Extracellular Ca²⁺-sensing receptor is a promiscuous divalent cation sensor that responds to lead

The extracellular Ca²⁺-sensing receptor (CaR) responds to polycations, including Ca²⁺ and neomycin. This receptor is a physiological regulator of systemic Ca²⁺ metabolism and may also mediate the toxic effects of hypercalcemia. A number of divalent cations, including Pb²⁺, Co²⁺, Cd²⁺, and Fe²⁺, are toxic to the kidney, brain, and other tissues where the CaR is expressed. To determine which divalent cations can activate the CaR, we expressed the human CaR in HEK-293 cells and measured activation of phospholipase A₂ (PLA₂) and the mitogen-activated protein kinase p42ERK in response to potential agonists for the receptor. HEK-293 cells expressing the nonfunctional mutant CaR R796W served as controls. Extracellular Ca²⁺, Ba²⁺, Cd²⁺, Co²⁺, Fe²⁺, Gd³⁺, Ni²⁺, Pb²⁺, and neomycin activated the CaR, but Hg²⁺ and Fe³⁺ did not. We analyzed the kinetics of activation of p42ERK and PLA₂ by the CaR in response to Ca²⁺, Co²⁺, and Pb²⁺. The EC₅₀ values ranged from ~0.1 mM for Pb²⁺ to ~4.0 mM for Ca²⁺. The Hill coefficients were >3, indicating multiple cooperative ligand binding sites or subunits. Submaximal concentrations of Ca²⁺ and Pb²⁺ were additive for activation of the CaR. The EC₅₀ for Ca²⁺ or Pb²⁺ was reduced four- to fivefold by the presence of the other ion. These divalent cations also activated PLA₂ via the CaR in Madin-Darby canine kidney cells that stably express the CaR. We conclude that many divalent cations activate the CaR and that their effects are additive. The facts that the CaR is a promiscuous polycation sensor and that the effects of these ions are additive to activate it suggest that the CaR may contribute to the toxicity of some heavy metals such as Pb²⁺, Cd²⁺, Co²⁺, and Fe²⁺ for the kidney and other tissues where it is expressed.