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, neurotransmitters 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 controls and consis- [7-3H]dopamine, 0.1 ,M L-[7-3H]norepinephrine, or 0.1 ,uM tently derived from brain regions specific to each neurotrans- [1,2-3H(N)]5HT (New England Nuclear) with or without mitter system. Dopamine transport was observed after in- inhibitors. Unlabeled substrates and inhibitors, except for jection of forebrain and brainstem mRNA, while 5HT trans- N-methyl-D-aspartate (Cambridge Research Biochemicals, port was significantly elevated only in oocytes injected with Cambridge, U.K.) were obtained from Sigma. GABA trans- brainstem mRNA. Only norepinephrine failed to be accumu- port assays contained 100 ,uM aminooxyacetic acid, a GABA- lated above background rates observed with uninjected transaminase (EC 2.6.1.19) inhibitor, while monoamine up- oocytes. Finally, enhanced choline transport was induced by take was assayed in the presence of 100 ,uM L-ascorbate to limit catechol and indole oxidation. To examine the Na+ Table 1. Regional distribution of neurotransmitter transport in X. dependency of transport, equimolar choline chloride was laevis oocytes substituted for NaCl in the incubation buffer. Incubations were conducted at 22°C for 60 min unless otherwise indi- Transport activity, cated. Uptake was terminated by aspirating assay medium Substrate RNA source Exp., n pmol/hr from oocytes, followed by three gentle 1-ml washes in L-Glutamate Forebrain 6 2.33 ± 0.51* Ca2+-containing oocyte medium (22°C), all within 60 sec. Cerebellum 13 1.73 ± 0.22* Oocytes were then solubilized in 500 ,ul of 1% NaDodSO4 and Brainstem 3 1.42 ± 0.49* transferred to 5 ml of EcoScint (National Diagnostics, Mann- Spinal cord 3 0.94 ± 0.08* ville, NJ) for scintillation spectrometry. All experiments Lung 4 0.25 ± 0.02 were conducted on four or more oocytes per condition. Mean Noninjected 14 0.16 ± 0.03 transport activities from three or more experiments, per- GABA Forebrain 8 0.56 ± 0.11* formed on separate batches of oocytes, are reported as Cerebellum 4 0.44 ± 0.11* pmol/hr (mean ± SEM). Significant (P - 0.05) increases in Brainstem 3 0.79 ± 0.32* induced uptake relative to that observed in parallel incuba- Spinal cord 3 1.07 ± 0.36* tions with noninjected oocytes were determined with a Noninjected 12 0.22 ± 0.04 one-tailed Student's t test for unpaired samples. Glycine Forebrain 4 0.55 ± 0.14 Cerebellum 2 0.52 ± 0.16 RESULTS Brainstem 5 1.59 ± 0.22* Spinal cord 4 0.96 ± 0.19* In initial studies, two-electrode voltage clamp analysis of Noninjected 9 0.48 ± 0.06 uninjected and injected oocytes showed resting membrane Dopamine Forebrain 9 0.028 ± 0.002* potentials of -50 to -70 mV. As frequently observed (18), Brainstem 3 0.026 ± 0.002* oocytes injected with rat brain mRNA expressed brisk elec- Noninjected 7 0.011 ± 0.002 trophysiological responses to 5HT and GABA (data not Norepinephrine Forebrain 3 0.014 ± 0.003 shown), confirming the integrity of mRNA preparations. An Brainstem 3 0.019 ± 0.005 initial evaluation of L-[3H]glutamate transport in cerebellar Noninjected 4 0.025 ± 0.008 RNA-injected (40 ng) oocytes established that uptake could be Serotonin Forebrain 3 0.050 ± 0.009 readily observed within single oocytes incubated for 60 min at Brainstem 5 0.053 ± 0.005* 22°C, with levels exceeding by >10-fold the basal accumula- Noninjected 4 0.036 ± 0.008 tion of L-glutamate in uninjected oocytes. A highly significant Choline Forebrain 4 0.94 ± 0.05* linear correlation (r > 0.99) between the quantity of mRNA Brainstem 3 1.10 ± 0.33* injected (0.4-40 ng) and L-glutamate uptake velocity was Spinal cord 4 0.90 ± 0.09* observed. Indeed, 1 ng of injected cerebellar poly(A)+ RNA Noninjected 8 0.66 ± 0.06 was sufficient to induce a 2-fold elevation in L-[3H]glutamate Each experiment was performed with a different batch of oocytes, transport above uninjected controls, while injections with with at least four oocytes per experiment (n). Transport activity is highly diluted solutions yielded insignificant