Jeremiah N. Sims, EJun Yun, Jonathan Chu, Mansoor A. Siddiqui, Sanjay A. Desai
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A robust fluorescence-based assay for human erythrocyte Ca++ efflux suitable for high-throughput inhibitor screens
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.
期刊介绍:
The journal publishes papers in the field of biophysics, which is defined as the study of biological phenomena by using physical methods and concepts. Original papers, reviews and Biophysics letters are published. The primary goal of this journal is to advance the understanding of biological structure and function by application of the principles of physical science, and by presenting the work in a biophysical context.
Papers employing a distinctively biophysical approach at all levels of biological organisation will be considered, as will both experimental and theoretical studies. The criteria for acceptance are scientific content, originality and relevance to biological systems of current interest and importance.
Principal areas of interest include:
- Structure and dynamics of biological macromolecules
- Membrane biophysics and ion channels
- Cell biophysics and organisation
- Macromolecular assemblies
- Biophysical methods and instrumentation
- Advanced microscopics
- System dynamics.