Membrane distribution of the glycine receptor alpha 3 studied by optical super-resolution microscopy

Abstract

In this study, the effect of glycine receptor (GlyR) alpha 3 alternative RNA splicing on the distribution of receptors in the membrane of human embryonic kidney 293 cells is investigated using optical super-resolution microscopy. Direct stochastic optical reconstruction microscopy is used to image both alpha 3K and alpha 3L splice variants individually and together using single-and dual-color imaging. Pair correlation analysis is used to extract quantitative measures from the resulting images. Autocorrelation analysis of the individually expressed variants reveals clustering of both variants, yet with differing properties. The cluster size is increased for alpha 3L compared to alpha 3K (mean radius 92 +/- 4 and 56 +/- 3 nm, respectively), yet an even bigger difference is found in the cluster density (9,870 +/- 1,433 and 1,747 +/- 200 mu m(-2), respectively). Furthermore, cross-correlation analysis revealed that upon co-expression, clusters colocalize on the same spatial scales as for individually expressed receptors (mean co-cluster radius 94 +/- 6 nm). These results demonstrate that RNA splicing determines GlyR alpha 3 membrane distribution, which has consequences for neuronal GlyR physiology and function.