Flow cytometric determination of PMCA-mediated Ca2+-extrusion in individual red blood cells

Abstract

Differences among red blood cells in the activity of the plasma membrane Ca2+-ATPase (PMCA) can impact cell signaling and survival. However, no method has been reported that measures this activity directly in individual cells. We have designed a novel assay for PMCA activity that uses the fluorescent Ca2+-reporter Fluo4 and flow cytometric analysis. The method recognizes the extrusion of Ca2+ from the cell after a short Ca2+-loading pulse, which avoids the problem of ATP depletion and ascertains activity at Vmax capacity. Our assay is responsive to known PMCA inhibitors, and while not intended for quantitative kinetic analysis of Ca2+-pumping, it can be used to determine qualitative differences between red blood cell populations that vary in PMCA activity. Using this assay, we confirmed that a normal red blood cell population shows heterogeneity with respect to the PMCA Vmax. We report a novel assay of PMCA activity in red blood cells that can provide qualitative information on PMCA activity in individual cells. © 2007 International Society for Analytical Cytology.