STRUCTURE OF GABA-A RECEPTOR ANION-SELECTIVE CHANNEL

Project: Research project

Project Details

Description

The long term goal of this research is to elucidate the structural basis of
ion conduction and gating in the GABAA receptor. The GABAA receptors are
the major post synaptic receptors for GABA. The binding of GABA triggers
the opening of an anion-selective channel, which is the basis of GABA's
role as the major inhibitory neurotransmitter in the brain. The GABAergic
system is essential for normal brain function and has been implicated in
the treatment and etiology of epilepsy and anxiety. The GABAA receptor is
the target of two classes of neuropsychiatric drugs, the benzodiazepines
and the barbiturates. Although the pharmacological, electrophysiological,
and molecular biological properties of the GABAA receptors have been
extensively studied, the structures of these receptors are not well-
determined beyond the sequences of their subunits. The subunits all have
similar sequences that include four hydrophobic, putative membrane-spanning
segments, named M1, M2, M3, and M4. By analogy with the homologous
acetylcholine receptor, the subunits likely form pseudosymmetrical,
pentameric rings around a central channel. The goal of this project is to
identify the residues of these segments that line the GABAA channel lumen.
Residues, initially in the alpha1 subunit M1 and M2 segments, will be
mutated to cysteine, one at a time. The mutant alpha1 subunit will be
coexpressed with the beta1 subunit in Xenopus oocytes. Only mutants which
have near-normal function when expressed in oocytes will be studied
further. These will be challenged with small, negatively charged,
sulfhydryl reagents, including iodoacetate and methanethiosulfonate-
ethylsulfonate. These reagents covalently attach a negatively charged
group to the sulfhydryl of cysteine. They are highly polar and are much
more likely to react with cysteines expressed on the receptor surface,
which includes residues lining the channel lumen. If a cysteine faces the
channel lumen and reacts with these reagents, the channel conductance
should be irreversibly altered. Reagents will be added to the
extracellular and intracellular sides of the membrane, and the effects will
be monitored by two-electrode voltage clamping or by patch clamping.
Reagents will be added in the presence and absence of GABA. By this
approach, I will identify the residues that line the anion channel, and
determine their secondary structure, the position of the ion selectivity
filter, and the position of the gate.
StatusFinished
Effective start/end date3/5/942/28/95

Funding

  • National Institute of Neurological Disorders and Stroke

ASJC

  • Cellular and Molecular Neuroscience
  • Biophysics
  • Structural Biology
  • Physiology
  • Pharmacology

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