Effect of membrane potential on surface Ca2+ receptor activation in rat osteoclasts

Osteoclasts are known to possess a divalent cation-sensitive receptor, the Ca2+ receptor (CaR). The latter monitors changes in the local Ca2+ concentration generated as a result of hydroxyapatite dissolution. CaR activation elevates cytosolic [Ca2+] and thereby inhibits osteoclastic bone resorption. Recent studies have used Ni2+ as a surrogate CaR agonist to elicit changes in cytosolic [Ca2+]. This article examines the effects of membrane potential changes on the kinetics of the cytosolic [Ca2+] signal resulting from such Ni(2+)-induced CaR activation. Membrane potential was altered through variations in the extracellular [K] in combination with applications of the K+ ionophore, valinomycin. Membrane potential changes were confirmed by independent electrophysiological patch clamp studies of whole osteoclasts. The application of valinomycin produced a distinct, sustained elevation of cytosolic [Ca2+] in single fura 2-loaded cells, a "primary" response. This response was independent of valinomycin concentration (between 5 nM to 5 microM) and persisted in Ca(2+)-free, EGTA-containing solutions. It also persisted both in high (105 mM) and low (5 mM) extracellular [K+]. A gradual "secondary" elevation of cytosolic [Ca2+] then followed with the continued application of valinomycin, but this was eliminated by sequestering the extracellular [Ca2+] or by increasing extracellular [K+] from 5 to 105 mM. In a separate set of experiments, the presence of 5 microM [valinomycin]-([K+] = 5 mM) prolonged the cytosolic [Ca2+] signal elicited by 50 microM-[Ni2+] application. These prolonged kinetics persisted in low extracellular [Ca2+] (zero-added Ca2+), but reverted to a rapid time-course in the presence of 105 mM-[K+] or at higher [Ni2+] (500 microM and 5 mM). The experiments thus indicate that membrane voltage modifies the kinetics of CaR activation by Ni2+ and therefore suggests that the CaR is an integral protein in the osteoclast surface membrane.