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

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 18α-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-C₁₈ 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.