Expression of neurotransmitter transport from rat brain mRNA in ...

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

presently employed in our society, including cocaine, amphetamines, and tricyclic antidepressants (10, 11). In sharp contrast to the detail with which other proteins involved in signal transduction are understood, and despite a wealth of bioenergetic and kinetic studies on transport itself (12, 13), our understanding of the molecular principles guiding neurotransmitter uptake is considerably limited. Due, perhaps, to their low abundance and poor stability in vitro (14), purification strategies have as yet yielded little structural data. Only within the past few years has a Na'-dependent GABA transporter from rat brain been reconstituted and purified (15). A cDNA clone for the Na'-glucose cotransporter from rabbit intestine has been isolated (16) and found to possess no sequence identity with cloned eukaryotic or prokaryotic facilitated metabolite carriers, pumps, or channels. Thus, it is likely that distinct gene families underlie the different modes of transport across biological membranes. To establish an in vitro system suitable for the expression, characterization, and molecular cloning of neurotransmitter transport proteins, we have exploited the ability of Xenopus laevis oocytes to faithfully translate, process, and insert membrane proteins derived from nonamphibian mRNA (17, 18). Herein, we report the expression of four major classes of brain transport activities-those for catecholamine (dopamine) and indoleamine (5HT) transport, excitatory and inhibitory amino acid uptake (L-glutamate, GABA, and glycine), and acetylcholine catabolite transport (choline). Given the abundant glutamatergic granule cells present in the rodent cerebellum (19, 20), we have also pursued a more detailed analysis of L-glutamate transport activity derived from cerebellar mRNA and have determined the size of mRNA species encoding cerebellar L-glutamate and GABA transport activities.

To permit a molecular characterization of ABSTRACT neurotransmitter transporter proteins, we have studied uptake activities induced in Xenopus laevis oocytes after injection of adult rat forebrain, cerebellum, brainstem, and spinal cord poly(A)+ RNA. L-Glutamate uptake could be observed as early as 24 hr after injection, was linearly related to the quantity of mRNA injected, and could be induced after injection of as little as 1 ng of cerebellar mRNA. Transport of radiolabeled L-glutamate, y-aminobutyric acid, glycine, dopamine, serotonin, and choline could be measured in single microinjected oocytes with a regional profile consistent with the anatomical distribution of particular neurotransmitter synthesizing soma. Forebrain L-glutamate and dopamine uptake, as well as cerebellar L-glutamate transport, were found to be Na+dependent. Cerebellar mRNA-induced L-glutamate transport was both time and temperature-dependent, was saturable by substrate, suggesting a single activity with an apparent transport Km of 14.2 ,uM and a Vmax of 15.2 pmol/hr per oocyte, and was sensitive to inhibitors of brain L-glutamate transport. Thus, the oocyte L-glutamate transport induced by injection of adult rat cerebellar mRNA appears essentially identical to the high-affinity, Na'-dependent L-glutamate uptake found in brain slices and nerve terminals. Experiments with sizefractionated cerebellar mRNA reveal single, comigrating peaks for cerebellar L-glutamate and y-aminobutyric acid transport, with peak activity obtained in fractions of -2.7 kilobases, suggesting the presence of single or similarly sized mRNAs encoding each of these activities. Response to neurotransmitters at postsynaptic receptors depends upon both the concentration of neurotransmitter reached in the synaptic cleft and the duration such concentrations are maintained. For neurons to maintain rapid and efficient chemical communication with effector sites, neurotransmitters must have a brief extracellular lifetime, paralleling the rise and fall of presynaptic excitation. Most neurotransmitters are inactivated by specific, pharmacologically distinguishable active transport activities, analogous to the noradrenergic carrier first described at peripheral sympathetic synapses (1, 2). Like peripheral synapses, brain nerve terminals conduct high-affinity (Km < 25 kkM) Na'dependent transport of norepinephrine, dopamine, and serotonin (5HT) (3-5) and actively accumulate the excitatory amino acids L-glutamate and L-aspartate, and the inhibitory neurotransmitters y-aminobutyric acid (GABA) and glycine (6, 7). Just as the inhibition of peripheral catecholamine transport enhances sympathetic transmission (1), the blockade of central transport mechanisms for L-glutamate and GABA increases their synaptic efficacy (8, 9). The importance of active cotransport proteins for the regulation of synaptic neurotransmission is perhaps best revealed by inspection of the catalogue of monoamine transport inhibitors

