Distinct, Developmentally Regulated Brain mRNAs Direct the Synthesis of Neurotransmitter Transporters

Abstract

The Xenopus laevis oocyte expression system was utilized to define developmental and structural properties of neurotransmitter transporter mRNAs and the pharmacological characteristics of encoded carriers independent of the complexities of brain tissue preparations. Poly(A)+ RNA from dissected brain regions of neonatal and adult rats was microinjected into Xenopus oocytes and the expression of Na(+)-dependent neurotransmitter transporters determined 48 h later. Transport studies conducted with oocytes injected with RNAs derived from juvenile rat tissues indicate a region- and transporter-specific, postnatal increase in mRNA abundance as a major factor in the developmental changes observed for brain high-affinity amino acid uptake systems. Both L-glutamic acid (Glu) and gamma-aminobutyric acid (GABA) uptake systems were detectable by day 3 in postnatal forebrain mRNA and became progressively enriched during the next 2 weeks of forebrain development. In contrast, brainstem Glu and GABA transporter enrichment was 60-70% of adult values by day 3 and exceeded adult levels by day 10. Parallel determinations of L-glutamic acid decarboxylase mRNA abundance during development argue for distinct regulatory influences on mRNAs directing transmitter synthesis and reuptake. Glycine uptake could not be detected at any point of forebrain development and exhibited a gradual postnatal rise to adult levels over the first 3 postnatal weeks of brainstem development. Uptake studies conducted with well-characterized inhibitors of Glu, GABA, dopamine, and choline transport (D-aspartate, nipecotic acid, nomifensine, and hemicholinium-3, respectively) revealed that oocyte transporters encoded by adult rat brain mRNAs retained antagonist sensitivities exhibited by in vitro brain preparations. In addition, a differential regional sensitivity to the Glu transport antagonist dihydrokainate (1 mM) was observed, lending support to previous reports of region-specific Glu transporter subtypes. To determine the structural diversity present among brain transporter mRNAs, poly(A)+ RNA was size-fractionated on linear (10-31%) sucrose density gradients prior to oocyte injection. These experiments revealed two mRNA size classes (2.4-3.0 kb, 4.0-4.5 kb) independently capable of directing the synthesis of Glu, GABA, and glycine transporters. In regions other than the cerebellum, Glu and GABA transporter activities migrated as single, yet distinct, peaks of 4.0-4.5 kb. In contrast, both Glu and GABA transporters exhibited major peaks of activity at 2.5-3.0 kb with size-fractionated cerebellar mRNA. Brainstem glycine uptake exhibited a broad sedimentation profile, with peaks apparent at 2.4 and 4.0 kb. Taken together, these findings indicate previously unappreciated complexity in mRNA structure and regulation which underlies the expression of amino acid neurotransmitter uptake systems in the rodent CNS.

Regional Distribution and Developmental Variation of the Glycine Transporters GLYT1 and GLYT2 in the Rat CNS

“…However, after cloning several glycine transporters, there is no evidence for a 2.4 kb mRNA coding for a glycine transporter. Taking together the size and localization of the 4 kb mRNA described previously (Blakely et al, 1991a), it is likely that this same gene product codes for GLYT1. However, this study failed to detect the 8.0 kb band corresponding to the GLYT2 mRNA.…”

Analysis of the Transmembrane Topology and Membrane Assembly of the GAT-1 γ-Aminobutyric Acid Transporter

“…Xenopus laevis Oocyte Expression-X. laevis were purchased from Xenopus (Ann Arbor, MI) and oocytes were dissected and prepared as described previously (26 Protease Protection Assay-Proteinase K (Boehringer Mannheim) was added to aliquots of oocyte homogenates (0.2 mg/ml final) in the presence or absence of 1% Triton X-100 and incubated on ice for 1 h. Protease was inactivated by addition of 10 mM phenylmethylsulfonyl fluoride (Sigma) in Me 2 SO, dilution with 10 volumes of 1% SDS, 0.1 M Tris, pH 8.0, and boiling for 5-10 min. Samples were diluted with 4 volumes of 1.25 ϫ RIPA buffer (187.5 mM NaCl, 1.25% Triton X-100, 1.25% deoxycholate, 62.5 mM Tris, pH 8.0, 2.5 mM EDTA, 0.125% SDS) and set at 4°C with rocking ϳ12-16 h. Samples were centrifuged at 14,000 ϫ g for 15 min and proteins immunoprecipitated from the supernatant as described below.…”

Molecular Approaches to Drug Abuse Research Volume II: Structure, Function, and Expression

“…Regional enrichment of transporter mRNAs followed well the distribution of soma predicted to synthesize each carrier. In addition, pharmacologic sensitivities of oocyte-expressed transporters were similar (Blakely et al 1991a) if not identical to those observed in brain membrane preparations (table 1). Using mRNA prepared from different brain regions during development, Blakely and colleagues (1991a) revealed a postnatal rise in Glu, GABA, and Gly transporter transcript abundance paralleling the postnatal rise in brain transport activity for these substrates.…”