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

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 -