From the Institut für Biochemie,
Universität Erlangen-Nürnberg, Fahrstrasse 17, D-91054
Erlangen, Germany, the § Max-Planck-Institut für
Hirnforschung, Deutschordenstrasse 46, D-60528 Frankfurt, Germany, the
¶ Abteilung Organisation Komplexer Genome and the
Abteilung
Molekulare Genomanalyse, Deutsches Krebsforschungszentrum, Im
Neuenheimer Feld 280, D-69120 Heidelberg, Germany
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ABSTRACT |
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The neuronal glycine receptor is a
ligand-gated chloride channel composed of ligand binding and
structural
polypeptides. Homology screening of a human fetal brain
cDNA library resulted in the identification of two alternative
splice variants of the glycine receptor
3 subunit. The amino acid
sequence predicted for the
3L variant was largely identical to the
corresponding rat subunit. In contrast, the novel splice variant
3K
lacked the coding sequence for 15 amino acids located within the
cytoplasmic loop connecting transmembrane spanning region 3 (TM3) and
TM4. Using P1 artificial chromosome (PAC) clones, the structure of the
GLRA3 gene was elucidated and its locus assigned to human chromosomal bands 4q33-q34 by fluorescence in situ
hybridization. Two transcripts of 2.4 and 9 kilobases, corresponding to
3L and
3K, respectively, were identified and found to be widely
distributed throughout the human central nervous system. Structural
analysis of the GLRA3 gene revealed that the
3K
transcript resulted from a complex splice event where excision of the
novel exon 8A comprising the alternative sequence of 45 base pairs
coincides with the persistence of a large intronic sequence in the
3'-untranslated region. Functional expression in HEK 293 cells of
3L
and
3K subunits resulted in the formation of glycine-gated chloride
channels that differed significantly in desensitization behavior, thus
defining the cytoplasmic loop as an important determinant of channel
inactivation kinetics.
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INTRODUCTION |
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Glycine serves as a major inhibitory neurotransmitter throughout
the mammalian central nervous system (1). The strychnine-sensitive glycine receptor (GlyR)1 is a
pentameric assembly of ligand binding and structural
subunits
displaying significant sequence homology to nicotinic acetylcholine
receptor (2),
-aminobutyric acid receptor type A
(GABAAR), and serotonin receptor type 3 (5-HT3R) subunits (1). As members of a superfamily of
ligand-gated ion channels, these polypeptides share topological
features including a large N-terminal extracellular domain followed by
four transmembrane spanning regions (TM1-TM4). While the N-terminal
domain carries structural determinants essential for agonist and
antagonist binding (3), TM2 is thought to form the inner wall of the
chloride channel (4).
The glycine receptor subunit of rodent central nervous system
exists in different subtypes (
1-
4) encoded by distinct genes (1).
In the murine genome, the corresponding loci have been localized on
chromosomes 11 (
1), X (
2,
4), and 8 (
3), respectively (5-8). Further diversity is achieved by alternative splicing of
primary transcripts encoding the
1 and
2 subunits (9, 10). In the
human, highly homologous
1 and
2 subunits have been identified by
cDNA cloning (11) and assigned to the chromosomal regions 5q31.3
(12, 13) and Xp21.2-22.1 (11), respectively. In both, man and mouse
mutant lines, mutations of GlyR subunit genes result in hereditary
motor disorders characterized by exaggerated startle responses and
increased muscle tone. Pathological alleles of the Glra1
gene are associated with the murine phenotypes oscillator (spdot) and spasmodic (spd) (5,
14-16). A mutant allele of Glrb has been found to underly
the molecular pathology of the spastic mouse (spa), where the intronic insertion of a LINE-1 transposable
element results in aberrant splicing of Glrb primary
transcripts (17, 18). Resembling the situation in the mouse, a variety
of GLRA1 mutant alleles have been shown to cause the human
neurological disorder hyperekplexia or startle disease (12, 13). In
contrast, mutations of the human GLRB gene in hyperekplexia
have not yet been reported (19). By analogy, the gene encoding the GlyR
3 subunit has to be considered a candidate gene for human and murine neurological disorders.
