Probing Dynamic Heterogeneity in Aggregated Ion Channels in Live Cells

N-Methyl-d-aspartate (NMDA) receptor is a crucial ion channel requiring the concurrent binding of both agonists glycine and glutamate along with the membrane depolarization for its opening and mediates calcium influx, which triggers the signal transduction important for synaptic transmission and plasticity. The efficiency and regulations of such cellular physiological processes are crucially relying on inherent molecular spatial organization and interactions of these channels with ligands as well as trafficking in the confined local environment. To decipher their organization, we have combined two imaging microscopic approaches, photobleaching step counting and single particle tracking analysis, that allow detection of unique organized patterns of NMDA receptor ion channel in the cell membrane. A broad range of photobleaching steps is observed ranging from 1 to 16. Single particle tracking analysis is performed to see the surface mobility of NMDA receptors and measured diffusion coefficient for all the trajectories, which on correlating with photobleaching steps shows that the higher diffusion coefficient corresponds to the lower number of bleaching steps and vice versa. These studies clearly indicate that NMDA receptors are organized on the cell membrane in clusters with different stoichiometry. Our findings for the clustering effect of NMDA receptor ion channel on the live cell membrane represent an important step forward for understanding the functioning of NMDA receptor ion channels.