[Ca.sup.2+] influx through the osteoclastic plasma membrane ryanodine receptor

We predict that the type 2 ryanodine receptor isoform (RyR-2) located in the osteoclastic membrane functions as a [Ca.sup.2+] influx channel and as a divalent cation ([Ca.sup.2+]) sensor. Cytosolic [Ca.sup.2+] measurements revealed [Ca.sup.2+] influx in osteoclasts at depolarized membrane potentials. The cytosolic [Ca.sup.2+] change was, as expected, not seen in [Ca.sup.2+]-free medium and was blocked by the RyR modulator ryanodine. In contrast, at basal membrane potentials (~25 mV) ryanodine triggered extracellular [Ca.sup.2+] influx that was blocked by [Ni.sup.2+]. In parallel, single-channel recordings obtained from inside-out excised patches revealed a divalent cation-selective ~60-pS conductance in symmetric solutions of Ba-aspartate [Ba-Asp; reversal potential ([E.sub.rev]) ~0 mV]. In the presence of a [Ba.sup.2+] gradient, i.e., with Ba-Asp in the pipette and Na-Asp in the bath, channel conductance increased to ~120 pS and [E.sub.rev] shifted to 21 mV. The conductance was tentatively classified as a RyR-gated [Ca.sup.2+] channel as it displayed characteristic metastable states and was sensitive to ruthenium red and a specific anti-RyR antibody, [Ab.sup.34]. To demonstrate that extracellular [Ca.sup.2+] sensing occurred at the osteoclastic surface rather than intracellularly, we performed protease protection assays using pronase. Preincubation with pronase resulted in markedly attenuated cytosolic [Ca.sup.2+] signals triggered by either [Ni.sup.2+] (5 mM) or [Cd.sup.2+] (50 [micro]M). Finally, intracellular application of antiserum [Ab.sup.34] potently inhibited divalent cation sensing. Together, these results strongly suggest the existence of 1) a membrane-resident [Ca.sup.2+] influx channel sensitive to RyR modulators; 2) an extracellular, as opposed to intracellular, divalent cation activation site; and 3) a cytosolic CaM-binding regulatory site for RyR. It is likely therefore that the surface RyR-2 not only gates [Ca.sup.2+] influx but also functions as a sensor for extracellular divalent cations. osteoclast; calcium receptor; calcium channels; osteoporosis; calcium