Abstract
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+.
Highlights
From the Department of Medical Biochemistry, University of Calgary, Health Science Centre, Calgary, Alberta T2N 4Nl, Canada
We concluded that the Ca 2 + extrusion mode of the exchanger operates in a brief burst (10-30 s) of high velocity activity followed by a nearly complete inactivation, a novel feature for Na-Ca( + K) exchangers. As this may present a mechanism to prevent lowering of cytosolic free Ca 2 + to undesirably low values when rods are saturated with light for prolonged periods of time, this study examines 1) the activity of the plasma membrane NaCa + K exchanger on time scales between a few seconds and 40 min, 2) the contribution of internal Ca 2 + stores to Ca 2 + homeostasis under those conditions, and 3) factors that determine at which free Ca2 + concentration the Ca 2 + efflux mode of the exchanger inactivates
Exchange-The objective of the experiments reported in this study was to analyze compartmentalization of Ca 2 + fluxes in isolated intact bovine rod outer segments (ROS) and its relationship to the observed inability of the Ca 2 + extrusion mode of the ROS Na-Ca+K exchanger to lower free cytosolic Ca2 + to the low values expected from the coupling stoichiometry of 4Na:(lCa+1K) [11, 12, 14]
Summary
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. A kinetic equilibrium between the above two Ca 2 + fluxes keeps cytosolic relatively high, Light causes a decrease in Ca 2 + influx, while continued Ca2 + efflux via the exchanger lowers cytosolic Ca 2 + , and this is thought to mediate, at least in part, the process of light adaptation in both retinal rods and cones [2, 3]. The Na-Ca+K exchanger is expected to lower cytosolie free Ca 2 + below 1 nM when Ca 2 + influx via the cGMP-gated channels is interrupted for a prolonged time, e.g. when rods are saturated under bright daylight illumination
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