transport. given as mean ± SEM. To explore the distribution and diversity of transporter *Represents substrate transport that was significantly above nonin- expression, experiments were conducted to examine the jected levels (P s 0.05). Downloaded by guest on September 25, 2021 9848 Neurobiology: Blakely et al. Proc. Natl. Acad. Sci. USA 85 (1988) injection of mRNA derived from brain regions containing large populations of cholinergic neurofs, spinal cord, brainstem, and forebrain. To more precisely characterize the transport activities J.U induced in oocytes by brain mRNAs, we focused upon L-glutamate transport arising from the injection of adult rat 2 ~~~~~~~~~~~~~3.0 2.0-~~~~~~~~~~ cerebellar mRNA. L-Glutamate was chosen based on its 2.0~~~~~~~~~~~~. pervasive role in synaptic excitation in the vertebrate brain 1.0~~~~~~~~~~~~~~~~0 and its robust transport activity in injected oocytes (Table 1); o 2~~~~~~~~~~~~~~.0 the cerebellum offers an abundant class of a single type of glutamatergic neuron, the granule cells. Within 24 hr after injection, cerebellar poly(A)+ RNA could be shown to induce oocyte L-glutamate transport, an activity that was maintained Forebrain Cerebellum Forebrain at relatively constant levels for up to 5 days (Fig. lA). [3~~~~~~~~~~~~~~0 L-Glutamate accumulation at 220C increased linearly with FIG. 2. NA' dependency of oocyte L-glutamate and dopamine time and plateaued between 60 and 80 min (Fig. 1B). Eadie- transport. (A) Na' dependence of oocyte L-glutamate transport after Hofstee transformation of data from assays conducted at injections (40 ng) offorebrain and cerebellar poly(A)+I RNA, assayed 220C for 60 min with various concentrations of substrate for 60 min at 22°C with 1.0 ,uM L-[3H]glutamate, 2 days after RNA suggests a single population of induced L-glutamate trans- injection. (B) As in A, except that forebrain RNA-injected oocytes with an apparent Km of 14.2 uM and a Vmax of 15.2 were assayed for [Hldopamine transport at 0.1,M. Data are values porters of three separate experiments, conducted on different batches of pmol/hr (Fig. 1C). oocytes, with at least four oocytes per condition (mean ± SEM). High-affinity L-glutamate uptake in brain slices and syn- Solid bars, injected oocytes with 96 mM NaCI; hatched bars, injected aptosomes is dependent upon extracellular Na' and is oocytes with 96 mM choline chloride; open bars, uninjected oocytes inhibited by submillimolar concentrations of certain acidic with 96 mM NaCI. Substitution of choline chloride for NaCI reduced both L-glutamate and dopamine transport activity in injected oocytes to uninj.ected control levels (P < 0.05, Student's t test); NaCI A substitution had no significant effect on uptake with uninjected 6 4 3.0- oocytes (data not shown). Is0

I 2.0- amino acid analogues or by incubations conducted at reduced 6 substitution of choline 10 temperatures (27, 28). Equimolar 0. induced W 1.0' chloride for NaCl reduced L-glutamate transport by 0 both cerebellar and forebrain mRNA to levels observed with a- uninjected oocytes (Fig. 2A). Similarly, dopamine uptake, 0 1 2 3 4 5 6 measured with forebrain mRNA-injected oocytes, was also Days Post-Injection found to bseparteeerimeto Na+ substitution (Fig. 2B). Assays conducted in thelaMpresence of 100 L-Cysteinate and B L-aspartate, relatively potent L-glutamate transport ionhibi- tors, reduced specific L-glutamate uptake by 76 and 70%, 0 respectively (Fi2 3). In contrast, 10i utM N-methyl- 0. D-aspartate, a selective L-glutamate receptor agonist, pro- duced no significant inhibition. As well, specitnc L-glutamate 00. -0

40 60 80 100 140 * 100 gML-.M 100- L IN Incubation Time (min) 0 Li giM l-Asp [] 100 uM L-Cyst C 1.5- * 100 pMNINI)A r_ 4C Km= 14.2 ,M E] Vmax = 15.2 pmol/hr ._

