©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Role of N-Glycosylation in the Targeting and Activity of the GLYT1 Glycine Transporter (*)

Luis Olivares , Carmen Aragón , Cecilio Giménez , Francisco Zafra (§)

From the (1) Centro de Biologa Molecular ``Severo Ochoa,'' Facultad de Ciencias, Universidad Autónoma de Madrid, Consejo Superior de Investigaciones Cientficas, 28049-Madrid, Spain

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

To elucidate the role of N-glycosylation in the function of the high affinity glycine transporter GLYT1, we have investigated the effect of the glycosylation inhibitor tunicamycin as well as the effect of the disruption of the putative glycosylation sites by site-directed mutagenesis. SDS-polyacrylamide gel electrophoresis of proteins from GLYT1-transfected COS cells reveals a major band of 80-100 kDa and a minor one of 57 kDa. Treatment with tunicamycin produces a 40% inhibition in transport activity and a decrease in the intensity of the 80-100-kDa band, whereas the 57-kDa band decreases in size to yield a 47-kDa protein corresponding to the unglycosylated form of the transporter. Simultaneous mutation of Asn-169, Asn-172, Asn-182, and Asn-188 to Gln also produces the 47-kDa form of the protein, indicating that there are no additional sites for N-glycosylation. Progressive mutation of the potential glycosylation sites produces a progressive decrease in transport activity and in size of the protein, indicating that the four putative glycosylation sites are actually glycosylated. N-Glycosylation of the GLYT1 is not indispensable for the transport activity itself, as demonstrated by enzymatic deglycosylation of the transporter. Analysis of surface proteins by biotinylation and by immunofluorescence demonstrates that a significant portion of the unglycosylated GLYT1 mutant remains in the intracellular compartment. This suggests that the carbohydrate moiety of glycine transporter GLYT1 is necessary for the proper trafficking of the protein to the plasma membrane.


INTRODUCTION

The re-uptake of neurotransmitter amino acids into presynaptic nerve terminals or the neighboring fine glial processes provides one way of clearing the extracellular space of the neuroactive substances and so constitutes an efficient mechanism by which the postsynaptic action can be terminated (1, 2, 3, 4) . This process is carried out by integral membrane proteins that use a chemical or electrochemical gradient as the driving force for the uphill movements of the transmitter across the plasma membrane. Some of the Na- and Cl-dependent neurotransmitter transporters have been purified from mammalian brain (5, 6, 7) , and more recently, cDNA clones encoding transporters for -aminobutyric acid (8, 9, 10, 11, 12) , catecholamines (13, 14, 15, 16, 17) , serotonin (18, 19) , glycine (20, 21, 22, 23, 24, 25) , glutamate (26, 27, 28) , and proline (29) have been isolated.

Glycine is an important inhibitory neurotransmitter in the central nervous system of vertebrates, mainly in the spinal cord and the brain stem. In addition, glycine could potentiate the action of glutamate, the main excitatory neurotransmitter in the brain, on postsynaptic N-methyl- D-aspartate receptors. Specific high affinity transport systems for glycine have been identified in nerve terminals and glial cells (30, 31, 32, 33, 34, 35, 36, 37) . To date, two different glycine transporters have been cloned. The first one, GLYT1 (20, 21, 22, 25) , presents three isoforms produced by alternative splicing and/or alternative promoter usage (23, 25) (termed GLYT1a, GLYT1b, and GLYT1c, according to Liu et al. (24) and Kim et al. (25) ). More recently, the existence of a second glycine transporter (GLYT2) (24) has been reported, which is present specifically in the brain stem and spinal cord (24, 38) , brain areas where strychnine-sensitive glycine receptors are more represented. The hydropathic profile of these proteins suggests the presence of 12 transmembrane segments. On the basis of cDNA sequence of GLYT1, there are seven potential glycosylation sites for N-linked glycosylation, but only four are supposed to be extracellular, located in the loop between putative transmembrane segments three and four (22) . The presence of N-glycosylation sites in all the transporters cloned so far suggests that glycosylation must play an important role in the function of these proteins. Moreover, it has been recently demonstrated that the removal of oligosaccharides by enzymatic methods from a purified glycine transporter affects the transport activity (39) .

In the present study, we have used site-directed mutagenesis to investigate which of the potential glycosylation sites of GLYT1 are actually utilized and the consequences of the disruption of these sites on the targeting and transport activity of the protein.


