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

J Neurosci Res. 2009 Jun;87(8):1737-47. doi: 10.1002/jnr.21991.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Chemical Fractionation / methods*
  • Cytoskeletal Proteins / metabolism
  • Extracellular Space / metabolism
  • Hippocampus / metabolism
  • Hippocampus / ultrastructure
  • Mass Spectrometry / methods*
  • Membrane Proteins / metabolism
  • Mice
  • Mice, Inbred C57BL
  • Mice, Knockout
  • Neural Inhibition / physiology
  • Neurochemistry / methods*
  • Neurons / metabolism
  • Neurons / ultrastructure
  • Protein Structure, Tertiary / physiology
  • Protein Subunits / chemistry
  • Protein Subunits / metabolism
  • Proteomics / methods*
  • Receptors, GABA-A / chemistry*
  • Receptors, GABA-A / metabolism
  • Receptors, GABA-A / physiology*
  • Solubility
  • Synaptic Membranes / metabolism
  • Synaptic Membranes / ultrastructure
  • Synaptic Transmission / physiology
  • gamma-Aminobutyric Acid / metabolism

Substances

  • Cytoskeletal Proteins
  • Gabra5 protein, mouse
  • Gabrb3 protein, mouse
  • Membrane Proteins
  • Protein Subunits
  • Receptors, GABA-A
  • radixin
  • gamma-Aminobutyric Acid