6 U~~~~ pi- * u O; 0.5- C 0 Z wz 40- cd 0.0 4.0 8.0 12.0 16.0 u Velocity (pmol/hr) - 20 FIG. 1. Characterization of L-glutamate transport in Xenopus oocytes injected with cerebellar poly(A)+ RNA (40 ng). (A) Expres- sion as a function of days after RNA injection. Incubations were 60 Cerebellum min long at 22°C with 1.0 ,uM L-[3H]glutamate. (B) Relationship of assay time to L-glutamate transport. Assays were conducted 2 days FIG. 3. Effects of inhibitors and reduced temperature on L- after RNA injection as described in A. (A and B) *, Injected oocytes; glutamate transport from cerebellar mRNA-injected oocytes. Data o, uninjected oocytes. (C) Eadie-Hofstee transformation of assay are plotted as a percentage of transport activity obtained with determinations with 20 nM L-[3H]glutamate and various concentra- oocytes incubated for 60 min at 22°C, after subtraction of basal tions ofunlabeled substrate. Incubations were 60 min at 22°C. Assays transport in uninjected oocytes. Data are values of at least three were done 2 days after injection. Data are values of at least three separate experiments, conducted on different batches of oocytes, separate experiments, conducted on different batches of oocytes, with at least four oocytes per condition (mean + SEM). NMDA, with at least four oocytes per point (mean + SEM). N-methyl-D-aspartate; L-Cyst, L-cysteinate; 4 C, 4°C. Downloaded by guest on September 25, 2021 Neurobiology: Blakely et al. Proc. Natl. Acad. Sci. USA 85 (1988) 9849

A 0.3 - DISCUSSION

0.3 0.8 1.7 2.0 4.3 5.1 c The X. laevis oocyte offers many advantages for the in vitro 0.2 - functional reconstitution of low-abundance membrane proteins, such as ion channels, pumps, and receptors (18), and rtCi has been shown to be suitable as a screening system for the ci 0.1 - rTQ isolation of cDNA clones (16, 29-31). With the oocyte .0 system, we have expressed transport activities for five of the u.i ...... major neurotransmitters in the vertebrate central nervous 0 5 10 15 20 25 30 3'5 system, L-glutamate, GABA, glycine, dopamine, and 5HT, 0 as well as uptake of the acetylcholine metabolite, choline. A B 4.0 2.7 good correlation was observed between the regional distri- + bution of active mRNA and the cellular origin of chemically 3.0 L-Glutamate defined neurons. Thus, all regional mRNAs gave rise to the E0. transport of L-glutamate and GABA, compounds implicated 2.0 in synaptic transmission throughout the nervous system, 0. while glycine uptake was restricted to mRNA isolated from 0 1.0 regions containing putative glycinergic neurons, brainstem ICu and spinal cord (26). The higher L-glutamate transport rates 0.0 ...... observed with forebrain and cerebellar mRNA are consistent 0 5 10 15 20 25 30 3'5 with the large numbers ofglutamatergic soma in these regions, including many hippocampal, thalamic, and cortical neurons, C 2.0 and the abundant cerebellar granule cells (26). In contrast, S. 2.7 mRNA derived from lung, a tissue not known for high-affinity 1.5- GABA Na'-dependent L-glutamate uptake, failed to induce L- E0 glutamate transport above levels observed with uninjected a F- 1.0 oocytes. Although widespread neurotransmitter system, a E- high density of GABAergic neurons is found in the dorsal Cu0 0.5 spinal cord of the rat (32) and is a likely mRNA source of the caudal enrichment of GABA transport we observed. Sarthy 0.0 .. .. (33) described the ability ofjuvenile rat brain RNA to induce 0 5 10 15 20 25 30 Na+-dependent GABA transport in oocytes, with activity Fractions Collected considerably greater than we observed with mRNA derived FIG. 4. Sucrose-density gradient size-fractionation of cerebellar from adult forebrain. This discrepancy may simply represent poly(A)+ RNA. Poly(A)+ RNA (100 ug) was fractionated over a an ontogenetic decline in forebrain GABA transporter mRNA. linear (10-31%) sucrose gradient. (A) Optical density measurement Brain regions rich in monoamine neurons (34, 35) provided of fractionated RNA at 254 nm. Sizes (in kb) of selected fractions are suitable sources for monoamine transporter expression. Our designated by arrows above the gradient. (B) L-Glutamate transport division of brainstem and forebrain transects the mesenceph- activity of selected fractions. Transport was assayed at 220C, for 60 alon at the caudal margin of the superior colliculus, placing min, with 1 AM L-[3H]glutamate, 2 days after oocyte