EXPERIMENTAL PROCEDURES

Materials [2-H]Glycine (1757.5 GBq/mmol) was supplied by DuPont NEN. Ligase and restriction enzymes were from Boehringer Mannheim. Taq polymerase was from Perkin-Elmer Corp. DEAE-dextran, pGEX-2, pSVL, and glutathione S-Sepharose 4B were from Pharmacia Biotech Inc. (Upsala, Sweden). Sulfo- N-succinimidyl-6-biotinamido hexanoate was from Pierce. Recombinant N-glycosidase F was from New England Biolabs. Citifluor, FITC() -coupled goat anti-rabbit IgG, peroxidase-linked anti-rabbit IgG, and ECL reagent were from Amersham Corp. (Bucks, United Kingdom). Immobilized streptavidin and cholic acid were obtained from Sigma. Cholic acid was recrystallized and neutralized as described (7) . All other reagents were obtained in the purest form available. Methods

Expression in COS Cells

Transient expression of COS cells was carried out using DEAE-dextran with dimethyl sulfoxide according to the method of Kaufman (40) with minor modifications. COS cells were grown in 24-well plates at 37 °C and 5% COin high glucose Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin G, and 100 µg/ml streptomycin sulfate. Cells were used 2 days after transfection.

Glycine Transport Assay

Glycine transport into reconstituted liposomes was measured using an inwardly directed NaCl gradient in the presence of a negative membrane potential, as described (7, 41) . Transport assays in transfected COS cells were performed at 37 °C in HEPES-buffered saline (150 m M NaCl, 10 m M HEPES, 1 m M CaCl, 10 m M glucose, 5 m M KCl, 1 m M MgCl, pH 7.4) as described (42) . All incubations were carried out in triplicate. Each experiment was repeated at least three times with different cell cultures. For estimating statistical differences, the data were compared using the Student's t test; differences at the 0.05 level were considered statistically significant.

Solubilization and Reconstitution Procedure

For each reconstitution experiment, transfected cells from one dish (10-cm diameter) were used. Cells were scraped and collected by centrifugation. Solubilization of cells was performed in the presence of 25% saturated ammonium sulfate and sodium cholate at a 1:1 detergent/protein ratio. After 10 min on ice, the mixture was centrifuged for 5 min. The supernatant was reconstituted with asolectin/brain lipids, and transport was measured as described (41) .

Preparation of Glutathione S-Transferase-Glycine Transporter Fusion Proteins

Antibodies against a fusion protein of the glutathione S-transferase and the carboxyl terminus of GLYT1 were produced as described (43) . In brief, a PCR fragment corresponding to the 76 amino acids of the carboxyl terminus of GLYT1 was cloned in the BamHI- EcoRI restriction sites of pGEX-2 plasmid, keeping the correct reading frame. Transformant Escherichia coli XL-1 were selected, and the expression of the fusion protein was induced by isopropylthiogalactoside. The protein was purified in a glutathione-Sepharose 4B column following the instructions of the supplier.

Rabbit Immunization

Two rabbits were immunized with GST-GLYT1 fusion protein. Each rabbit received intradermic injection of the fusion protein in complete Freund's adjuvant (100 µg at day 0). Subsequent immunizations were done subcutaneously with the same amount of protein in incomplete Freund's adjuvant at days 14, 28, 42, and 56. The antibodies have been characterized elsewhere.()

Preparation of SDS Extracts

SDS extracts from whole rat brains or specific regions were prepared by homogenizing and solubilizing the tissue in PBS medium (137 m M NaCl, 0.9 m M CaCl, 2.68 m M KCl, 1.47 m M KHPO, 0.49 m M MgCl, 7.37 m M NaHPO, pH 7.4) with SDS (10 mg/ml) and 1 m M phenylmethanesulfonyl fluoride and removing unsolubilized material by centrifugation (50,000 g, 10 min, 10 °C).

Electrophoresis and Blotting

SDS-PAGE was done in the presence of 2-mercaptoethanol. The gels were run slowly (overnight) at constant current starting at 30 V. After electrophoresis, samples were transferred by electroblotting onto a nitrocellulose membrane in a semidry electroblotting system (Life Technologies, Inc.) at 1.2 mA/cmfor 2 h. The transfer buffer consisted of 192 m M glycine and 25 m M Tris-HCl, pH 8.3. Nonspecific protein binding to the blot was blocked by the incubation of the filter with 3% non-fat milk protein in 10 m M Tris-HCl, pH 7.5, 150 m M NaCl for 4 h at 25 °C. The blot was then probed with the indicated dilutions of crude antisera overnight at 4 °C. After washing, blots were then probed with a peroxidase-linked anti-rabbit IgG, and bands were visualized with the ECL detection method (Amersham Corp.) and quantified by densitometry (Molecular Dynamics Image Quant v. 3.0).