MATERIALS AND METHODS Preparation of poly(A)+ RNA and Size Fractionation. Except for spinal cords, which were processed fresh, all brain regions were frozen in liquid nitrogen after dissection from adult male Sprague-Dawley rats. Brainstem and spinal cord were divided by a transection at the obex; forebrain was separated from brainstem by a transection at the caudal margin of the occipital cortex. RNA was prepared from both fresh and frozen tissue by the guanidine isothiocyanate/cesium chloride method (21). Poly(A)+ RNA was obtained from total RNA by oligo(dT)cellulose (Collaborative Research) chromatography (22) and stored at -700C until further use. Size fractionation of poly(A)+ RNA (100 Ag) was performed by centrifugation (41,000 rpm, 20C, 16 hr, TH-641 rotor, Sorvall) on linear [1031% (wt/vol)] sucrose density gradients in LiDodSO4 (23). Fractions were acetate precipitated, resuspended in 10 ill of sterile H20, and stored at -70'C prior to injection. mRNA sizes across the gradient were estimated by comparison with the migration of 18S and 28S rRNAs from brain poly(A)- RNA in an identical gradient run in parallel and by alkaline gel electrophoresis and autoradiography of oligo(dT)-primed 3 P-

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Neurobiology: Blakely et al.

Proc. Natl. Acad. Sci. USA 85 (1988)

labeled, reverse-transcription products (24) synthesized from selected fractions. Oocyte Dissection and Injection. Ovarian follicles from X. laevis (Nasco, Fort Atkinson, WI) were surgically removed into calcium-free oocyte medium (96 mM NaCl/2 mM KCl/5 mM MgCl/5 mM Hepes, pH 7.5), dissected into small fragments, and incubated with collagenase (2 mg/ml; type 1A, Sigma) for 2 hr at 22°C. Collagenase-treated oocytes were washed and plated in culture dishes in Ca2+-supplemented (0.6 mM) oocyte medium. Between 12 and 24 hr after collagenase treatment, oocytes were microinjected with 40 nl of total or size-fractionated poly(A)+ RNA and incubated in Ca2'-supplemented oocyte medium for 2 days, except where noted. Two-electrode voltage clamp (25) of oocytes in Ca2+containing medium was used to examine resting membrane potentials and to verify, by eliciting responses to 5HT, acetylcholine, or GABA, translation of membrane proteins from injected mRNA. Transport Assays. Uptake experiments were initiated by transferring single injected or noninjected oocytes to 1.8-ml polypropylene microcentrifuge tubes containing 500 ,ul of 1.0 ,M L-[3,4-3H]glutamate, 1 ,M [2,3-3H]GABA, 1 ,M [23H]glycine, 1 ,uM [methyl-3H]choline chloride, 0.1 ,uM 3,4[7-3H]dopamine, 0.1 ,M L-[7-3H]norepinephrine, or 0.1 ,uM [1,2-3H(N)]5HT (New England Nuclear) with or without inhibitors. Unlabeled substrates and inhibitors, except for N-methyl-D-aspartate (Cambridge Research Biochemicals, Cambridge, U.K.) were obtained from Sigma. GABA transport assays contained 100 ,uM aminooxyacetic acid, a GABAtransaminase (EC 2.6.1.19) inhibitor, while monoamine uptake was assayed in the presence of 100 ,uM L-ascorbate to limit catechol and indole oxidation. To examine the Na+ dependency of transport, equimolar choline chloride was substituted for NaCl in the incubation buffer. Incubations were conducted at 22°C for 60 min unless otherwise indicated. Uptake was terminated by aspirating assay medium from oocytes, followed by three gentle 1-ml washes in Ca2+-containing oocyte medium (22°C), all within 60 sec. Oocytes were then solubilized in 500 ,ul of 1% NaDodSO4 and transferred to 5 ml of EcoScint (National Diagnostics, Mannville, NJ) for scintillation spectrometry. All experiments were conducted on four or more oocytes per condition. Mean transport activities from three or more experiments, performed on separate batches of oocytes, are reported as pmol/hr (mean SEM). Significant (P 0.05) increases in induced uptake relative to that observed in parallel incubations with noninjected oocytes were determined with a one-tailed Student's t test for unpaired samples. ±