Here we describe the cloning and characterization of two splice
variants of the human 3 subunit and the corresponding
GLRA3 gene which was mapped to the chromosomal region
4q33-q34. Functional expression in HEK 293 cells of GlyR
3L
and GlyR
3K resulted in the formation of glycine-gated chloride
channels that significantly differed in desensitization behavior.
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EXPERIMENTAL PROCEDURES |
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Isolation of GlyR 3 cDNA Clones--
A human fetal brain
library (CLONTECH, Heidelberg, Germany) was used
for isolation of cDNA clones. After plating of ca. 9 × 106 plaque forming units, screening was performed under
intermediate stringency conditions using a complex cDNA probe which
covered the whole open reading frames of rat
1,
2,
3 and human
1 and
2 cDNAs. Hybridization was carried out at 52 °C in 1 mM EDTA, 0.5 M
NaH2PO4/Na2HPO4, 7%
SDS (w/v) and 100 mg/ml denaturated salmon sperm DNA for 12 h.
Filters were washed two times in 2× SSC and 0.1% SDS at 52 °C for
20 min. Of the positive clones, a randomly selected sample was further
analyzed by Southern blot hybridization. To this end, human
1- and
2-specific cDNA probes were generated. The
1 probe amplified
by PCR corresponded to nucleotide positions
30 to 92, while an
2-specific probe was generated by EcoRV and
PvuII restriction digest of an
2 cDNA clone and
covered nucleotides 1162-1262 encoding the cytoplasmic loop between
TM3 and TM4. Hybridization and washing conditions were as given above,
except that the temperature was 65 °C. Two of the resulting clones,
p7 (3 kb) and p12 (1 kb), that hybridized to neither the
1 nor the
2 probe were characterized further. All other DNA manipulations were
according to standard procedures (20). DNA sequencing was performed on
the ABI PRISM 377 automated DNA sequencer.
Isolation and Characterization of Genomic PAC Clones-- Genomic P1 artificial chromosome (PAC) clones were obtained by screening spotted filters of a human high density PAC-library (catalog no. 704) provided by the Resource Center of the German Human Genome Project (Berlin, Germany). The radiolabeled 3-kb insert of clone p7 was employed as screening probe at hybridization and washing conditions as given above (65 °C). Ten genomic PAC clones obtained were subjected to both, PCR characterization using exonic primers and Southern blot hybridization to cDNA probes. Four PAC clones (ZP2.1, LLNLP704H07287Q; ZP3.2, LLNLP704P0954Q1; ZP5.1, LLNLP704L4250Q; and ZP10.1, LLNLP704O21267Q) were further analyzed as they turned out to represent a complete GLRA3 contig. These clones served as templates for direct, automated DNA sequencing with appropriate cDNA primers.
Northern Blot Analysis--
Regional expression of the GlyR 3
subunit was analyzed using prefabricated RNA blots of human central
nervous system tissues (human brain MTN blot II;
CLONTECH, Heidelberg, Germany). Two probes covering
those nucleotide positions that encode the cytoplasmic loop between TM3
and TM4 were used for isoform selective detection of both mRNA
variants,
3L and
3K. Among the probes used, oligonucleotide hsa3-ish-ins (CCATATCTGAGAAACGGTAAAACTTCTCAGTGCAAAAGCTTCTGT) contains the alternatively spliced cDNA stretch of 45 bp characteristic for
the GlyR
3L transcript variant. In contrast, GlyR
3K transcripts were detected employing a radiolabeled
3K-cDNA amplimer from clone p7, covering nucleotides 981 to 1187. The cDNA probe was radiolabeled by random primed incorporation (PrimeIt; Stratagene, Heidelberg, Germany) of [
-32P]ATP, while the
oligonucleotide hsa3-ish-ins was 5'-end-labeled with polynucleotide
kinase (Promega, Mannheim, Germany) using [
-32P]ATP.
Following the manufacturer instructions, overnight hybridizations were
performed using ExpressHyb hybridization solution
(CLONTECH) at 68 °C for oligonucleotide and
70 °C for cDNA probes. Subsequently, membranes were washed three
times at 32 °C for 10 min, and once at 54 °C for 20 min in 2×
SSC and 0.1% SDS. Using intensifying screens, x-ray films were exposed
at
70 °C for 20 h (blot A), 52 h (blot B), and 5.5 h (blot C) as depicted in Figs. 2, A-C.
