Ca²⁺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 Ca²⁺influx channel and as a divalent cation (Ca²⁺) sensor. Cytosolic Ca²⁺measurements revealed Ca²⁺influx in osteoclasts at depolarized membrane potentials. The cytosolic Ca²⁺change was, as expected, not seen in Ca²⁺-free medium and was blocked by the RyR modulator ryanodine. In contrast, at basal membrane potentials (~25 mV) ryanodine triggered extracellular Ca²⁺influx that was blocked by Ni²⁺. 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_rev) ~0 mV]. In the presence of a Ba²⁺gradient, i.e., with Ba-Asp in the pipette and Na-Asp in the bath, channel conductance increased to ~120 pS and E_revshifted to 21 mV. The conductance was tentatively classified as a RyR-gated Ca²⁺channel as it displayed characteristic metastable states and was sensitive to ruthenium red and a specific anti-RyR antibody, Ab³⁴. To demonstrate that extracellular Ca²⁺sensing occurred at the osteoclastic surface rather than intracellularly, we performed protease protection assays using pronase. Preincubation with pronase resulted in markedly attenuated cytosolic Ca²⁺signals triggered by either Ni²⁺(5 mM) or Cd²⁺(50 μM). Finally, intracellular application of antiserum Ab³⁴potently inhibited divalent cation sensing. Together, these results strongly suggest the existence of 1) a membrane-resident Ca²⁺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 Ca²⁺influx but also functions as a sensor for extracellular divalent cations.