NMDA Receptor Activity in Circulating Red Blood Cells: Methods of Detection.

Abundance and activity of N-methyl-D-aspartate (NMDA) in circulating red blood cells contributes to the maintenance of intracellular Ca2+ in these cells and, by doing that, controls red cell volume, membrane stability, and O2 carrying capacity. Detection of the NMDA receptor activity in red blood cells is challenging as the number of its copies is low and shows substantial cell-to-cell heterogeneity. Receptor abundance is reliably assessed using the radiolabeled antagonist ([3H]MK-801) binding technique. Uptake of Ca2+ following the NMDA receptor activation is detected in cells loaded with Ca2+-sensitive fluorescent dye Fluo-4 AM. Both microfluorescence live-cell imaging and flow cytometry may be used for fluorescence intensity detection. Automated patch clamp is currently used for recording of electric currents triggered by the stimulation of the NMDA receptor. These currents are mediated by the Ca2+-sensitive K+ (Gardos) channels that open upon Ca2+ uptake via the active NMDA receptor. Furthermore, K+ flux through the Gardos channels induced by the NMDA receptor stimulation in red blood cells may be detected using unidirectional K+(86Rb+) influx.