injection (40 n1). the mesencephalic dopamine neurons largely in the forebrain (C) GABA transport activity of selected fractions. Transport of with those from the hypothalamus, while the bulk of seroto- [3H]GABA was measured as for L-[3H]glutamate in B, with 1.0 AuM neurons substrate. Data represent pooled values from three separate exper- nergic raphe largely reside within our brainstem iments conducted with different batches of oocytes. division. Like synaptosomal dopamine uptake (5), dopamine transport in forebrain mRNA-injected oocytes is Na+- accumulation was reduced to levels obtained with uninjected dependent. The absolute levels of dopamine and 5HT uptake oocytes when incubations were conducted at 40C rather than were considerably lower than the levels found for amino acid 220C (Fig. 3). Thus, the oocyte L-glutamate transport activity transport, despite incubations with substrate concentrations induced by cerebellar mRNA is both time and temperature- closer to their endogenous transport Km [-0.1 ,uM (36)]. dependent, saturable by micromolar substrate concentra- These observations are consistent with the low abundance of tions, Na'-dependent, and inhibited by agents known to monoamine soma in the rodent central nervous system block brain L-glutamate uptake. relative to glutamatergic and GABAergic neurons. Our in- To determine the sizes of mRNA encoding cerebellar ability to detect norepinephrine transport may thus be a transport activities, poly(A)+ RNA was fractionated on a consequence of the even lower density of noradrenergic linear (10-31%) sucrose-density gradient (Fig. 4A) and frac- neurons in the rodent central nervous system (11), as well as tions assayed for both L-glutamate and GABA transport the -10 times lower affinity of norepinephrine for the activity (Fig. 4 B and C). The major activities were found to dopamine transporter (3, 36). Further studies will ascertain if migrate as single peaks between 2.5 and 3.0 kilobases (kb), oocyte dopamine and 5HT transporters display the pharma- with peak transport for both arising from fractions of 2.7 kb. cological sensitivities of those localized to hypothalamic and Approximately 90% of both the L-glutamate and GABA mesencephalic dopamine or raphe 5HT neurons (11). Choline transport activities present in unfractionated cerebellar transport was observed after injection of mRNA from spinal mRNA was recovered from the gradient. As fractions 19-25 cord, brainstem, and forebrain, conceivably a reflection of contain <15% of the fractionated mRNA, yet >95% of the cholinergic neurons in these regions, such as those of striatal L-glutamate and GABA transport activity across the gradient, and septal nuclei and of hindbrain motor neurons (37). a substantial enrichment of transport activity over unfraction- Although the anatomical distribution of transport activities ated mRNA was obtained. Enrichment was reflected as well appears highly consistent with the regional localization of in comparably higher specific uptake per ng of injected neurons synthesizing and releasing a particular neurotrans- mRNA. Thus, 40 ng of total cerebellar mRNA induced on mitter, glial mRNAs may also contribute to the induced average L-glutamate transport of 1.73 pmol/hr and GABA transport activities, especially for the amino acids (38). In this transport of0.44 pmol/hr (Table 1), while in fraction 23, -6 ng regard, oocyte studies with mRNA derived from rodents gave 3.05 pmol/hr and 1.33 pmol/hr, respectively. deficient in the putative neuronal sources ofthese signals (19) Downloaded by guest on September 25, 2021 9850 Neurobiology: Blakely et al. Proc. Natl. Acad. Sci. USA 85 (1988) may allow for finer cellular distinctions. Specific subtypes of cally to neurotransmitter transport proteins become avail- both L-glutamate and GABA transporters have also been able, we should be poised to determine the degree to which proposed (39, 40). It may be possible with specific inhibitors these similarities represent the conserved elements of a gene of glial and neuronal GABA transport to pharmacologically