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RESULTS In initial studies, two-electrode voltage clamp analysis of uninjected and injected oocytes showed resting membrane potentials of -50 to -70 mV. As frequently observed (18), oocytes injected with rat brain mRNA expressed brisk electrophysiological responses to 5HT and GABA (data not shown), confirming the integrity of mRNA preparations. An initial evaluation of L-[3H]glutamate transport in cerebellar RNA-injected (40 ng) oocytes established that uptake could be readily observed within single oocytes incubated for 60 min at 22°C, with levels exceeding by >10-fold the basal accumulation of L-glutamate in uninjected oocytes. A highly significant linear correlation (r > 0.99) between the quantity of mRNA injected (0.4-40 ng) and L-glutamate uptake velocity was observed. Indeed, 1 ng of injected cerebellar poly(A)+ RNA was sufficient to induce a 2-fold elevation in L-[3H]glutamate transport above uninjected controls, while injections with highly diluted solutions yielded insignificant transport. To explore the distribution and diversity of transporter expression, experiments were conducted to examine the

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uptake of several neurotransmitters after injection of mRNA derived from dissected brain regions. Substrates were chosen that possess well-characterized Na'-dependent transport systems (11), typically assayed with brain slices or synaptosomes in vitro. In accordance with the density and most likely neuroanatomic distribution of cell bodies synthesizing these activities, significant transport above uninjected controls was observed for virtually all substrates (Table 1). Thus, Lglutamate and GABA, the principal excitatory and inhibitory neurotransmitters in the vertebrate brain (26), were transported after injections of mRNA derived from all brain regions. L-Glutamate transport was highest with forebrain and cerebellar mRNA. Lung MRNA, found to direct the expression of electrophysiologically assayable acetylcholine receptors (data not shown), failed to significantly induce L-glutamate transport activity. Unlike L-glutamate transport, GABA uptake was most pronounced in oocytes injected with brainstem and spinal cord mRNA. Glycine, thought to act as an inhibitory neurotransmitter in hindbrain regions (26), was only accumulated above controls in oocytes injected with brainstem and spinal cord mRNA. Dopamine and 5HT transport was considerably lower than observed for the amino acids, yet clearly elevated above controls and consistently derived from brain regions specific to each neurotransmitter system. Dopamine transport was observed after injection of forebrain and brainstem mRNA, while 5HT transport was significantly elevated only in oocytes injected with brainstem mRNA. Only norepinephrine failed to be accumulated above background rates observed with uninjected oocytes. Finally, enhanced choline transport was induced by Table 1. Regional distribution of neurotransmitter transport in X.