Site-directed Mutagenesis

A PCR-based site-directed mutagenesis strategy was followed using as template the rB20a clone that had been previously subcloned in the XhoI- XbaI sites of pBluescript. Glycosylation sites were eliminated sequentially, starting with mutations N182Q and N188Q (mutant N2). Mutant N2 was used as template for mutation N172Q (mutant N3), and mutant N3 was used as template for mutation N169Q (mutant N4). This mutagenesis strategy is a modification of the method of Higuchi (44) and is summarized in Fig. 1. The ``left'' PCR product for the N2, N3, and N4 mutants was generated by using oligonucleotides T3 (ATTAACCCTCACTAAAG) (sequence from the vector) and B1 (CACTTTCCCTGAAGACTTGACTCCTCG) (sequence complementary to bases 715-741 of rB20a). The ``right'' PCR product for the N2 mutant was generated by using the oligonucleotides A2 (CTGTCTGGCCAACTGTCTCACCTGTTCCAATACACCTTG) (bases 535-573 of rB20a) and KV1 (GGATGCCATGGTGATGAGG) (complementary to bases 930-948 of rB20a). For mutant N3, the ``right'' PCR was produced with oligonucleotides A3 (TTCCAATCTCACCCAAGGCTCCCGGCCC) (bases 501-528) and KV1. For mutant N4, the ``right'' PCR was produced with oligonucleotides A4 (GGATGCTTCCCAACTCACCCAAGGCTCCC) (bases 495-523) and KV1. Underlined bases correspond to mutated codons. For every mutant, the corresponding ``left'' and ``right'' PCR products were purified, denatured (94 °C, 10 min), annealed each other (50 °C, 5 h), and elongated with Taq polymerase (72 °C, 10 min) in a volume of 50 µl. Then, DNA contained in 5 µl of the elongation reaction was reamplified by PCR using the external oligonucleotides T3 and KV1. The amplified products were digested with XhoI and NcoI, and the XhoI -NcoI fragment of the wild type rB20a clone was exchanged for the mutated one. The XhoI site was located between the T3 oligonucleotide and the starting ATG of rB20a, and the NcoI restriction site was located in the KV1 oligonucleotide. This procedure yields 50% mutants and 50% of wild type. Mutant clones were identified by sequencing, and then the full-length clone was subcloned in the XhoI- XbaI sites of the pSVL expression vector.

Immunofluorescence of Transfected Cells

48 h after transfection, COS cells were washed three times in PBS, fixed for 20 min at room temperature in 2% paraformaldehyde in PBS, and rewashed three times in PBS. The fixed cells were then incubated for 1 h at room temperature in PBS containing 1% bovine serum albumin and 0.02% digitonin. After that, fixed cells were incubated for 2 h at room temperature in the same medium containing rabbit anti-GLYT1 antibody diluted 1:1000. The cells were washed, and bound primary antibodies were detected with FITC-coupled goat anti-rabbit IgG for 60 min at room temperature. Cells were thoroughly washed, and the cover glasses were mounted in 90% glycerol plus Citifluor. Samples were visualized on a microscope Zeiss Axioskop.

Cell Surface Labeling

Cell surface proteins of transfected COS cells were labeled with the cell membrane impermeable reagent sulfo- N-succinimidyl-6-biotinamido hexanoate as described (45) . In brief, cells were washed with PBS and incubated for 30 min in the presence of 0.5 mg/ml sulfo- N-succinimidyl-6-biotinamido hexanoate in ice-cold PBS. Then, cells were washed, and the excess of reagent was quenched with 10 m M lysine in PBS for 10 min. Cells were lysed in immunoprecipitation buffer (150 m M NaCl, 50 m M HEPES-Tris, 5 m M EDTA, 1% Triton X-100, 0.25% deoxycholate, 0.1% SDS, pH 7.4), and biotinylated proteins were precipitated with agarose-streptavidin. Precipitated proteins were fractionated by SDS-PAGE and analyzed by Western blot for immunoreactivity with anti-GLYT1 antibodies, as described above.

Protein Determination

Protein concentration was measured by the method of Bradford (46) or Peterson (47) using bovine serum albumin as a standard.


