Type A ¿-aminobutyric receptors (GABAAR) are Cl--preferring ligand gated ion channels
that mediate fast phasic synaptic inhibition in the adult brain and are are drug targets for barbiturates,
benzodiazepines, intravenous anesthetics, and neurosteroids. Consistent with their essential role in regulating
neuronal excitability, modification in GABAAR activity contribute to anxiety, autism, depression, epilepsy,
substance abuse and schizophrenia. The receptors are heteropentamers that can be assembled from a(1-6),
ß(1-3), ¿(1-3), d, e, ¿ and p subunits. Surprisingly, to date our comprehension of the molecular structure of native
GABAARs subtypes, and how these proteins are targeted to inhibitory synapses is rudimentary. However, these
issues are of fundamental importance given the critical role GABAARs play in determining neuronal excitability,
in neuropsychiatric diseases and as drug targets.
To address these issues, we will determine the structure of the principal GABAAR subtypes assembled
from a1 and a2 subunits in the brain. We will then assess which proteins co-purify with these receptor subtypes.
The roles that the most abundant “receptor-associated proteins” play in the synaptic targeting of distinct GABAAR
subtypes will then be examined. Our studies will be facilitated by the novel mouse lines we have developed in
which the N-terminus of the a1 and a2 subunits have been modified with distinct fluorescent reporters;
pHlourin/9E10-a2 (pHa2) and mKate/FLAG-a1 (mKa1). These additions are functionally silent but allow
sequential affinity purification of individual GABAAR subtypes, and analysis of their structure using blue-native
polyacrylamide gel electrophoresis (BN-PAGE) followed by liquid chromatography coupled mass spectroscopy
(LC-MS/MS). Preliminary experiments using these new tools have allowed us to formulate a central hypothesis
that will be tested here; a2- and a1-subunits are assembled into two distinct GABAAR subtypes; ‘a2/a1’
containing equimolar amounts of both a subunits (a2a1ß¿2), and ‘a1/a1’ which does not contain an a2
subunit (a1ß¿2). These subtypes are targeted to distinct subsets of synapses by spectrins, which are
intimately associated with these GABAAR subtypes. Our experiments will focus on the following specific
Aim 1. To test the hypothesis that neurons assemble GABAAR subtypes containing a2a1ß¿2 and
a1ß¿2 subunits. Aim 2. To test the hypothesis that the a2/a1 and a1/a1 GABAAR subtypes are associated
with distinct spectrins. Aim 3. To test the hypothesis that spectrins facilitate the synapse-specific
targeting of GABAARs.
Collectively, our study will provide novel insights into the structure of native GABAARs and the processes
that regulate their accumulation at inhibitory synapses, information that may lead to improved understanding of,
and treatments for epilepsy and other neuropsychiatric disorders.