37] Measurement of sodium-calcium exchange activity in plasma membrane vesicles

Abstract

Publisher Summary This chapter describes the techniques, and some of the pitfalls, of measuring Na–Ca exchange activity in osmotically sealed plasma membrane vesicles. The discussion is focused on sarcolemmal membranes obtained from cardiac muscle, but the techniques are applicable to other types of vesicle preparations as well. Na–Ca exchange activity is assayed by measuring the Ca 2+ movements, which occur when transmembrane Na + concentration gradients are generated by appropriate loading and dilution procedures. Ca 2+ uptake will occur when the vesicles are loaded internally with NaCI and then diluted into an isosmotic, Na + -free medium containing CaCI 2 . Na + -dependent Ca 2+ efflux can be measured by equilibrating the vesicles with CaCI 2 in a Na + -free medium, and then diluting the Ca 2+ -loaded vesicles into an isosmotic medium containing NaCl. Na-Ca exchange is a carrier-mediated transport process in which transmembrane movements of calcium ions in one direction are directly coupled to sodium ion movements in the opposite direction. It is found primarily in plasma membranes of excitable cells, such as muscle and nerve where it appears to function as a mechanism for extruding Ca 2+ from the cell.