RESULTS AND DISCUSSION

To determine whether the carbohydrate moiety of GLYT1 plays a role in the function of the protein, we carried out initial experiments by using the general inhibitor of glycosylation tunicamycin. When COS cells transfected with the wild type GLYT1 expression vector were treated with 10 µg/ml tunicamycin for 24 h, a reduction in the transport activity from 33.8 pmol/10 min/mg of protein to 20.1 pmol/10 min/mg of protein was observed. A similar decrease in transport activity has been previously described for norepinephrine and -aminobutyric acid transporters in different cell types after treatment with tunicamycin (48, 49) . Fig. 2 shows immunoblotting data with a specific antibody raised against the carboxyl terminus of GLYT1. When proteins from GLYT1-transfected COS cells were probed with this antibody, two bands of 57 and 80-100 kDa appeared in controls. Molecular mass of the GLYT1 transporter expressed in COS cells slightly differs from that of native GLYT1 from rat spinal cord membranes,indicating differences in host cell-specific posttranslational processing, a phenomenon that has been also shown for the dopamine transporter (50) . After treatment with tunicamycin, a new band of 47 kDa was observed, while the 80-100-kDa band decreased in intensity, and the 57-kDa band almost disappeared. The broad shape of the upper band is characteristic of glycoproteins, reflecting in some cases heterogeneity in the glycosylation pattern (51) . This experiment suggests that the 57-kDa band of the wild type corresponds to a partially glycosylated intermediate that disappears in the absence of further glycosylation to become the 47-kDa fully deglycosylated protein. This intermediate lasted much shorter than the upper form, whose amount was only reduced by 39% after a 24-h treatment. A 24-h half-life has been reported for the fully glycosylated form of the closely related norepinephrine transporter (49) . However, the interpretation of the effects of tunicamycin is complicated by the fact that tunicamycin affects glycosylation of many other proteins and reduces protein synthesis. Thus, the observed effects on the transport activity could be indirectly mediated by changing the activity of ion channels or the (Na-K)-ATPase function and hence altering ion concentrations both inside and outside of the cell.

To address the role of glycosylation on the GLYT1 function in a more selective and specific way, we performed site-directed mutagenesis of the potential glycosylation sites of GLYT1 and studied the effect of such modification on the properties of the protein in transfected COS cells. We disrupted 2, 3, or all 4 predicted extracellular N-glycosylation sites by substituting glutamine for asparagine in the consensus sites. The resulting mutants were called N2, N3, or N4. This stands for substitution of two (Asn-182 and Asn-188), three (Asn-172, Asn-182, and Asn-188), or four (Asn-169, Asn-172, Asn-182, and Asn-188) putative glycosylation sites, respectively. Besides the inability of glutamine to function as acceptor of N-glycosylation, this is a highly conservative amino acid substitution that should have minimal structural effects on the protein.

Time courses of [2-H]glycine transport in COS cells transfected with the wild type or the mutated GLYT1 expression vector are presented in Fig. 3. The results show that transport activity could be detected in all cases, confirming that each of these forms of the transporter was expressed in the cells. However, progressive mutation of the potential glycosylation sites produced a progressive decrease in transport activity. When the four potential glycosylation sites were mutated, only approximately 30% of the activity was retained (average percent of initial rate steady state of the wild type activity). All the active or partially active transporter forms retained the characteristic sodium and chloride dependence (Table I).

Fig. 4 shows a kinetic analysis of the N4 mutant using a wide range of substrate concentrations (0.2 µ M to 2 m M). The Eadie-Hofstee plot of the data (Fig. 4 B) revealed that the transport characteristics of the glycosylation-defective N4 mutant were significantly different from those of the wild type. Whereas the apparent Kof the transport process did not change significantly (195 µ M in the wild type versus 167 µ M in the N4 mutant), the Vdecreased dramatically (19.2 nmol/4 min/mg of protein in wild type versus 3.5 nmol/4 min/mg of protein in N4 mutant). According to enzyme kinetics, Vis an indicator of total enzyme activity. Thus, in the case of uptake kinetics, a decrease in Vof the transport system could be explained by defects in the synthesis and/or turnover of the protein, by a decrease in the number of available transport systems at the plasma membrane, or by a reduced functioning of the transporter.

