Distinct properties of murine alpha 5 gamma-aminobutyric acid type a receptors revealed by biochemical fractionation and mass spectroscopy

Abstract

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.