose following receptor activation, sometimes displaying an oscillatory pattern. Our data suggest that PMCA-mediated cycling of Ca2+ across the plasma membrane leads to localized increases in [Ca2+](out) that increase the excitability of CaR.