laevis oocytes Substrate L-Glutamate

RNA source Forebrain Cerebellum Brainstem Spinal cord Lung

Exp., n 6 13 3 3 4 14 8 4 3 3 12 4 2 5

Transport activity, pmol/hr 2.33 ± 0.51* 1.73 ± 0.22* 1.42 ± 0.49* 0.94 ± 0.08*

0.25 ± 0.02 0.16 ± 0.03 GABA 0.56 ± 0.11* 0.44 ± 0.11* 0.79 ± 0.32* 1.07 ± 0.36* 0.22 ± 0.04 Forebrain Glycine 0.55 ± 0.14 Cerebellum 0.52 ± 0.16 Brainstem 1.59 ± 0.22* 4 Spinal cord 0.96 ± 0.19* Noninjected 9 0.48 ± 0.06 Dopamine Forebrain 9 0.028 ± 0.002* Brainstem 3 0.026 ± 0.002* 7 Noninjected 0.011 ± 0.002 Norepinephrine Forebrain 3 0.014 ± 0.003 Brainstem 3 0.019 ± 0.005 4 Noninjected 0.025 ± 0.008 Serotonin Forebrain 3 0.050 ± 0.009 Brainstem 5 0.053 ± 0.005* 4 Noninjected 0.036 ± 0.008 Choline Forebrain 4 0.94 ± 0.05* Brainstem 3 1.10 ± 0.33* 4 Spinal cord 0.90 ± 0.09* 8 Noninjected 0.66 ± 0.06 Each experiment was performed with a different batch of oocytes, with at least four oocytes per experiment (n). Transport activity is given as mean ± SEM. *Represents substrate transport that was significantly above noninjected levels (P s 0.05).

Noninjected Forebrain Cerebellum Brainstem Spinal cord Noninjected

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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 induced in oocytes by brain mRNAs, we focused upon L-glutamate transport arising from the injection of adult rat cerebellar mRNA. L-Glutamate was chosen based on its pervasive role in synaptic excitation in the vertebrate brain and its robust transport activity in injected oocytes (Table 1); 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 at relatively constant levels for up to 5 days (Fig. lA). L-Glutamate accumulation at 220C increased linearly with time and plateaued between 60 and 80 min (Fig. 1B). EadieHofstee transformation of data from assays conducted at 220C for 60 min with various concentrations of substrate suggests a single population of induced L-glutamate transporters with an apparent Km of 14.2 uM and a Vmax of 15.2 pmol/hr (Fig. 1C). High-affinity L-glutamate uptake in brain slices and synaptosomes is dependent upon extracellular Na' and is inhibited by submillimolar concentrations of certain acidic

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[3~~~~~~~~~~~~~~0 FIG. 2. NA' dependency of oocyte L-glutamate and dopamine transport. (A) Na' dependence of oocyte L-glutamate transport after injections (40 ng) of forebrain and cerebellar poly(A)+I RNA, assayed for 60 min at 22°C with 1.0 ,uM L-[3H]glutamate, 2 days after RNA injection. (B) As in A, except that forebrain RNA-injected oocytes were assayed for [Hldopamine transport at 0.1,M. Data are values of three separate experiments, conducted on different batches of oocytes, with at least four oocytes per condition (mean ± SEM). Solid bars, injected oocytes with 96 mM NaCI; hatched bars, injected oocytes with 96 mM choline chloride; open bars, uninjected oocytes 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 substitution had no significant effect on uptake with uninjected oocytes (data not shown).

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amino acid analogues or by incubations conducted at reduced temperatures (27, 28). Equimolar substitution of choline chloride for NaCl reduced L-glutamate transport induced by both cerebellar and forebrain mRNA to levels observed with uninjected oocytes (Fig. 2A). Similarly, dopamine uptake, measured with forebrain mRNA-injected oocytes, was also found to bseparte eerimeto Na+ substitution (Fig. 2B). Assays laM conducted in the L-Cysteinate and presence of 100 L-aspartate, relatively potent L-glutamate transport ionhibitors, reduced specific L-glutamate uptake by 76 and 70%, respectively (Fi2 3). In contrast, 10i utM N-methylD-aspartate, a selective L-glutamate receptor agonist, produced no significant inhibition. As well, specitnc L-glutamate

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FIG. 1. Characterization of L-glutamate transport in Xenopus oocytes injected with cerebellar poly(A)+ RNA (40 ng). (A) Expression as a function of days after RNA injection. Incubations were 60 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 after RNA injection as described in A. (A and B) *, Injected oocytes; o, uninjected oocytes. (C) Eadie-Hofstee transformation of assay determinations with 20 nM L-[3H]glutamate and various concentrations of unlabeled substrate. Incubations were 60 min at 22°C. Assays were done 2 days after injection. Data are values of at least three separate experiments, conducted on different batches of oocytes, with at least four oocytes per point (mean + SEM).

