Analysis of GBBA(A) receptor assembly in mammalian cell lines and hippocampal neurons using gamma 2 subunit green fluorescent protein chimeras

Citation
Jt. Kittler et al., Analysis of GBBA(A) receptor assembly in mammalian cell lines and hippocampal neurons using gamma 2 subunit green fluorescent protein chimeras, MOL CELL NE, 16(4), 2000, pp. 440-452
Citations number
42
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Neurosciences & Behavoir
Journal title
MOLECULAR AND CELLULAR NEUROSCIENCE
ISSN journal
1044-7431 → ACNP
Volume
16
Issue
4
Year of publication
2000
Pages
440 - 452
Database
ISI
SICI code
1044-7431(200010)16:4<440:AOGRAI>2.0.ZU;2-Y
Abstract
Type A gamma -aminobutyric acid receptors (GABA(A)), the major sites of fas t synaptic inhibition in the brain, ave believed to be predominantly compos ed 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-G FPN receptors expressed in A293 cells. Furthermore, this chimera allowed th e visualization of receptor membrane targeting and endocytosis in live cell s. In contrast, incorporation of GFP at the C-terminus reduced subunit stab ility, impairing assembly with receptor alpha and beta subunits. Using gamm a 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 tha t the assembly and membrane targeting of GABA(A) receptors composed of alph a1 beta2 gamma 2L-GFPN subunits follow similar itineraries in heterologous systems and neurons.