family and to more clearly articulate their role(s) in synaptic distinguish subtype mRNAs, although such tools are not neurotransmission. presently available for L-glutamate carriers (41). Thus, GABA transport from oocytes injected with unfractionated juvenile rat brain mRNA display both diaminobutyric acid We thank Janet Burton for excellent technical assistance during and /-alanine sensitivity (33), inhibitors reputedly selective the course of this research. This work was supported by The Howard for neuronal and glial carriers, respectively (38). Hughes Medical Institute (R.D.B and S.G.A.) and the Pew Foun- We focused upon the expression of L-glutamate uptake dation (M.B.R.). from mRNA derived from cerebellum, with hopes of char- 1. Iversen, L. L. (1967) The Uptake and Storage of Noradrenaline in acterizing a homogeneous population of well-defined gluta- Sympathetic Nerves (Cambridge Univ. Press, Cambridge). matergic neurons, the granule cells (19, 20). These studies 2. Axelrod, J. (1971) Science 173, 598-606. revealed L-glutamate transport to be Na'- and time- 3. Coyle, J. T. & Snyder, S. (1969) J. Pharmacol. Exper. Ther. 170, 221- 231. dependent, and to be both temperature-sensitive and satura- 4. Ross, S. M. & Renyi, A. L. (1967) Life Sci. 6, 1407-1415. ble at micromolar substrate concentrations. Cerebellar 5. Holz, R. W. & Coyle, J. T. (1974) Mol. Pharmacol. 10, 746-758. mRNA-induced L-glutamate uptake was also inhibited by 6. Snyder, S. H., Young, A. B., Bennett, J. P. & Mulder, A. H. (1973) Fed. compounds known to block Na'-dependent high-affinity Proc. Fed. Am. Soc. Exp. Biol. 32, 2039-2047. 7. Fagg, G. E. & Lane, J. D. (1979) Neuroscience 4, 1015-1036. L-glutamate uptake in synaptosomes and brain slices (27, 28), 8. Lodge, D., Curtis, D. R. & Johnston, G. A. R. (1978) J. Neurochem. 31, while N-methyl-D-aspartate, a selective L-glutamate receptor 1525-1528. agonist, was inactive. Thus, these studies confirm the use of 9. Johnston, G. A. R., Lodge, D., Bornstein, J. C. & Curtis, D. R. (1980) the Xenopus oocyte system for in vitro reconstitution ofNa'- J. Neurochem. 34, 241-243. 10. Maxwell, R. A. & White, H. L. (1978) in Handbook ofPsychopharma- neurotransmitter cotransporters, permitting an investigation cology, eds. Iversen, L. L., Iversen, S. D. & Snyder, S. H. (Plenum, of their intra- and extracellular regulation in single cells. New York), Vol. 14, pp. 83-155. Aoshima et al. (42) have described an induction of intestinal 11. Cooper, J. R., Bloom, F. E. & Roth, R. H. (1986) The Biochemical Basis and renal Nat/amino acid transport in both Xenopus and of Neuropharmacology (Oxford Univ. Press, New York), 5th Ed. 12. Kanner, B. I. (1983) Biochim. Biophys. Acta 726, 293-316. Cynops oocytes. Interestingly, no evidence for acidic amino 13. Stein, W. D. (1986) Transport and Diffusion Across Cell Membranes acid transport could be demonstrated by these investigators. (Academic, New York). Although their electrophysiological approach does not di- 14. Koepsell, H. (1986) Rev. Physiol. Biochem. Pharmacol. 104, 65-137. measure amino acid transport, we have also failed to 15. Radian, R., Bendahan, A. & Kanner, B. I. (1986) J. Biol. Chem. 261, rectly 15437-15441. detect L-glutamate transport with peripheral (lung) mRNA. 16. Hediger, M. A., Coady, M. J., Ikeda, T. S. & Wright, E. M. (1987) Nonetheless, this expression system appears well suited for Nature (London) 330, 379-381. investigation of transport proteins in both neural and non- 17. Gurdon, J. B. & Wickens, M. P. (1983) Methods Enzymol. 101, 370-386. neural tissues, emphasized by the expression (43) 18. Snutch, T. P. (1988) Trends Neurosci. 11, 251-256. particularly 19. Young, A. B., Oster-Granite, M. L., Herndon, R. M. & Snyder, S. H. and cDNA cloning (16) of Na'-glucose transporter from (1974) Brain Res. 73, 1-13. rabbit intestine. 20. Shepherd, G. M. (1979) The Synaptic Organization ofthe Brain (Oxford Expression cloning of neurotransmitter transporters would Univ. Press, New York) be facilitated the presence of single, small mRNAs 21. MacDonald, R. J., Swift, G. H., Przybyla, A. E. & Chirgwin, J. M. greatly by (1987) Methods Enzymol. 