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Expression of Neurotransmitter Transport from Rat Brain Mrna in Xenopus Laevis Oocytes RANDY D

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Expression of Neurotransmitter Transport from Rat Brain Mrna in Xenopus Laevis Oocytes RANDY D Proc. Natl. Acad. Sci. USA Vol. 85, pp. 9846-9850, December 1988 Neurobiology Expression of neurotransmitter transport from rat brain mRNA in Xenopus laevis oocytes RANDY D. BLAKELY*, MICHAEL B. ROBINSONt, AND SUSAN G. AMARA* *Section of Molecular Neurobiology, Howard Hughes Medical Institute Research Laboratories, Yale University School of Medicine, 333 Cedar Street, P.O. Box 3333, New Haven, CT 06510; and tDepartments of Pediatrics and Pharmacology, Children's Seashore House, Philadelphia, PA 19104 Communicated by Charles F. Stevens, October 3, 1988 ABSTRACT To permit a molecular characterization of presently employed in our society, including cocaine, am- neurotransmitter transporter proteins, we have studied uptake phetamines, and tricyclic antidepressants (10, 11). activities induced in Xenopus laevis oocytes after injection of In sharp contrast to the detail with which other proteins adult rat forebrain, cerebellum, brainstem, and spinal cord involved in signal transduction are understood, and despite a poly(A)+ RNA. L-Glutamate uptake could be observed as early wealth of bioenergetic and kinetic studies on transport itself as 24 hr after injection, was linearly related to the quantity of (12, 13), our understanding ofthe molecular principles guiding mRNA injected, and could be induced after injection ofas little neurotransmitter uptake is considerably limited. Due, per- as 1 ng of cerebellar mRNA. Transport of radiolabeled haps, to their low abundance and poor stability in vitro (14), L-glutamate, y-aminobutyric acid, glycine, dopamine, seroto- purification strategies have as yet yielded little structural data. nin, and choline could be measured in single microinjected Only within the past few years has a Na'-dependent GABA oocytes with a regional profile consistent with the anatomical transporter from rat brain been reconstituted and purified (15). distribution of particular neurotransmitter synthesizing soma. A cDNA clone for the Na'-glucose cotransporter from rabbit Forebrain L-glutamate and dopamine uptake, as well as intestine has been isolated (16) and found to possess no cerebellar L-glutamate transport, were found to be Na+- sequence identity with cloned eukaryotic or prokaryotic fa- dependent. Cerebellar mRNA-induced L-glutamate transport cilitated metabolite carriers, pumps, or channels. Thus, it is was both time and temperature-dependent, was saturable by likely that distinct gene families underlie the different modes substrate, suggesting a single activity with an apparent trans- of transport across biological membranes. port Km of 14.2 ,uM and a Vmax of 15.2 pmol/hr per oocyte, and To establish an in vitro system suitable for the expression, was sensitive to inhibitors of brain L-glutamate transport. characterization, and molecular cloning of neurotransmitter Thus, the oocyte L-glutamate transport induced by injection of transport proteins, we have exploited the ability of Xenopus adult rat cerebellar mRNA appears essentially identical to the laevis oocytes to faithfully translate, process, and insert high-affinity, Na'-dependent L-glutamate uptake found in membrane proteins derived from nonamphibian mRNA (17, brain slices and nerve terminals. Experiments with size- 18). Herein, we report the expression of four major classes of fractionated cerebellar mRNA reveal single, comigrating peaks brain transport activities-those for catecholamine (dopa- for cerebellar L-glutamate and y-aminobutyric acid transport, mine) and indoleamine (5HT) transport, excitatory and inhib- with peak activity obtained in fractions of -2.7 kilobases, itory amino acid uptake (L-glutamate, GABA, and glycine), suggesting the presence of single or similarly sized mRNAs and acetylcholine catabolite transport (choline). Given the encoding each of these activities. abundant glutamatergic granule cells present in the rodent cerebellum (19, 20), we have also pursued a more detailed Response to neurotransmitters at postsynaptic receptors analysis of L-glutamate transport activity derived from cere- depends upon both the concentration of neurotransmitter bellar mRNA and have determined the size of mRNA species reached in the synaptic cleft and the duration such concen- encoding cerebellar L-glutamate and GABA transport activi- trations are maintained. For neurons to maintain rapid and ties. efficient chemical communication with effector sites, neuro- transmitters must have a brief extracellular lifetime, paral- MATERIALS AND METHODS leling the rise and fall of presynaptic excitation. Most neurotransmitters are inactivated by specific, pharmacolog- Preparation ofpoly(A)+ RNA and Size Fractionation. Except