We predict that the type 2 ryanodine receptor isoform (RyR-2) located in the osteoclastic membrane functions as a Ca(2+) influx channel and as a divalent cation (Ca(2+)) sensor. Cytosolic Ca(2+) measurements revealed Ca(2+) influx in osteoclasts at depolarized membrane potentials. The cytosolic Ca(2+) change was, as expected, not seen in Ca(2+)-free medium and was blocked by the RyR modulator ryanodine. In contrast, at basal membrane potentials (approximately 25 mV) ryanodine triggered extracellular Ca(2+) influx that was blocked by Ni(2+). In parallel, single-channel recordings obtained from inside-out excised patches revealed a divalent cation-selective approximately 60-pS conductance in symmetric solutions of Ba-aspartate [Ba-Asp; reversal potential (E(rev)) approximately 0 mV]. In the presence of a Ba(2+) gradient, i.e., with Ba-Asp in the pipette and Na-Asp in the bath, channel conductance increased to approximately 120 pS and E(rev) shifted to 21 mV. The conductance was tentatively classified as a RyR-gated Ca(2+) channel as it displayed characteristic metastable states and was sensitive to ruthenium red and a specific anti-RyR antibody, Ab(34). To demonstrate that extracellular Ca(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(2+) signals triggered by either Ni(2+) (5 mM) or Cd(2+) (50 microM). Finally, intracellular application of antiserum Ab(34) potently inhibited divalent cation sensing. Together, these results strongly suggest the existence of 1) a membrane-resident Ca(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(2+) influx but also functions as a sensor for extracellular divalent cations.