Schnetkamp P P, Li X B, Basu D K, Szerencsei R T
Department of Medical Biochemistry, University of Calgary, Alberta, Canada.
J Biol Chem. 1991 Dec 5;266(34):22975-82.
Regulation of free cytosolic Ca2+ concentration in the rod outer segments (ROS) isolated from bovine retinas was examined with the fluorescent Ca(2+)-indicating dye fluo-3. In situ calibration of cytosolic fluo-3 was done in the presence of the Ca2+ ionophore A23187 and yielded a dissociation constant of 500 nM for the Ca(2+)-fluo-3 complex. Ca2+ influx in Ca(2+)-depleted ROS was completely abolished when internal Na+ was removed suggesting that Ca2+ influx exclusively occurred via Na-Ca-K exchange. The most striking observation was that Na-Ca-K exchange could mediate a rapid increase in cytosolic free Ca2+ over the most of the usable indicating range of fluo-3 (from 10 nM to 2 microM), even when exposed to free external Ca2+ concentrations as low as 10 nM. From a comparison between changes in free Ca2+ and changes in total Ca2+, we conclude that physiologically occurring changes in cytosolic free Ca2+ are mediated by exchange fluxes less than 1% of the maximal Na-Ca-K exchange flux. The Na-Ca-K exchanger could mediate both K(+)-dependent and K(+)-independent Ca2+ influx; Li+ caused a complete inhibition of K(+)-independent Ca2+ influx, but had no effect on K(+)-dependent Ca2+ influx. We examined the complex interactions of alkali cations with Ca2+ influx and discuss the results in terms of a three-site model for the Na-Ca-K exchanger (Schnetkamp, P. P. M. and Szerencsei, R. T. (1991) J. Biol. Chem. 266, 189-197). Ca2+ competed with one Mg2+ ion or two Na+ ions for binding to a common site. High K+ concentration greatly diminished the ability of Na+ and Mg2+ to compete with Ca2+ for this common site on the exchanger protein. As a result, high internal K+ induced a conformation of the exchange protein that kinetically favoured Ca2+ extrusion.
用荧光钙指示剂fluo-3检测了从牛视网膜分离的视杆外段（ROS）中游离胞质Ca2+浓度的调节情况。在Ca2+离子载体A23187存在的情况下对胞质中的fluo-3进行原位校准，得到Ca(2+)-fluo-3复合物的解离常数为500 nM。当去除内部Na+时，Ca(2+)-耗尽的ROS中的Ca2+内流完全被消除，这表明Ca2+内流仅通过Na-Ca-K交换发生。最引人注目的观察结果是，即使暴露于低至10 nM的游离外部Ca2+浓度下，Na-Ca-K交换也能在fluo-3的大部分可用指示范围内（从10 nM到2 μM）介导胞质游离Ca2+的快速增加。通过比较游离Ca2+的变化和总Ca2+的变化，我们得出结论，生理条件下胞质游离Ca2+的变化是由小于最大Na-Ca-K交换通量1%的交换通量介导的。Na-Ca-K交换器可以介导依赖K+和不依赖K+的Ca2+内流；Li+完全抑制不依赖K+的Ca2+内流，但对依赖K+的Ca2+内流没有影响。我们研究了碱金属阳离子与Ca2+内流的复杂相互作用，并根据Na-Ca-K交换器的三位点模型讨论了结果（施内特坎普，P.P.M.和瑟伦采伊，R.T.（1991年）《生物化学杂志》266，189 - 197）。Ca2+与一个Mg2+离子或两个Na+离子竞争结合一个共同位点。高K+浓度大大降低了Na+和Mg2+与Ca2+竞争交换蛋白上这个共同位点的能力。结果，高内部K+诱导了交换蛋白的一种构象，从动力学角度有利于Ca2+的排出。