ically distinguishable active transport activities, analogous to for spinal cords, which were processed fresh, all brain regions the noradrenergic carrier first described at peripheral sym- were frozen in liquid nitrogen after dissection from adult male pathetic synapses (1, 2). Like peripheral synapses, brain Sprague-Dawley rats. Brainstem and spinal cord were divided nerve terminals conduct high-affinity (Km < 25 kkM) Na'- by a transection at the obex; forebrain was separated from dependent transport of norepinephrine, dopamine, and sero- brainstem by a transection at the caudal margin ofthe occipital tonin (5HT) (3-5) and actively accumulate the excitatory cortex. RNA was prepared from both fresh and frozen tissue amino acids L-glutamate and L-aspartate, and the inhibitory by the guanidine isothiocyanate/cesium chloride method (21). neurotransmitters y-aminobutyric acid (GABA) and glycine Poly(A)+ RNA was obtained from total RNA by oligo(dT)- (6, 7). Just as the inhibition of peripheral catecholamine cellulose (Collaborative Research) chromatography (22) and transport enhances sympathetic transmission (1), the block- stored at -700C until further use. Size fractionation of ade of central transport mechanisms for L-glutamate and poly(A)+ RNA (100 Ag) was performed by centrifugation GABA increases their synaptic efficacy (8, 9). The impor- (41,000 rpm, 20C, 16 hr, TH-641 rotor, Sorvall) on linear [10- tance of active cotransport proteins for the regulation of 31% (wt/vol)] sucrose density gradients in LiDodSO4 (23). synaptic neurotransmission is perhaps best revealed by Fractions were acetate precipitated, resuspended in 10 ill of inspection ofthe catalogue ofmonoamine transport inhibitors sterile H20, and stored at -70'C prior to injection. mRNA sizes across the gradient were estimated by comparison with The publication costs of this article were defrayed in part by page charge the migration of18S and 28S rRNAs from brain poly(A)- RNA payment. This article must therefore be hereby marked "advertisement" in an identical gradient run in parallel and by alkaline gel in accordance with 18 U.S.C. §1734 solely to indicate this fact. electrophoresis and autoradiography of oligo(dT)-primed 3 P- 9846 Downloaded by guest on September 25, 2021 Neurobiology: Blakely et al. Proc. Natl. Acad. Sci. USA 85 (1988) 9847 labeled, reverse-transcription products (24) synthesized from uptake of several neurotransmitters after injection of mRNA selected fractions. derived from dissected brain regions. Substrates were chosen Oocyte Dissection and Injection. Ovarian follicles from X. that possess well-characterized Na'-dependent transport laevis (Nasco, Fort Atkinson, WI) were surgically removed systems (11), typically assayed with brain slices or synapto- into calcium-free oocyte medium (96 mM NaCl/2 mM KCl/5 somes in vitro. In accordance with the density and most likely mM MgCl/5 mM Hepes, pH 7.5), dissected into small neuroanatomic distribution of cell bodies synthesizing these fragments, and incubated with collagenase (2 mg/ml; type activities, significant transport above uninjected controls was 1A, Sigma) for 2 hr at 22°C. Collagenase-treated oocytes were observed for virtually all substrates (Table 1). Thus, L- washed and plated in culture dishes in Ca2+-supplemented glutamate and GABA, the principal excitatory and inhibitory (0.6 mM) oocyte medium. Between 12 and 24 hr after neurotransmitters in the vertebrate brain (26), were trans- collagenase treatment, oocytes were microinjected with 40 nl ported after injections of mRNA derived from all brain of total or size-fractionated poly(A)+ RNA and incubated in regions. L-Glutamate transport was highest with forebrain Ca2'-supplemented oocyte medium for 2 days, except where and cerebellar mRNA. Lung MRNA, found to direct the noted. Two-electrode voltage clamp (25) of oocytes in Ca2+- expression of electrophysiologically assayable acetylcholine containing medium was used to examine resting membrane receptors (data not shown), failed to significantly induce potentials and to verify, by eliciting responses to 5HT, L-glutamate transport activity. Unlike L-glutamate transport, acetylcholine, or GABA, translation of membrane proteins GABA uptake was most pronounced in oocytes injected with from injected mRNA. brainstem and spinal cord mRNA. Glycine, thought to act as Transport Assays. Uptake experiments were initiated by an inhibitory neurotransmitter in hindbrain regions (26), was transferring single injected or noninjected oocytes to 1.8-ml only accumulated above controls in oocytes injected with polypropylene microcentrifuge tubes containing 500 ,ul of 1.0 brainstem and spinal cord mRNA. Dopamine and 5HT ,M L-[3,4-3H]glutamate, 1 ,M [2,3-3H]GABA, 1 ,M [2- transport was considerably lower than observed for the 3H]glycine, 1 ,uM [methyl-3H]choline chloride, 0.1 ,uM 3,4- amino acids, yet clearly elevated above
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