FISH and Image Processing-- PAC clones ZP3.2 and ZP5.1 were labeled with digoxigenin-11-dUTP using standard nick translation protocols, and FISH was performed to normal human metaphase chromosomes separately for each PAC clone (21). For FISH, 200 ng of each labeled PAC clone were combined with 10 µg of human Cot1 DNA and 10 µg of salmon sperm DNA in 10 µl hybridization solution. Following denaturation, the DNA was allowed to pre-anneal for 30 min at 37 °C. After hybridization at 37 °C overnight, posthybridization washes were performed to a stringency of 0.5× SSC at 60 °C. Hybridized probe was detected by anti-digoxigenin fluorescein isothiocyanate (FITC) and chromosomes were counterstained with 4,6-diamidino-2-phenylindol-dihydrochloride (DAPI). The experiments were analyzed by epifluorescence microscopy. Digitized images were obtained separately for DAPI and FITC with a cooled charge-coupled device camera (Photometrics, Tucson, AZ).
SSCP Screening for GLRA3 Mutant Alleles-- PCR amplimers covering exonic regions and flanking intronic sequences of the GLRA3 gene were generated from peripheral leukocyte DNA and subjected to mutational analysis by SSCP screening. Amplifications (30 cycles) were carried out in a total volume of 50 µl containing 25-50 ng of genomic DNA, 10 pmol of each primer, 50 mM each dNTP, 20 mM Tris-HCl, pH 8.3, 2 mM MgCl2, 50 mM KCl, and 1 unit of Taq polymerase with an annealing temperature of 55 °C. For denaturation, 10 µl of the PCR reaction were mixed with 1.5 µl of 0.5 M NaOH, 10 mM EDTA, 1.5 ml of 50% sucrose, 0.1 M EDTA, 0.1% xylene cyanol, 0.1% bromphenol blue, and subsequently heated. Denaturated fragments were separated for 1.5-2 h on a 12% polyacrylamide gel at the temperatures of 10, 15, 20, and 25 °C. DNA was detected by silver staining (Amersham Pharmacia Biotech, Freiburg, Germany).
Functional Expression of 3 Subunit Variants--
To generate
an appropriate expression construct, the GlyR
3K open reading frame
was amplified from clone p7 using the PCR primers hsa3-S1-exp
(ACAGAATTCCGTATCATGGCCCACGTGA) and hsa3-A10-exp (ACAGGATCCCCCAGAGACTTAATCTTG). Restriction sites for
EcoRI and BamHI (underlined nucleotide positions)
were introduced into hsa3-S1-exp and hsa3-A10-exp and used for
subcloning of the amplimers obtained into the multiple cloning site of
the expression vector pRk5. The cDNA clone p12, corresponding to
GlyR
3L, lacks the 5' end of the open reading frame. Thus, two PCRs
were required for amplification of a complete GlyR
3 open reading
frame. Nucleotide positions
8 to 1186 were amplified from clone p7
using the primers hsa3-S1-Exp and hsa3-A10-1167
(CCTTTGCTTGTAGACATGGT). The sequence harboring the alternatively
spliced part of the GlyR
3L cDNA was amplified employing
oligonucleotides hsa3-S8-999 (TTTTCAGCACTTCTGGAG) and hsa3-A10-exp and
clone p12 as template. Both amplimers were cut at a common
PvuII restriction site following nucleotide position 1006, and the fragments resulting were ligated to yield a complete GlyR
3L
open reading frame. The GlyR
3L fragment obtained was subcloned into
the EcoRI and BamHI restriction sites of the
expression vector pRK5. Both expression constructs, differing in the
45-bp insert of GlyR
3L, were verified by complete sequencing. Human embryonic kidney cells (HEK-293 cells, CRL 1573; ATCC, Manassas, VA)
were transfected (22) with the human GlyR
3K and GlyR
3L expression constructs.
