Glycine Receptor Oligomerization Characterized by Number and Brightness Analysis

Abstract

Glycine receptors (GlyR) are ligand-gated chloride channels that mediate synaptic inhibition in the CNS. A loss of GlyR function by single site mutations is the major cause hyperekplexia, a rare neuromotor disorder that is characterized by exaggerated startle reflexes. The V280M mutation in the α1 subunit causes heperekplexia but, surprisingly, results in a gain of function of the receptor. The mutant receptor shows spontaneous activation and prolonged deactivation. It has been suggested that V280M might change GlyRs oligomerization and the formation of clusters. We tested this idea by using Number and Brightness (N&B) analysis to determine GlyR oligomerization. A SNAP tag was fused to the N-terminal of wt and V280M human α1 GlyR. Receptors were expressed in HEK-293 cells. Cells were incubated with the Atto-488 fluorescent probe that covalently labels SNAP tag-containing receptors. N&B analysis measures the variance in fluorescence as receptors diffuse on the surface of the basolateral cell membrane. Larger variance corresponds to larger diffusing entities - multimers of GlyRs. Our tentative stoichiometry assignment is monomer, dimer, 4-mer and 8-mer. Using outside-out patch clamp recordings of macroscopic currents, we found that the SNAP-tagged wt and V280M GlyRs had similar glycine-sensitivity and kinetics compared to the corresponding untagged receptors. Using fluorescence measurements, we found that SNAP-tagged wt receptors tended to form dimers and 4-mers as the dominant species. In the presence of 3 mM glycine ,monomers were predominant. Addition of 1 µM strychnine antagonized the effect of glycine. SNAP-tagged V280M receptors exhibit a higher level of oligomerization than wt receptors. In preliminary data from 12 cells, the 8-mer species was dominant. Further experiments are needed to determine whether changes in receptor clustering underlie the hyperekplexia phenotype of V280M α1 GlyRs.