Distinct properties of murine alpha 5 gamma-aminobutyric acid type a receptors revealed by biochemical fractionation and mass spectroscopy.
Gamma-aminobutyric acid type A receptors (GABA(A)Rs) that contain the alpha 5 subunit are expressed predominantly in the hippocampus, where they regulate learning and memory processes. Unlike conventional postsynaptic receptors, GABA(A)Rs containing the alpha 5 subunit (alpha 5 GABA(A)Rs) are localized primarily to extrasynaptic regions of neurons, where they generate a tonic inhibitory conductance. The unique characteristics of alpha 5 GABA(A)Rs have been examined with pharmacological, immunostaining, and electrophysiological techniques; however, little is known about their biochemical properties. The aim of this study was to modify existing purification and enrichment techniques to isolate alpha 5 GABA(A)Rs preferentially from the mouse hippocampus and to identify the alpha 5 subunit by using tandem mass spectroscopy (MS/MS). The results showed that the detergent solubility of the alpha 5 subunits was distinct from that of alpha1 and alpha2 subunits, and the relative distribution of the alpha 5 subunits in Triton X-100-soluble fractions was correlated with that of the extracellular protein radixin but not with that of the postsynaptic protein gephyrin. Mass spectrometry identified the alpha 5 subunit and showed that this subunit associates with multiple alpha, beta, and gamma subunits, but most frequently the beta 3 subunit. Thus, the alpha 5 subunits coassemble with similar subunits as their synaptic counterparts yet have a distinct detergent solubility profile. Mass spectroscopy now offers a method for detecting and characterizing factors that confer the unique detergent solubility and possibly cellular location of alpha 5 GABA(A)Rs in hippocampal neurons.
Ju
Young H
YH
Department of Physiology, University of Toronto, Toronto, Ontario, Canada.
Guzzo
Angelina
A
Chiu
Mary W
MW
Taylor
Paul
P
Moran
Michael F
MF
Gurd
James W
JW
MacDonald
John F
JF
Orser
Beverley A
BA
D000818:Animals; D005591:Chemical Fractionation / Q000379:methods*; D003598:Cytoskeletal Proteins / Q000378:metabolism; D005110:Extracellular Space / Q000378:metabolism; D006624:Hippocampus / Q000378:metabolism / Q000648:ultrastructure; D013058:Mass Spectrometry / Q000379:methods*; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009433:Neural Inhibition / Q000502:physiology; D009448:Neurochemistry / Q000379:methods*; D009474:Neurons / Q000378:metabolism / Q000648:ultrastructure; D017434:Protein Structure, Tertiary / Q000502:physiology; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D011963:Receptors, GABA-A / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D012995:Solubility; D013570:Synaptic Membranes / Q000378:metabolism / Q000648:ultrastructure; D009435:Synaptic Transmission / Q000502:physiology; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
D003598:Cytoskeletal Proteins; C496250:Gabra5 protein, mouse; C546270:Gabrb3 protein, mouse; D008565:Membrane Proteins; D021122:Protein Subunits; D011963:Receptors, GABA-A; C071776:radixin; D005680:gamma-Aminobutyric Acid