Since GABA (4-aminobutyrate) is both a major inhibitory neurotransmitter in brain (extracellular concentration is regulated), and a key intermediate linking nitrogen and carbohydrate metabolism in cells from neurons to bacteria (intracellular concentration is regulated), its translocation mechanism is of significant interest. Therefore, the long- term goal is to elucidate the mechanism(s) by which GABA is translocated across biological membranes. Since several GABA transporters are now cloned, key GAPS IN KNOWLEDGE include the need: (A) to identity a candidate translocation pathway, and (B)to learn how the structure of that pathway determines transport function. The leading candidate is an amphipathic helical structure called CAR, which (i) exhibits channel-like properties in GABA transporters from the APC Superfamily, and (ii) is also present in unrelated GABA transporters from the Neurotransmitter Transporter Superfamily. This research therefore uses bacterial and human GABA transporters as parallel models to test broadly the central hypothesis that CAR is part of a translocation pathway. The approach is to learn whether CAR exhibits key structural and functional properties expected for a translocation pathway.
The specific aims are: (1) to test (via pharmacology, kinetics, and mutagenesis) the hypothesis that residues on the hydrophilic face of the CAR determine translocation rates and ligand recognition patterns; (2) to test the hypothesis that the """"""""signature cysteine"""""""" within CAR exists in a channel-like environment that acts as a bi-directional sieve for thiol-reagents of differing structure; (3) to test the hypotheses that CAR contains a long polar face that can be disrupted by offsetting helical periodicity via Ala-insertion mutagenesis, and to test (via sulfhydryl-specific site-directed radiolabeling) the sub-hypothesis that only a portion of the Ala- disruptable domain in CAR lies membrane-imbedded; and (4) to test the predictive hypothesis (via point mutagenesis and chimera construction) that the CAR-like element in a human GABA transporter also modulates key ligand recognition properties and translocation rates. The project is health-related since domain-level and specific structure-function detail will be strategically helpful in approaching disorders such as posthypoxic myoclonus and epilepsy which involve a neurological deficit that can be pharmacologically ameliorated by GABA transport inhibitors.
