Susan G. Amara

Vaccinia-T7 RNA polymerase expression system: Evaluation for the expression cloning of plasma membrane transporters

The vaccinia/T7 transient expression system, which results in rapid, high-level expression of proteins encoded by plasmids bearing T7 promoters, provides a powerful strategy for the expression cloning of membrane transporters. To test the feasibility of this approach, we introduced the rabbit Na+/glucose transporter by liposome-mediated transfection into vaccinia infected HeLa cells and determined the characteristics and sensitivity of induced [14C]alpha-methyl D-glucopyranoside uptake. We observed a rapid (4-12 h) expression of saturable (Kt = 342 microM) [14C]alpha-methyl D-glucopyranoside uptake following transfection, with substrate and inhibitor sensitivities of the native carrier, including Na+ and temperature dependence and appropriate phloridzin sensitivity (KI = 9.1 microM). The time-dependent increase in alpha-methyl D-glucopyranoside uptake coincided with a decline in endogenous Na+/D-aspartate transport. Maximal levels of expression achieved were nearly 10-fold higher than that reported for transient expression of Na+/glucose transporters in the COS cell system. Rate and dilution estimates demonstrates a sensitivity of detection of single clones diluted several thousand fold by nonspecific plasmid DNA. A further 3-fold increase in transport sensitivity was achieved after transfection of plasmid constructs bearing additional 5-T7 stem-loop and 3-T7 termination signals. When cell lines with low endogenous transport were coupled with substrates of high specific activity, as with measurements of induced [3H]gamma-aminobutyric acid uptake, we were able to detect expression from transporter bearing plasmids diluted as much as 10,000-fold by non-specific plasmid DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

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 γ‐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.

Sodium-dependent neurotransmitter reuptake systems

The recent cloning of the gamma-aminobutyric acid and norepinephrine Na(+)-dependent neurotransmitter transporters has led to new approaches for understanding the structure and function of these important synaptic proteins and provides a conceptual model in which to consider recent advances in the physiology, pharmacology and biochemistry of this gene family.

Calcitonin and Calcitonin-Related Peptide Genes

The neuroendocrine system serves critical regulator and communicative functions during development and in the maintenance of physiological homeostasis. To serve these functions with requisite specificity and nuance, many diverse regulatory peptides are produced by various strategies. Complex regulatory mechanisms operate to restrict the expression of genes encoding neuroendocrine peptides to precise groups in neurons in the neural tissues and in specific cell types in peripheral organs. An understanding of the mechanisms by which expression of neuroendocrine genes is regulated is critical to achieve insights into the molecular mechanisms important in developmental processes.