Gamma aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the mammalian brain. GABA binds to postsynaptic GABA-A receptors and gates (opens) a chloride ion-pore integral to the receptor complex. The resulting chloride flux across the membrane inhibits the postsynaptic neuron. The binding of GABA and the subsequent opening of the pore is collectively termed activation. The long term objective of this project is to gain insight into the molecular mechanism of GABA channel activation. Five classes of GABA channel subunits (alpha, beta, gamma, delta, and rho), with multiple isoforms in each class, have thus far been identified in the mammalian brain. The most recently cloned class of subunits, rho, is unique in that it forms homomeric channels when expressed in Xenopus laevis oocytes. Investigating homomeric channels greatly simplifies an analysis of the relationship between channel structure and function. In addition, homomeric rho GABA channels have very different activation and pharmacological properties than typical heteromeric GABA channels (e.g. alpha, beta, gamma). This project takes advantage of these differences between the two GABA channel subtypes to identify GABA channel activation domains. Based on information on the location of the agonist binding site of heteromeric GABA channels, site-directed mutagenesis and oocyte expression will be used to identify potential agonist binding site amino acids of rho-GABA channels. Single-channel kinetic analysis will verify if mutation of the identified amino acids disrupts agonist binding or later steps in channel activation. In addition, recombinant DNA techniques will be used to swap domains between the rho and beta subunit. (The beta subunit contains a major component of the GABA binding site in alpha-beta-gamma GABA channels.) Swapping increasingly smaller (as well as other) domains, coupled with site-directed mutagenesis, will identify the regions and amino acids that determine the activation properties of GABA channels. And lastly, experiments are proposed that use the binding-site mutants as probes to gain insight into the GABA channel gating mechanism. Dysfunctions of GABA-mediated inhibition have been implicated in some brain disorders, most notably epilepsy. In addition, a variety of clinically-prescribed drugs used to treat convulsive disorders (i.e., barbiturates and benzodiazepines) exert their therapeutic effects, at least in part, by altering GABA channel function. Insights into the molecular properties of GABA channels are crucial for understanding the mechanisms of these drugs and may aid in the design of novel, more effective, GABA channel modulators. Information derived from this project should make significant steps towards understanding the mechanism of GABA channel activation.
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