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Volume 270, Number 22, Issue of June 2, pp. 13231-13239, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.

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How Does the Retinal Rod Na-CaK Exchanger Regulate Cytosolic Free Ca?

Paul P. M. Schnetkamp

The roles of 1) inactivation of Na-Ca+K exchange and 2) Ca release from discs in regulation of cytosolic free Ca were examined in intact rod outer segments (ROS) purified from bovine retinas. Measurements of cytosolic free Ca (with fluo-3) were combined with Ca flux measurements (Ca) in ROS that contained about 600 µM total Ca. Na-induced Ca extrusion was measured in a Ca-free medium and did not lower cytosolic free Ca to below 1 nM as expected from a coupling stoichiometry of 4Na:(1Ca+1K). Instead, cytosolic free Ca was rapidly (20 s) lowered from about 1300 nM to 100-150 nM, while at the same time about 35% of total ROS Ca was removed. During the next 40 min cytosolic free Ca remained virtually steady, but total ROS Ca was reduced by a further 50% at a 100-fold lower rate than that observed for the initial fast phase. The steady cytosolic Ca concentration resulted from Ca 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 Ca concentration to about 400 nM, presumably caused by an increase in intracellular Na. It is suggested that cytosolic free Ca is not determined by the Na:Ca coupling ratio of the exchanger, but rather by a sensor on its cytoplasmic domain that controls inactivation of the Ca extrusion mode and is sensitive to intracellular Ca, Na, and K.

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