Selective amino acid substitutions convert the creatine transporter to a gamma-aminobutyric acid transporter.
The creatine transporter (CRT) is a member of a large family of sodium-dependent neurotransmitter and amino acid transporters. The CRT is closely related to the gamma-aminobutyric acid (GABA) transporter, GAT-1, yet GABA is not an effective substrate for the CRT. The high resolution structure of a prokaryotic homologue, LeuT has revealed precise details of the substrate binding site for leucine (Yamashita, A., Singh, S. K., Kawate, T., Jin, Y., and Gouaux, E. (2005) Nature 437, 215-223). We have now designed mutations based on sequence comparisons of the CRT with GABA transporters and the LeuT structural template in an attempt to alter the substrate specificity of the CRT. Combinations of two or three amino acid substitutions at four selected positions resulted in the loss of creatine transport activity and gain of a specific GABA transport function. GABA transport by the "gain of function" mutants was sensitive to nipecotic acid, a competitive inhibitor of GABA transporters. Our results show LeuT to be a good structural model to identify amino acid residues involved in the substrate and inhibitor selectivity of eukaryotic sodium-dependent neurotransmitter and amino acid transporters. However, modification of the binding site alone appears to be insufficient for efficient substrate translocation. Additional residues must mediate the conformational changes required for the diffusion of substrate from the binding site to the cytoplasm.
Dodd
Joanna R
JR
Molecular, Cellular, and Developmental Biology Section, School of Biological Sciences, University of Auckland, Private Bag 92019, Auckland Mail Centre, Auckland 1142, New Zealand.
Christie
David L
DL
D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D001693:Biological Transport, Active / Q000235:genetics; D019556:COS Cells; D002417:Cattle; D002522:Chlorocebus aethiops; D050485:GABA Plasma Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D020125:Mutation, Missense; D009557:Nipecotic Acids / Q000494:pharmacology; D051381:Rats; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity / Q000235:genetics
D050485:GABA Plasma Membrane Transport Proteins; D026901:Membrane Transport Proteins; D009557:Nipecotic Acids; C080679:creatine transporter; C030278:nipecotic acid