When the expression of GLYT1 was analyzed by Western blot, two bands were observed in the wild type and in the mutants N2 and N3, and a single band was observed in N4. The intensity of the upper band of these mutants was comparable with the wild type (except for N4 mutant). In addition, the mobility of the bands became progressively increased by increasing the number of mutated asparagines (Fig. 5, left blot). This experiment demonstrated that the lower band was indeed an intermediate in the synthesis of the transporter that was already glycosylated, corresponding probably to the endoplasmic reticulum form of the protein after accepting the core of 14 sugars from the dolychol-linked oligosaccharide (52) . The scale of sizes of the lower band, ranging from 57 kDa in the wild type, to 52 kDa in N2, to 49 kDa in N3, and to 47 kDa in N4 mutants, was compatible with this possibility (the molecular mass of each oligosaccharide core is approximately 2500 Da) and indicated that the four putative glycosylation sites are actually glycosylated and extracellularly located. The N4 mutant was expressed as a single band whose size coincides with that of the lower band observed in the presence of tunicamycin (Fig. 2), indicating that there are no additional sites for N-glycosylation. The two asparagines placed in the carboxyl-terminal part of the protein (Asn-578 and Asn-628), and that located close to the amino terminus (Asn-29) are therefore not used as glycosylation targets, as corresponds to the intracellular location of both amino and carboxyl termini of the protein (43) .


Figure 2: Effect of tunicamycin on GLYT1 expression. COS cells were transfected with GLYT1 (clone rB20a). After 48 h of incubation at 37 °C in the COincubator, cells were treated for 24 h with 10 µg/ml tunicamycin ( TN). Solvent (ethanol) was added to control cells ( CT). MT, mock-transfected cells. Cell protein was subjected to SDS-PAGE, electroblotted onto nitrocellulose, and incubated with anti-GLYT1 antibody (1:250). Bands were visualized with the ECL detection method.



Next, we investigated the reasons for a decrease in the transport activity with deglycosylation. In the experiment shown in Fig. 5( right blot), we measured the amount of protein reaching the plasma membrane. For that purpose, proteins from the cell surface were biotinylated with the impermeant reagent sulfo- N-succinimidyl-6-biotinamido hexanoate, and the biotinylated proteins were precipitated with immobilized streptavidin, followed by quantitation in Western blot with the anti-GLYT1 antibody. The results show that the protein arrived to the plasma membrane with progressively more difficulties as the degree of glycosylation decreased. This was especially clear in the mutant N3, where the amount of total cellular GLYT1 protein was similar to that in the wild type, whereas membrane GLYT1 was only 55% of the wild type (Fig. 5). In the case of N4 mutant, there was also an important decrease in the protein reaching the membrane. Considering as 100% the fraction of total protein that reached the membrane in the wild type (compare lane 2 in Fig. 5 versus lane 6), only 42% of total N4 protein arrived to the cell surface ( lane 5 in Fig. 5 versus lane 9).


Figure 5: Subcellular distribution of GLYT1 immunoreactivity in transfected COS cells. COS cells were transfected with the wild type GLYT1 (clone rB20a) ( WT), N2, N3, or N4 mutants or mock transfected ( C). After 48 h of incubation at 37 °C in the COincubator, total cell protein (total protein) was solubilized, or cell surface proteins were biotinylated and isolated with streptavidin-agarose beads (membrane protein). Proteins were subjected to SDS-PAGE, electroblotted onto nitrocellulose, and incubated with anti-GLYT1 antibody (1:250). Bands were visualized with the ECL detection method.



Further evidence of this difficulty of deglycosylated protein to be transported to the plasma membrane was obtained by immunofluorescence (Fig. 6). Whereas the plasma membrane was clearly labeled in wild type cells, fluorescence remained mainly in the endoplasmic reticulum in the N4 mutant. Considering together all these data, it can be concluded that glycosylation is necessary for proper targeting of GLYT1 to the plasma membrane, although a certain leakage exists, and a fraction of the deglycosylated protein can reach the cell surface.

