| Autor: |
Moonga, Baljit S., Li, Sun, Iqbal, Jameel, Davidson, Robert, Shankar, Vijai S., Bevis, Peter J. R., Inzerillo, Angela, Abe, Etsuko, Huang, Christopher L.-H., Zaidi, Mone |
| Zdroj: |
American Journal of Physiology - Renal Physiology; May 2002, Vol. 282 Issue: 5 pF921-F932, 12p |
| Abstrakt: |
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 Erevshifted 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. |
| Databáze: |
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