Schnetkamp P P
Department of Medical Biochemistry, University of Calgary, Alberta, Canada.
J Biol Chem. 1995 Jun 2;270(22):13231-9. doi: 10.1074/jbc.270.22.13231.
The roles of 1) inactivation of Na-Ca+K exchange and 2) Ca2+ release from discs in regulation of cytosolic free Ca2+ were examined in intact rod outer segments (ROS) purified from bovine retinas. Measurements of cytosolic free Ca2+ (with fluo-3) were combined with Ca2+ flux measurements (45Ca) in ROS that contained about 600 microM total Ca2+. Na(+)-induced Ca2+ extrusion was measured in a Ca(2+)-free medium and did not lower cytosolic free Ca2+ to below 1 nM as expected from a coupling stoichiometry of 4Na+:(1Ca(2+) + 1K+). Instead, cytosolic free Ca2+ was rapidly (20 s) lowered from about 1300 nM to 100-150 nM, while at the same time about 35% of total ROS Ca2+ was removed. During the next 40 min cytosolic free Ca2+ remained virtually steady, but total ROS Ca2+ was reduced by a further 50% at a 100-fold lower rate than that observed for the initial fast phase. The steady cytosolic Ca2+ concentration resulted from Ca2+ release from discs and subsequent removal across the plasma membrane by Na-Ca+K exchange operating at a greatly reduced rate. Addition of the alkali cation channel ionophore gramicidin led to a persistent increase in cytosolic free Ca2+ concentration to about 400 nM, presumably caused by an increase in intracellular Na+. It is suggested that cytosolic free Ca2+ is not determined by the Na+:Ca2+ coupling ratio of the exchanger, but rather by a sensor on its cytoplasmic domain that controls inactivation of the Ca2+ extrusion mode and is sensitive to intracellular Ca2+, Na+, and K+.
在从牛视网膜纯化的完整视杆细胞外段（ROS）中，研究了1）钠-钙钾交换失活和2）从盘膜释放钙离子在调节胞质游离钙离子方面的作用。利用荧光素-3测量胞质游离钙离子，并结合含有约600微摩尔总钙的ROS中的钙离子通量测量（45Ca）。在无钙培养基中测量钠诱导的钙离子外流，结果并未如4Na+：（1Ca2+ + 1K+）的耦合化学计量所预期的那样将胞质游离钙离子降低至1纳摩尔以下。相反，胞质游离钙离子迅速（20秒）从约1300纳摩尔降至100 - 150纳摩尔，同时约35%的ROS总钙离子被移除。在接下来的40分钟内，胞质游离钙离子基本保持稳定，但ROS总钙离子以比初始快速阶段低100倍的速率进一步降低了50%。稳定的胞质钙离子浓度是由盘膜释放钙离子以及随后通过以大幅降低速率运行的钠-钙钾交换穿过质膜移除钙离子所导致的。添加碱性阳离子通道离子载体短杆菌肽导致胞质游离钙离子浓度持续增加至约400纳摩尔，推测这是由细胞内钠离子增加引起的。研究表明，胞质游离钙离子不是由交换体的钠：钙耦合比率决定的，而是由其细胞质结构域上的一种传感器决定，该传感器控制钙离子外流模式的失活，并且对细胞内的钙离子、钠离子和钾离子敏感。
