학술논문
[Ca.sup.2+] influx through the osteoclastic plasma membrane ryanodine receptor
Document Type
Abstract
Author
Source
The American Journal of Physiology. May, 2002, Vol. 282 Issue 5, pF921, 12 p.
Subject
Language
ISSN
0002-9513
Abstract
We predict that the type 2 ryanodine receptor isoform (RyR-2) located in the osteoclastic membrane functions as a [Ca.sup.2+] influx channel and as a divalent cation ([Ca.sup.2+]) sensor. Cytosolic [Ca.sup.2+] measurements revealed [Ca.sup.2+] influx in osteoclasts at depolarized membrane potentials. The cytosolic [Ca.sup.2+] change was, as expected, not seen in [Ca.sup.2+]-free medium and was blocked by the RyR modulator ryanodine. In contrast, at basal membrane potentials (~25 mV) ryanodine triggered extracellular [Ca.sup.2+] influx that was blocked by [Ni.sup.2+]. 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.sub.rev]) ~0 mV]. In the presence of a [Ba.sup.2+] gradient, i.e., with Ba-Asp in the pipette and Na-Asp in the bath, channel conductance increased to ~120 pS and [E.sub.rev] shifted to 21 mV. The conductance was tentatively classified as a RyR-gated [Ca.sup.2+] channel as it displayed characteristic metastable states and was sensitive to ruthenium red and a specific anti-RyR antibody, [Ab.sup.34]. To demonstrate that extracellular [Ca.sup.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.sup.2+] signals triggered by either [Ni.sup.2+] (5 mM) or [Cd.sup.2+] (50 [micro]M). Finally, intracellular application of antiserum [Ab.sup.34] potently inhibited divalent cation sensing. Together, these results strongly suggest the existence of 1) a membrane-resident [Ca.sup.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.sup.2+] influx but also functions as a sensor for extracellular divalent cations. osteoclast; calcium receptor; calcium channels; osteoporosis; calcium