Ensemble and single particle fluorimetric techniques in concerted action to study the diffusion and aggregation of the glycine receptor α3 isoforms in the cell plasma membrane

The spatio-temporal membrane behavior of glycine receptors (GlyRs) is known to be of influence on receptor homeostasis and functionality. In this work, an elaborate fluorimetric strategy was applied to study the GlyR alpha 3K and L isoforms. Previously established differential clustering, desensitization and synaptic localization of these isoforms imply that membrane behavior is crucial in determining GlyR alpha 3 physiology. Therefore diffusion and aggregation of homomeric alpha 3 isoform-containing GlyRs were studied in HEK 293 cells. A unique combination of multiple diffraction-limited ensemble average methods and subdiffraction single particle techniques was used in order to achieve an integrated view of receptor properties. Static measurements of aggregation were performed with image correlation spectroscopy (ICS) and, single particle based, direct stochastic optical reconstruction microscopy (dSTORM). Receptor diffusion was measured by means of raster image correlation spectroscopy (RICS), temporal image correlation spectroscopy (TICS), fluorescence recovery after photobleaching (FRAP) and single particle tracking (SPT). The results show a significant difference in diffusion coefficient and cluster size between the isoforms. This reveals a positive correlation between desensitization and diffusion and disproves the notion that receptor aggregation is a universal mechanism for accelerated desensitization. The difference in diffusion coefficient between the clustering GlyR alpha 3L and the non-clustering GlyR alpha 3K cannot be explained by normal diffusion. SPT measurements indicate that the alpha 3L receptors undergo transient trapping and directed motion, while the GlyR alpha 3K displays mild hindered diffusion. These findings are suggestive of differential molecular interaction of the isoforms after incorporation in the membrane. (C) 2012 Elsevier B.V. All rights reserved.