Analysis of GABAA receptor assembly in mammalian cell lines and hippocampal neurons using gamma 2 subunit green fluorescent protein chimeras.
Type A gamma-aminobutyric acid receptors (GABAA), the major sites of fast synaptic inhibition in the brain, are believed to be predominantly composed of alpha, beta, and gamma subunits. To examine the membrane trafficking of GABAA receptors we have produced gamma 2L subunit chimeras with green fluorescent protein (GFP). Addition of GFP to the N-terminus of the gamma 2 subunit (gamma 2L-GFPN) was functionally silent for alpha 1 beta 2 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 gamma 2 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 GABAA receptors composed of alpha 1 beta 2 gamma 2L-GFPN subunits follow similar itineraries in heterologous systems and neurons.
Kittler
J T
JT
Medical Research Council Laboratory of Molecular Cell Biology, University College London, United Kingdom.
Wang
J
J
Connolly
C N
CN
Vicini
S
S
Smart
T G
TG
Moss
S J
SJ
D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D004705:Endocytosis / Q000502:physiology; D015870:Gene Expression / Q000502:physiology; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D006624:Hippocampus / Q000166:cytology*; D006801:Humans; D007202:Indicators and Reagents / Q000378:metabolism; D015640:Ion Channel Gating / Q000502:physiology; D007668:Kidney / Q000166:cytology; D008024:Ligands; D008164:Luminescent Proteins / Q000235:genetics; D008322:Mammals; D051379:Mice; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011963:Receptors, GABA-A / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013569:Synapses / Q000378:metabolism
D007202:Indicators and Reagents; D008024:Ligands; D008164:Luminescent Proteins; D011963:Receptors, GABA-A; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D011493:Protein Kinase C