Ca2+ influx through the osteoclastic plasma membrane ryanodine receptor

Tracy A. McGowan, Muniswamy Madesh, Yanqing Zhu, Lewei Wang, Mark Russo, Leo Deelman, Rob Henning, Suresh Joseph, Gyorgy Hajnoczky, Kumar Sharma

Research output: Contribution to journalArticle

38 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)F921-F932
JournalAmerican Journal of Physiology - Renal Physiology
Volume282
Issue number5 51-5
StatePublished - Jun 29 2002
Externally publishedYes

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Keywords

  • Calcium
  • Calcium channels
  • Calcium receptor
  • Osteoclast
  • Osteoporosis

ASJC Scopus subject areas

  • Physiology
  • Urology

Cite this

McGowan, T. A., Madesh, M., Zhu, Y., Wang, L., Russo, M., Deelman, L., Henning, R., Joseph, S., Hajnoczky, G., & Sharma, K. (2002). Ca2+ influx through the osteoclastic plasma membrane ryanodine receptor. American Journal of Physiology - Renal Physiology, 282(5 51-5), F921-F932.