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FIG. 3. Effects of inhibitors and reduced temperature on Lglutamate transport from cerebellar mRNA-injected oocytes. Data are plotted as a percentage of transport activity obtained with oocytes incubated for 60 min at 22°C, after subtraction of basal transport in uninjected oocytes. Data are values of at least three separate experiments, conducted on different batches of oocytes, with at least four oocytes per condition (mean + SEM). NMDA, N-methyl-D-aspartate; L-Cyst, L-cysteinate; 4 C, 4°C.


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FIG. 4. Sucrose-density gradient size-fractionation of cerebellar poly(A)+ RNA. Poly(A)+ RNA (100 ug) was fractionated over a linear (10-31%) sucrose gradient. (A) Optical density measurement of fractionated RNA at 254 nm. Sizes (in kb) of selected fractions are designated by arrows above the gradient. (B) L-Glutamate transport activity of selected fractions. Transport was assayed at 220C, for 60 min, with 1 AM L-[3H]glutamate, 2 days after oocyte injection (40 n1). (C) GABA transport activity of selected fractions. Transport of [3H]GABA was measured as for L-[3H]glutamate in B, with 1.0 AuM substrate. Data represent pooled values from three separate experiments conducted with different batches of oocytes.

accumulation was reduced to levels obtained with uninjected oocytes when incubations were conducted at 40C rather than 220C (Fig. 3). Thus, the oocyte L-glutamate transport activity induced by cerebellar mRNA is both time and temperaturedependent, saturable by micromolar substrate concentrations, Na'-dependent, and inhibited by agents known to block brain L-glutamate uptake. To determine the sizes of mRNA encoding cerebellar transport activities, poly(A)+ RNA was fractionated on a linear (10-31%) sucrose-density gradient (Fig. 4A) and fractions assayed for both L-glutamate and GABA transport activity (Fig. 4 B and C). The major activities were found to migrate as single peaks between 2.5 and 3.0 kilobases (kb), with peak transport for both arising from fractions of 2.7 kb. Approximately 90% of both the L-glutamate and GABA transport activities present in unfractionated cerebellar mRNA was recovered from the gradient. As fractions 19-25 contain 95% of the L-glutamate and GABA transport activity across the gradient, a substantial enrichment of transport activity over unfractionated mRNA was obtained. Enrichment was reflected as well in comparably higher specific uptake per ng of injected mRNA. Thus, 40 ng of total cerebellar mRNA induced on average L-glutamate transport of 1.73 pmol/hr and GABA transport of 0.44 pmol/hr (Table 1), while in fraction 23, -6 ng gave 3.05 pmol/hr and 1.33 pmol/hr, respectively.

