Cloning, functional expression, and pharmacology of a GABA transporter from Manduca sexta.
Termination of synaptic transmission occurs by several mechanisms that include uptake of the neurotransmitter molecules into the presynaptic neuron by specialized membrane transport proteins. We have cloned a (DABA)-sensitive gamma-aminobutyric acid (GABA) transporter from a cDNA library from Manduca sexta embryo. The cDNA clone, MasGAT, shows high sequence homology to known mammalian GABA transporters. The transcript is about 5.5 kb with an open reading frame of 1793 bp. Injection of a 2.2-kb cRNA from this clone into Xenopus oocytes results in [3H]GABA transport. A Michaelis-Menten kinetic analysis shows that GABA transport occurs by a high-affinity and saturable process, suggesting that it is carrier-mediated. Ion substitution studies also show the transport process to be highly dependent on extracellular Na+ gradient, a finding that is consistent with properties of known mammalian neurotransmitter transporters. Although MasGAT shares certain pharmacological similarities with known mammalian GABA transporters, this transporter is pharmacologically distinct from the known mammalian GABA transporters.
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Department of Entomology, University of California, Riverside 92521, USA.
Ross
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Gill
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000378:metabolism*; D017931:DNA Primers; D004625:Embryo, Nonmammalian / Q000378:metabolism; D005260:Female; D050485:GABA Plasma Membrane Transport Proteins; D015870:Gene Expression; D015723:Gene Library; D007700:Kinetics; D018402:Manduca / Q000196:embryology / Q000378:metabolism*; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000378:metabolism*; D026901:Membrane Transport Proteins; D051379:Mice; D008969:Molecular Sequence Data; D009557:Nipecotic Acids / Q000494:pharmacology; D009865:Oocytes / Q000187:drug effects / Q000502:physiology; D027361:Organic Anion Transporters; D016133:Polymerase Chain Reaction; D011392:Proline / Q000031:analogs & derivatives*; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014982:Xenopus laevis; D005680:gamma-Aminobutyric Acid / Q000378:metabolism*
D002352:Carrier Proteins; D017931:DNA Primers; D050485:GABA Plasma Membrane Transport Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009557:Nipecotic Acids; D027361:Organic Anion Transporters; D012333:RNA, Messenger; D011994:Recombinant Proteins; C030278:nipecotic acid; D005680:gamma-Aminobutyric Acid; D011392:Proline; C010983:homoproline