Abstract
Two novel gamma-aminobutyric acid (GABA) transporters, GAT3 and GAT4, were cloned from the mouse neonatal brain cDNA library and expressed in Xenopus oocytes. Sequence analysis indicated they were members of the Na(+)-dependent neurotransmitter transporter family. The GABA uptake activities were measured in cRNA injected Xenopus oocytes. The Km for GABA uptake by GAT3 was 18 microM and by GAT4 was 0.8 microM. GAT3 also transports beta-alanine and taurine with Km of 28 and 540 microM, respectively. Similarly, GAT4 transports beta-alanine with Km of 99 microM and taurine with a Km of 1.4 mM. The newly cloned GABA transporters were compared with two previously cloned GABA transporters, GAT1 and GAT2, in terms of molecular and pharmacological properties. While GAT1 and GAT4 gene expression were neural specific, GAT2 and GAT3 mRNAs were detected in other tissues such as liver and kidney, in which GAT3 mRNA was especially abundant. The expression of GAT3 mRNA in mouse brain is developmentally regulated, and its mRNA is abundant in neonatal brain but not in adult brain. High affinity GABA transporters GAT1 and GAT4 were more sensitive to inhibition by nipecotic acid. Low affinity GABA transporters GAT2 and GAT3 were inhibited most effectively by betaine and beta-alanine, respectively. The differential tissue distribution and distinct pharmacological properties of those four GABA transporters suggest functional specialization in the mechanisms of GABA transmission termination.
Highlights
Two novel y-aminobutyric acid (GABA) transporters, GAT3andGAT4, were cloned from the mouse neonatal brain cDNAlibrary and expressed in Xenopus oocytes
GATl and GAT4gene expression were neural specific, GAT2 and GAT3 mRNAs were detected in other tissues such as liver and kidney, in which GAT3 mRNA was especially abundant
In mouse brain is developmentally regulated, and its mRNA is abundant in neonatal brain but not in adult brain
Summary
The RNA of the XL1-blue overnight cell culture was mixed with 200 p1 of phage gel was stained with ethidium bromide in 10 mM sodium phosphate stock and 1 pl of R408 helper phage (106-7pfu/ml) and incubated a t buffer (pH 7.2), for 30 min. The mixture was incubated a t 37 "C for 15 Prehybridization for 2 h and hybridization overnight with specific min, and 50pl of the mixture were spread on LB-agar plates contain- probes were carried out in the samehybridization buffer A single positive plaque was picked up with sodium phosphate buffer (pH 7.2), and 1 mM EDTA) twice for 15 min each, with washing solution 2
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