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.