TY - JOUR
T1 - Analysis of GABAA receptor assembly in mammalian cell lines and hippocampal neurons using γ2 subunit green fluorescent protein chimeras
AU - Kittler, Josef T.
AU - Wang, Jianfeng
AU - Connolly, Christopher N.
AU - Vicini, Stefano
AU - Smart, Trevor G.
AU - Moss, Stephen J.
PY - 2000
Y1 - 2000
N2 - Type A ?-aminobutyric acid receptors (GABA(A)), the major sites of fast
synaptic inhibition in the brain, are believed to be predominantly
composed of a, ß, and ? subunits. To examine the membrane trafficking of
GABA(A) receptors we have produced ?2L subunit chimeras with green
fluorescent protein (GFP). Addition of GFP to the N-terminus of the ?2
subunit (?2L-GFPN) was functionally silent for a1ß2?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 a and ß subunits.
Using ?2L-GFPN we were able to demonstrate that targeting of the ?2
subunit to GABAergic synapses in hippocampal neurons was dependent upon
coassembly with receptor a and subunits. Together our results
demonstrate that the assembly and membrane targeting of GABA(A)
receptors composed of a1ß2?2L-GFPN subunits follow similar itineraries
in heterologous systems and neurons.
AB - Type A ?-aminobutyric acid receptors (GABA(A)), the major sites of fast
synaptic inhibition in the brain, are believed to be predominantly
composed of a, ß, and ? subunits. To examine the membrane trafficking of
GABA(A) receptors we have produced ?2L subunit chimeras with green
fluorescent protein (GFP). Addition of GFP to the N-terminus of the ?2
subunit (?2L-GFPN) was functionally silent for a1ß2?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 a and ß subunits.
Using ?2L-GFPN we were able to demonstrate that targeting of the ?2
subunit to GABAergic synapses in hippocampal neurons was dependent upon
coassembly with receptor a and subunits. Together our results
demonstrate that the assembly and membrane targeting of GABA(A)
receptors composed of a1ß2?2L-GFPN subunits follow similar itineraries
in heterologous systems and neurons.
U2 - 10.1006/mcne.2000.0882
DO - 10.1006/mcne.2000.0882
M3 - Article
SN - 1044-7431
VL - 16
SP - 440
EP - 452
JO - Molecular and Cellular Neuroscience
JF - Molecular and Cellular Neuroscience
IS - 4
ER -