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.
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