Cloning, functional expression, and pharmacology of a GABA transporter from Manduca sexta

Abstract

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.