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. C aR activation elevates cytosolic [Ca2+] and thereby inhibits osteoclas tic bone resorption. Recent studies have used Ni2+ as a surrogate CaR agonist to elicit changes in cytosolic [Ca2+]. This article examines t he effects of membrane potential changes on the kinetics of the cytoso lic [Ca2+] signal resulting from such Ni2+-induced CaR activation. Mem brane 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 electrophysio logical patch clamp studies of whole osteoclasts. The application of v alinomycin 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 mu M) and persisted in Ca2+-free, EGTA-containing solutions. It also persis ted both in high (105 mM) and low (5 mM) extracellular [K+]. A gradual ''secondary'' elevation of cytosolic [Ca2+] then followed with the co ntinued application of valinomycin, but this was eliminated by sequest ering the extracellular [Ca2+] or by increasing extracellular [K+] fro m 5 to 105 mM. In a separate set of experiments, the presence of 5 mu M [valinomycin]-([K+] = 5 mM) prolonged the cytosolic [Ca2+] signal el icited by 50 mu M-[Ni2+] application. These prolonged kinetics persist ed in low extracellular [Ca2+] (zero-added Ca2+), but reverted to a ra pid time-course in the presence of 105 mM-[K+] or at higher [Ni2+] (50 0 mu M 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 membran e. (C) 1995 Wiley-Liss, Inc.