The effect of the deglycosylation on the transport activity itself was studied in an experiment of solubilization, reconstitution, and deglycosylation of the transporter. GLYT1-transfected COS cells underwent detergent extraction, and the solubilized proteins reconstituted into liposomes. The reconstituted proteins were treated with N-glycosidase F, an enzyme which hydrolyzes most types of N-linked carbohydrate groups from glycoproteins. Fig. 7 shows the effect of N-glycosidase F both on the specific glycine transport activity (Fig. 7 A) and on the electrophoretic mobility of the transporter (Fig. 7 B). Results show that N-glycosidase F treatment does not affect transport of glycine. When deglycosylated proteins were analyzed by Western blot, a single band of 47 kDa appeared. The size of the band agrees with that observed in the expressed N4 mutant. These results indicate that after 3 or 5 h of incubation, the deglycosidase was able to remove almost completely the N-linked oligosaccharide chains of the reconstituted GLYT1 (Fig. 7 B). From these data, we can conclude that glycosylation is not involved in the GLYT1 transport activity itself. Modifications of the oligosaccharide structure of glycoproteins related to changes in the biological activity of transporter proteins have been described. The Na-Hantiporter of renal brush-border membrane (53) , the GLUT1 glucose transporter (54, 55) , the organic cation transporter of renal brush-border membrane (56) , and a purified glycine transporter probably corresponding to GLYT2 (39) are examples of such a correlation. Moreover, N-glycosylation plays an important role in the targeting to the membrane for the GLUT1 glucose transporter (57) , the renal Na-Pcotransporter (58) , or the serotonin transporter (59) . However, glycosylation is not required for the transport activity of the serotonin transporter (59) or the intestinal Na-glucose transporter (60, 61) .


Figure 7: Effect of glycosidase treatment on transport activity and electrophoretic mobility of reconstituted native GLYT1. Panel A, COS cells transfected with wild type GLYT1 were solubilized, reconstituted into liposomes, and treated with N-glycosidase F (500 units/µg of protein). 1, mock-transfected COS cells; 2, control non-treated GLYT1-transfected COS cells; 3, control GLYT1-transfected COS cells after 3 h at 37 °C; 4, GLYT1-transfected COS cells after 3 h of treatment with N-glycosidase F; 5, control GLYT1-transfected COS cells after 5 h at 37 °C; 6, GLYT1-transfected COS cells after 5 h of treatment with N-glycosidase F. The results are means ± S.E. of two triplicate determinations. Values were compared with control values. Result in bar 4 was not significant versus its control in bar 3. Result in bar 6 was not significant versus its control in bar 5. Uptake value for nontreated GLYT1-transfected cells is 20.02 pmol/15 min/mg of protein (100%). Panel B, 25 µg of protein of the same fractions used in A were electrophoresed, electroblotted onto a nitrocellulose membrane, and incubated with anti-GLYT1 antibody (1:250). Bands were visualized with the ECL detection method.



In summary, our data demonstrate that the mature form of the glycine transporter GLYT1 contains four Asn-linked glycosylation sites located extracellularly in the hydrophilic domain between the predicted third and fourth membrane-spanning segments. Although impairment of the transporter glycosylation either by tunicamycin treatment or by site-directed mutagenesis leads to a decrease in glycine transport, N-glycosylation of the GLYT1 glycine transporter is not absolutely essential for the transport activity itself. Instead, glycosylation appears to play an important role in the proper trafficking of the transporter to the plasma membrane.

  
Table: Ionic dependence of glycine transport mutants in COS cells

Cells were transfected with an expression vector containing the wild type GLYT1 (clon rB20a) or the indicated mutants. After 48 h of incubation at 37 °C in the COincubator, uptake was assayed at 37 °C in the presence of 0.2 µ M [H]glycine for 10 min in HEPES-buffered saline containing 150 m M NaCl (HBS) or a modified HBS where NaCl had been isotonically substituted by LiCl (-Na) or sodium gluconate (-Cl). The results are the means ± S.E. of two triplicate determinations. Values were compared with control values by using Student's t test.



FOOTNOTES

*
This work was supported by grants from the Spanish Dirección General de Investigación Cientfica y Técnica (PB92-0131), Boehringer Ingelheim Espaa S.A., the Biomed program of the European Union (BMH1-CT93-1110), and an institutional grant from the Fundación Ramón Areces. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Centro de Biologa Molecular ``Severo Ochoa,'' Facultad de Ciencias, Universidad Autónoma de Madrid, 28049-Madrid, Spain. Tel.: 34-1-3974592; Fax: 34-1-3974799.

The abbreviations used are: FITC, fluorescein isothiocyanate; PCR, polymerase chain reaction; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis.

Zafra, F., Gomeza, J., Olivares, L., Aragón, C., and Giménez, C. (1995) Eur. J. Neurosci., in press.


ACKNOWLEDGEMENTS

We thank Drs. K. E. Smith and R. L. Weinshank from Synaptic Pharmaceutical Corp. for sending the rB20a clone. We also thank E. Nez for expert technical assistance.


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