Analysis of GBBAA receptor assembly in mammalian cell lines and hippocampal neurons using γ2 subunit green fluorescent protein chimeras

Type A gamma -aminobutyric acid receptors (GABA(A)), the major sites of fast synaptic inhibition in the brain, ave believed to be predominantly composed of alpha, beta, and gamma subunits. To examine the membrane trafficking of GABA(A) receptors we have produced gamma 2L subunit chimeras with green fluorescent protein (GFP), Addition of GFP to the N-terminus of the gamma2 subunit (gamma 2L-GFPN) was functionally silent for alpha1 beta2 gamma 2L-GFPN receptors expressed in A293 cells. Furthermore, this chimera allowed the visualization of receptor membrane targeting and endocytosis in live cells. In contrast, incorporation of GFP at the C-terminus reduced subunit stability, impairing assembly with receptor alpha and beta subunits. Using gamma 2L-GFPN we were able to demonstrate that targeting of the gamma2 subunit to GABAergic synapses in hippocampal neurons was dependent upon coassembly with receptor alpha and beta subunits. Together our results demonstrate that the assembly and membrane targeting of GABA(A) receptors composed of alpha1 beta2 gamma 2L-GFPN subunits follow similar itineraries in heterologous systems and neurons.