152, 219-227. (<6 kb) encoding each activity, as described for the sub- 22. Jacobson, A. (1987) Methods Enzymol. 152, 254-261. stance K (29) and 5HT1c (30, 31) receptors and Na+-glucose 23. Sumikawa, K., Houghton, M., Bell, L., Richards, B. M. & Barnard, cotransporter (16). L-Glutamate and GABA transport assays E. A. (1982) Nucleic Acids Res. 10, 5809-5822. with sucrose size-fractionated 24. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular Cloning: A conducted gradient poly(A)+ Laboratory Manual (Cold Spring Harbor Lab., Cold Spring Harbor, NY). RNA revealed that both activities arise predominantly from 25. Kusano, K., Miledi, R. & Stinnakre, J. (1982) J. Physiol. (London) 328, fractions of 2.5-3.0 kb. Clearly, activities requiring the 143-170. translation of multiple, differently sized mRNAs are less 26. McGeer, P. L. & McGeer, E. G. (1981) in Basic Neurochemistry, eds. to be reconstituted after ofsingle fractions. As Siegel, G. J., Albers R. W., Agranoff, B. W. & Katzman, R. (Little, likely injection Brown, Boston), 3rd Ed., pp. 233-253. we achieved relatively full recovery of transport activity, 27. Balcar, V. J. & Johnston, G. A. R. (1972) J. Neurochem. 19, 2657-2666. L-glutamate and GABA transporters may each be encoded by 28. Bennet, J. P., Logan, W. J., & Snyder, S. H. (1973) J. Neurochem. 21, a single mRNA, like the Na+-glucose cotransporter. The 1533-1550. cerebellar and GABA trans- 29. Masu, Y., Nakayama, K., Tamaki, H., Harada, Y., Kuno, M. & coincidence of peak L-glutamate Nakanishi, S. (1987) Nature (London) 329, 836-838. port activities in a fraction of -2.7 kb suggests that similarly 30. Lubbert, H., Hoffman, B. J., Snutch, T., van Dyke, T., Levine, A. J., sized mRNAs encode the two carriers. This mRNA size is Hartig, P. R., Lester, H. A. & Davidson, N. (1987) Proc. Natl. Acad. sufficient to encode a transporter subunit no larger than -900 Sci. USA 84, 4332-4336. size-fractionation 31. Julius, D., MacDermott, A. B., Axel, R. & Jessell, T. M. (1988) Science amino acids. Analogously, electrophoretic 241, 558-564. of the Na+-glucose cotransporter revealed a single active 32. Mugniani, E. & Oertel, W. H. (1983) in Handbook of Chemical Neuro- peak of -2.3 kb (43), now understood to arise from a single anatomy, eds. Bjorklund, A. & Hokfelt, T. (Elsevier, New York), pp. mRNA (16). Deduced amino acid sequence of the cDNA 436-608. clone for this transporter suggests a core protein of662 amino 33. Sarthy, V. (1986) Mol. Brain Res. 1, 97-100. 34. Ungerstedt, U. (1971) Acta Physiol. Scand. (Suppl.) 367, 1-48. acids and a molecular mass of 73 kDa. Little structural 35. Steinbusch, H. M. W. (1981) Neuroscience 6, 577-618. information is available for neurotransmitter-specific active 36. Horn, A. S. (1978) Adv. Bioch. Psychopharmacol. 19, 25-34. cotransporters, although reconstitution and purification stud- 37. MacIntosh, F. C. (1981) in Basic Neurochemistry, eds. Siegel, G. J., ies with a brain Na'-GABA cotransport protein indicate a Albers, R. W., Agranoff, B. W. & Katzman, R. (Little, Brown, Boston), 3rd Ed., pp. 183-204. glycosylated subunit size of -80 kDa (15), similar in size to 38. Schousboe, A. (1981) Int. Rev. Neurobiol. 22, 1-45. the Na+-glucose cotransporter and clearly within the coding 39. Ferkany, J. & Coyle, J. T. (1986) J. Neurosci. Res. 16, 491-503. capacity of a 2.7-kb mRNA. Although finer distinctions can 40. Wood, J. D. & Sidhu, H. S. (1987) J. Neurochem. 49, 1202-1208. be bioenergetic studies (12, 13) predict structural 41. Roberts, P. J. & Watkins, J. C. (1975) Brain Res. 85, 120-125. drawn, 42. Aoshima, H., Tomita, K. & Sugio, S. (1988) Arch. Biochem. Biophys. similarities among the large number of active Na'-cotran- 265, 73-81. sport proteins, encompassing both metabolite and neuro- 43. Hediger, M. A., Ikeda, T., Coady, M., Gundersen, C. B. & Wright, transmitter substrates. As structural clues relating specifi- E. M. (1987) Proc. Natl. Acad. Sci. USA 84, 2634- 2637. Downloaded by guest on September 25, 2021