Mutational Probes for Structure-Function Relationships in the GABA Transporter:Studies on a Presynaptic Anticonvulsant Binding Site

摘要

This is a final report submitted to effect a change of institution by the author.The report reflects progress at Baylor College of Medicine on the project which is being continued at The University of Texas Medical Branch. The human neuronal GABA transporter, hGAT-1, has been cloned and functionally expressed in mouse fibroblasts. The cloned transporter exhibits all of the properties anticipated based on studies done with the rodent transporter. Chemicals that inhibit GABA transport are potent anticonvulsants that many have utility as counter measures against the convulsive effects of organophosphorus anticholinesterases. In order to elucidate the molecular mechanism of GABA transport and pharmacological inhibition of the transporter, we have constructed mutations to probe structural features thought to be important to function. These include the hydrophilic portions of the amphipathic helices, evolutionarily conserved peptide sequences, and sites of N-linked glycosylation. The effect of these mutations is currently being studied by expression in mouse fibroblasts. Early results indicated that Ser-543 and Tyr-551 of the transmembrane helix XII are important to function. Elimination of potential N-linked glycosylation sites at positrons 176, 181, and 184 does not totally abolish transport function.