Schnetkamp P P, Basu D K, Li X B, Szerencsei R T
Department of Medical Biochemistry, University of Calgary, Alberta, Canada.
J Biol Chem. 1991 Dec 5;266(34):22983-90.
Regulation of cytosolic free Ca2+ in the physiologically relevant submicromolar range was measured in isolated intact bovine rod outer segments (ROS) with the intracellular Ca(2+)-indicating dye fluo-3. Changes in free Ca2+ were compared with changes in total Ca2+ measured with 45Ca fluxes and a good qualitative correlation was observed. Ca2+ homeostasis in isolated bovine ROS was exclusively mediated via the Na-Ca-K exchanger. Free cytosolic Ca2+ concentration was lowered by an increase in the inward Na+ gradient, was raised by an increase in external K+, and was raised by depolarization of the plasma membrane. The simplest stoichiometry consistent with these qualitative observations is 4Na:(1Ca + 1K). The individual K:Ca, Na:Ca, and K:Na coupling ratios were deduced from quantitative changes in cytosolic free Ca2+ upon changes in the transmembrane Na+ and K+ gradients. The observed changes in free Ca2+ did not agree with changes in free Ca2+ calculated on the basis of the above fixed stoichiometry which may reflect the flexibility in the Ca:K coupling ratio observed before in flux experiments (Schnetkamp, P. P. M., Szerencsei, R. T., and Basu, D. K. (1991) J. Biol. Chem. 266, 198-206). The most dramatic discrepancy was observed for the Na:Ca coupling ratio: the expected very large changes in cytosolic free Ca2+ upon changes in the transmembrane Na+ gradient were not observed. Rapid Na(+)-induced Ca2+ extrusion was unable to lower cytosolic free Ca2+ below 100 nM, even under nonequilibrium conditions and despite the observation that Ca2+ influx via reverse Na-Ca-K exchange readily occurred at a free external Ca2+ concentration of 20 nM. We conclude that the Na(+)-dependent extrusion mode of the Na-Ca-K exchanger occurs in a brief (20-s) burst of high maximal velocity transport followed by a nearly complete inactivation of transport. The importance of our findings for Ca2+ homeostasis in functioning rod photoreceptors is discussed.
利用细胞内钙离子指示染料fluo-3，在分离的完整牛视杆外段（ROS）中测量了生理相关亚微摩尔范围内的胞质游离Ca2+调节情况。将游离Ca2+的变化与通过45Ca通量测量的总Ca2+变化进行比较，观察到良好的定性相关性。分离的牛视杆外段中的Ca2+稳态完全通过钠钙钾交换体介导。胞质游离Ca2+浓度因内向Na+梯度增加而降低，因细胞外K+增加而升高，因质膜去极化而升高。与这些定性观察结果一致的最简单化学计量比是4Na:(1Ca + 1K)。通过跨膜Na+和K+梯度变化时胞质游离Ca2+的定量变化推导出单个K:Ca、Na:Ca和K:Na耦合比。观察到的游离Ca2+变化与基于上述固定化学计量比计算的游离Ca2+变化不一致，这可能反映了之前通量实验中观察到的Ca:K耦合比的灵活性（施内坎普，P.P.M.， Szerencsei，R.T.和巴苏，D.K.（1991年）《生物化学杂志》266，198 - 206）。在Na:Ca耦合比方面观察到最显著的差异：未观察到跨膜Na+梯度变化时胞质游离Ca2+预期的非常大的变化。即使在非平衡条件下，快速的Na+诱导的Ca2+外排也无法将胞质游离Ca2+降低到100 nM以下，尽管观察到在细胞外游离Ca2+浓度为20 nM时，通过反向钠钙钾交换的Ca2+内流很容易发生。我们得出结论，钠钙钾交换体的Na+依赖性外排模式以短暂（20秒）的高最大速度运输爆发形式发生，随后运输几乎完全失活。讨论了我们的发现对视杆光感受器功能中Ca2+稳态的重要性。
