Shankar V S, Huang C L, Adebanjo O, Simon B, Alam A S, Moonga B S, Pazianas M, Scott R H, Zaidi M
St. George's Hospital Medical School, London, United Kingdom.
J Cell Physiol. 1995 Jan;162(1):1-8. doi: 10.1002/jcp.1041620102.
Osteoclasts are known to possess a divalent cation-sensitive receptor, the Ca2+ receptor (CaR). The latter monitors changes in the local Ca2+ concentration generated as a result of hydroxyapatite dissolution. CaR activation elevates cytosolic [Ca2+] and thereby inhibits osteoclastic bone resorption. Recent studies have used Ni2+ as a surrogate CaR agonist to elicit changes in cytosolic [Ca2+]. This article examines the effects of membrane potential changes on the kinetics of the cytosolic [Ca2+] signal resulting from such Ni(2+)-induced CaR activation. Membrane potential was altered through variations in the extracellular [K] in combination with applications of the K+ ionophore, valinomycin. Membrane potential changes were confirmed by independent electrophysiological patch clamp studies of whole osteoclasts. The application of valinomycin produced a distinct, sustained elevation of cytosolic [Ca2+] in single fura 2-loaded cells, a "primary" response. This response was independent of valinomycin concentration (between 5 nM to 5 microM) and persisted in Ca(2+)-free, EGTA-containing solutions. It also persisted both in high (105 mM) and low (5 mM) extracellular [K+]. A gradual "secondary" elevation of cytosolic [Ca2+] then followed with the continued application of valinomycin, but this was eliminated by sequestering the extracellular [Ca2+] or by increasing extracellular [K+] from 5 to 105 mM. In a separate set of experiments, the presence of 5 microM [valinomycin]-([K+] = 5 mM) prolonged the cytosolic [Ca2+] signal elicited by 50 microM-[Ni2+] application. These prolonged kinetics persisted in low extracellular [Ca2+] (zero-added Ca2+), but reverted to a rapid time-course in the presence of 105 mM-[K+] or at higher [Ni2+] (500 microM and 5 mM). The experiments thus indicate that membrane voltage modifies the kinetics of CaR activation by Ni2+ and therefore suggests that the CaR is an integral protein in the osteoclast surface membrane.
已知破骨细胞拥有一种对二价阳离子敏感的受体，即钙受体（CaR）。后者监测因羟基磷灰石溶解而产生的局部钙离子浓度变化。CaR激活会提高胞质钙离子浓度，从而抑制破骨细胞的骨吸收。最近的研究使用镍离子作为替代的CaR激动剂来引发胞质钙离子浓度的变化。本文研究了膜电位变化对由镍离子诱导的CaR激活所产生的胞质钙离子信号动力学的影响。通过改变细胞外钾离子浓度并结合应用钾离子载体缬氨霉素来改变膜电位。通过对整个破骨细胞进行独立的电生理膜片钳研究来确认膜电位变化。在单个负载fura 2的细胞中，应用缬氨霉素会产生明显的、持续的胞质钙离子浓度升高，即“初级”反应。这种反应与缬氨霉素浓度无关（5 nM至5 μM之间），并且在不含钙离子、含有乙二醇双四乙酸（EGTA）的溶液中持续存在。它在高（105 mM）和低（5 mM）细胞外钾离子浓度下也都持续存在。随后，随着缬氨霉素的持续应用，胞质钙离子浓度会逐渐出现“次级”升高，但通过螯合细胞外钙离子或通过将细胞外钾离子浓度从5 mM增加到105 mM可以消除这种升高。在另一组实验中，5 μM缬氨霉素（钾离子浓度 = 5 mM）的存在延长了应用50 μM镍离子所引发的胞质钙离子信号。这些延长的动力学在低细胞外钙离子浓度（零添加钙离子）下持续存在，但在105 mM钾离子存在下或更高镍离子浓度（500 μM和5 mM）时恢复为快速的时间进程。因此，这些实验表明膜电压会改变镍离子对CaR激活的动力学，因此表明CaR是破骨细胞表面膜中的一种整合蛋白。