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DISCUSSION The X. laevis oocyte offers many advantages for the in vitro functional reconstitution of low-abundance membrane proteins, such as ion channels, pumps, and receptors (18), and has been shown to be suitable as a screening system for the isolation of cDNA clones (16, 29-31). With the oocyte system, we have expressed transport activities for five of the major neurotransmitters in the vertebrate central nervous system, L-glutamate, GABA, glycine, dopamine, and 5HT, as well as uptake of the acetylcholine metabolite, choline. A good correlation was observed between the regional distribution of active mRNA and the cellular origin of chemically defined neurons. Thus, all regional mRNAs gave rise to the transport of L-glutamate and GABA, compounds implicated in synaptic transmission throughout the nervous system, while glycine uptake was restricted to mRNA isolated from regions containing putative glycinergic neurons, brainstem and spinal cord (26). The higher L-glutamate transport rates observed with forebrain and cerebellar mRNA are consistent with the large numbers of glutamatergic soma in these regions, including many hippocampal, thalamic, and cortical neurons, and the abundant cerebellar granule cells (26). In contrast, mRNA derived from lung, a tissue not known for high-affinity Na'-dependent L-glutamate uptake, failed to induce Lglutamate transport above levels observed with uninjected oocytes. Although a widespread neurotransmitter system, a high density of GABAergic neurons is found in the dorsal spinal cord of the rat (32) and is a likely mRNA source of the caudal enrichment of GABA transport we observed. Sarthy (33) described the ability of juvenile rat brain RNA to induce Na+-dependent GABA transport in oocytes, with activity considerably greater than we observed with mRNA derived from adult forebrain. This discrepancy may simply represent an ontogenetic decline in forebrain GABA transporter mRNA. Brain regions rich in monoamine neurons (34, 35) provided suitable sources for monoamine transporter expression. Our division of brainstem and forebrain transects the mesencephalon at the caudal margin of the superior colliculus, placing the mesencephalic dopamine neurons largely in the forebrain with those from the hypothalamus, while the bulk of serotonergic raphe neurons largely reside within our brainstem division. Like synaptosomal dopamine uptake (5), dopamine transport in forebrain mRNA-injected oocytes is Na+dependent. The absolute levels of dopamine and 5HT uptake were considerably lower than the levels found for amino acid transport, despite incubations with substrate concentrations closer to their endogenous transport Km [-0.1 ,uM (36)]. These observations are consistent with the low abundance of monoamine soma in the rodent central nervous system relative to glutamatergic and GABAergic neurons. Our inability to detect norepinephrine transport may thus be a consequence of the even lower density of noradrenergic neurons in the rodent central nervous system (11), as well as the -10 times lower affinity of norepinephrine for the dopamine transporter (3, 36). Further studies will ascertain if oocyte dopamine and 5HT transporters display the pharmacological sensitivities of those localized to hypothalamic and mesencephalic dopamine or raphe 5HT neurons (11). Choline transport was observed after injection of mRNA from spinal cord, brainstem, and forebrain, conceivably a reflection of cholinergic neurons in these regions, such as those of striatal and septal nuclei and of hindbrain motor neurons (37). Although the anatomical distribution of transport activities appears highly consistent with the regional localization of neurons synthesizing and releasing a particular neurotransmitter, glial mRNAs may also contribute to the induced transport activities, especially for the amino acids (38). In this regard, oocyte studies with mRNA derived from rodents deficient in the putative neuronal sources of these signals (19)

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may allow for finer cellular distinctions. Specific subtypes of both L-glutamate and GABA transporters have also been proposed (39, 40). It may be possible with specific inhibitors of glial and neuronal GABA transport to pharmacologically distinguish subtype mRNAs, although such tools are not presently available for L-glutamate carriers (41). Thus, GABA transport from oocytes injected with unfractionated juvenile rat brain mRNA display both diaminobutyric acid and /-alanine sensitivity (33), inhibitors reputedly selective for neuronal and glial carriers, respectively (38). We focused upon the expression of L-glutamate uptake from mRNA derived from cerebellum, with hopes of characterizing a homogeneous population of well-defined glutamatergic neurons, the granule cells (19, 20). These studies revealed L-glutamate transport to be Na'- and timedependent, and to be both temperature-sensitive and saturable at micromolar substrate concentrations. Cerebellar mRNA-induced L-glutamate uptake was also inhibited by compounds known to block Na'-dependent high-affinity L-glutamate uptake in synaptosomes and brain slices (27, 28), while N-methyl-D-aspartate, a selective L-glutamate receptor agonist, was inactive. Thus, these studies confirm the use of the Xenopus oocyte system for in vitro reconstitution of Na'neurotransmitter cotransporters, permitting an investigation of their intra- and extracellular regulation in single cells. Aoshima et al. (42) have described an induction of intestinal and renal Nat/amino acid transport in both Xenopus and Cynops oocytes. Interestingly, no evidence for acidic amino acid transport could be demonstrated by these investigators. Although their electrophysiological approach does not directly measure amino acid transport, we have also failed to detect L-glutamate transport with peripheral (lung) mRNA. Nonetheless, this expression system appears well suited for investigation of transport proteins in both neural and nonneural tissues, particularly emphasized by the expression (43) and cDNA cloning (16) of Na'-glucose transporter from rabbit intestine. Expression cloning of neurotransmitter transporters would be greatly facilitated by the presence of single, small mRNAs (