Resumen
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 (Erev) ∼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 Erev shifted 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 extra-cellular 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 Ab34 potently 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.
Idioma original | English (US) |
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Páginas (desde-hasta) | F921-F932 |
Publicación | American Journal of Physiology - Renal Physiology |
Volumen | 282 |
N.º | 5 51-5 |
Estado | Published - 2002 |
Publicado de forma externa | Sí |
ASJC Scopus subject areas
- Physiology