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Flow cytometric determination of PMCA-mediated Ca2+-extrusion in individual red blood cells
Kitty de Jong1, Frans A Kuypers
1Children's Hospital Oakland Research Institute, Oakland, California, USA. kdejong@chori.org
Background:
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.
Methods:
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.
Results:
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.
Conclusion:
We report a novel assay of PMCA activity in red blood cells that can provide qualitative information on PMCA activity in individual cells.
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