A robust fluorescence-based assay for human erythrocyte Ca++ efflux suitable for high-throughput inhibitor screens

Abstract

Intracellular calcium is maintained at very low concentrations through the action of PMCA Ca⁺⁺ extrusion pumps. Although much of our knowledge about these Ca⁺⁺ extrusion pumps derives from studies with human erythrocytes, kinetic studies of Ca⁺⁺ transport for these cells are limited to radioisotope flux measurements. Here, we developed a robust, microplate-based assay for erythrocyte Ca⁺⁺ efflux using extracellular fluorescent Ca⁺⁺ indicators. We optimized Ca⁺⁺ loading with the A23187 ionophore, established conditions for removal of the ionophore, and adjusted fluorescent dye sensitivity by addition of extracellular EGTA to allow continuous tracking of Ca⁺⁺ efflux. Efflux kinetics were accelerated by glucose and inhibited in a dose-dependent manner by the nonspecific inhibitor vanadate, revealing that Ca⁺⁺ pump activity can be tracked in a 384-well microplate format. These studies enable radioisotope-free kinetic measurements of the Ca⁺⁺ pump and should facilitate screens for specific inhibitors of this essential transport activity.