Intracellular Ca2+ sequestration and release in intact bovine retinal rod outer segments. Role in inactivation of Na-Ca+K exchange

Abstract

Intracellular Ca2+ sequestration and Ca2+ release was analyzed in intact rod outer segments (ROS) purified from bovine retinas. Ca2+ influx in Ca(2+)-depleted and fully bleached ROS was mediated exclusively by the Na-Ca+K exchanger and was measured both as a rise in cytosolic free Ca2+ with fluo 3 and as a total transmembrane Ca2+ flux with 45Ca. Ca2+ fluxes across the ROS plasma membrane were not completely reversible, in small part due to inactivation of the Ca2+ extrusion mode of the Na-Ca+K exchanger but mostly due to sequestration of cytosolic Ca2+ into the intradiskal space. Ca2+ release from the intradiskal space into the cytosol could be induced by low concentrations of the Ca2+ ionophore A23187 which gave rise to increases in cytosolic free Ca2+ by several hundred nanomolar (low A23187 concentrations did not affect the Ca2+ permeability of the ROS plasma membrane). We have used intracellular Ca2+ release induced by A23187 to examine inactivation of the rapid Ca2+ efflux mode mediated by the plasma membrane Na-Ca+K exchanger. Inactivation of Na-Ca+K exchange appeared to be dependent on intradiskal Ca2+ as it was abolished by selective Ca2+ permeabilization of the disk membrane by A23187. We discuss possible physiological roles for Ca2+ sequestration and release from ROS disks.