MUTAGENIC ANALYSIS OF NEUROTRANSMITTER TRANSPORTERS

The proposed research focuses on mutagenic analysis of the GABA (gamma-
aminobutyric acid) transporter. This work should be of general interest:
1) as a model system to clarify the mechanism by which presynaptic
neurotransmitter reuptake proteins regulate synaptic transmission, and 2)
as a model system in which to clarify the role of presynaptic
neurotransmitter reuptake protein in central nervous system diseases. Neurotransmitter reuptake into the presynaptic nerve terminal is the
dominant mechanism for terminating synaptic transmission mediated by a wide
array of the small molecule neurotransmitters (e.g., epinephrine,
norepinephrine, serotonin, taurine, aspartate, glutamate, glycine, and
GABA). Such neurotransmitters are removed from the synaptic cleft by
specific, high-affinity, (Na+ and C1-)-dependent transport proteins. Although transport proteins from this class are directly relevant to
several commonly occurring medical conditions - including drug dependence
(norepinephrine transporter/cocaine receptor), clinical depression
(serotonin transporter/antidepressant receptor), and epilepsy (GABA
transporter/anticonvulsant receptor) - it has not been possible until
recently to study any of these transporters in molecular detail. To date,
information concerning the relationship between the function of these
transporters and their molecular structure is completely lacking. The
proposed research will make a significant contribution in this area by
using mutagenesis to probe structural aspects of the GABA transporter that
are functionally important. The applicant has already developed the expression systems required to use
mutagenesis as a means of probing structure/function relationships in the
GABA transporter, and is pursuing a research plan to: 1) Engineer specific
alterations into the GABA transporter using oligonucleotide-directed
mutagenesis; 2) Evaluate the functional consequence of mutation by
expression in mouse fibroblasts. In broadest terms, the applicant's proposal is significant in that the
methodologies developed specifically to the study of structure/function
relationships in the GABA transporter should be widely applicable to the
genetic characterization of other neurotransmitter reuptake proteins. More
specifically, structural information gained from mutagenesis of the GABA
transporter may speed the development of novel anticonvulsants acting to
inhibit the GABA transporter in brain.