Electrophysiological Recording--
Transfected cells were
viewed with an inverted microscope (Axiovert 35, Zeiss, Jena, Germany)
and continuously perfused (1 ml min1) at room temperature
(21-25 °C) with an extracellular bath solution containing: 137 mM NaCl, 5.4 mM KCl, 1.8 mM
CaCl2, 1 mM MgCl2, 11 mM EGTA, 10 mM HEPES, adjusted to pH 7.2 with
NaOH. Membrane currents were obtained from cells using an EPC-9
amplifier (HEKA Elektronik, Lambrecht, Germany) linked to an Atari STE
computer controlled by HEKA software. The membrane potential was
clamped at
70 mV in all experiments and agonist-induced whole-cell
currents were sampled at 20 Hz. Electrodes were pulled from
borosilicate glass capillaries (Hilgenberg, Malsfeld, Germany) with a
Zeitz DMZ Universal Puller (Zeitz Instruments, Augsburg, Germany) to yield tip resistance of 2-4 megohm. Pipettes were filled with a
solution containing: 120 mM CsCl, 20 mM
tetraethylammonium chloride, 1 mM CaCl2, 2 mM MgCl2, 11 mM EGTA, and 10 mM HEPES (pH 7.2). Series resistances after whole-cell
formation were compensated for 50-90%. Superfusion was performed with
a DAD-12 (Adams and List, Westbury, NY) drug application system.
Dose-response curves of agonist-induced peak currents were normalized
to the maximum value and data were fitted with the sigmoidal Hill
equation using the Levenberg-Marquardt algorithm. Results are expressed
as means ± standard deviation. Double exponential curves were
fitted to the desensitizing phase of the GlyR responses.
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RESULTS |
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cDNA Cloning and Characterization of GlyR 3
Transcripts--
To identify novel variants of the ligand binding GlyR
subunit from human central nervous system, a fetal brain cDNA
library was screened using a complex probe composed of various human
and rodent
subunit cDNAs (
1-
3). From a random sample
comprising 20 out of >100 hybridizing clones, transcripts of the human
GlyR
1 and
2 subunit genes were excluded by Southern blot
hybridization to
1 and
2 cDNA fragments. While none of the
clones hybridized to the
1 fragment, 18 clones were recognized by
the
2-specific probe. This is consistent with a prevalence of the
2 subunit in the human fetal central nervous system, resembling the
situation in the rodent (23, 24). The nonhybridizing clones p7 and p12 were found to contain inserts of 3069 and 940 base pairs, respectively. As revealed by DNA sequencing, clone p12 was highly homologous to the
rat GlyR
3 subunit cDNA (25) representing a 3' partial clone
that lacked a 5' segment of 733 nucleotides as counted from the
translation start (Fig. 1). In contrast,
clone p7 contained a complete open reading frame as well as large parts
of 5'- and 3'-untranslated regions (UTRs). Compared with clone p12 and
the rat
3 subunit cDNA, clone p7 lacked a stretch of 45 bp
corresponding to nucleotides 1072-1116 of the coding sequence (Fig.
1), indicative of alternative splicing. This predicts a loss of 15 amino acids positioned within the cytoplasmic loop connecting TM3 and
TM4 of the subunit polypeptide, hence referred to as GlyR
3K.
Moreover, the sequences of both clones differ within the 3'-UTR
starting with nucleotide position 1649. This would be compatible with
the occurrence of two distinct transcripts resulting from a complex splice event where excision of the alternative sequence of 45 bp
coincides with a splice event in the 3'-UTR. A search for internal homologies revealed that a 31-bp long internal repeat existed within
the GlyR
3L transcript. The sequence of nucleotide positions 692-723 is identical to the reverse sequence comprising nucleotides 1642-1672, predicting a putative loop structure with a 31-bp stem length. In the GlyR
3K transcript, this motif does not exist as both
variants differ starting from nucleotide position 1649. Except for the
deletion present in GlyR
3K, the human GlyR
3 open reading frame
was 88% identical at the nucleotide and over 98% identical at amino
acid level to the rat
3 subunit. Of seven amino acid substitutions,
one affects the signal peptide, five are localized within the
cytoplasmic loop, and an additional substitution is in the C-terminal
part. Thus, interspecies sequence divergency is highest in the
cytoplasmic loop, reminiscent of other GlyR
subunit variants (2,
6). A search for transcription regulator motifs identified a consensus
sequence of the neuron-restrictive silencer element situated within the
5'-UTR, 145 bp in front of the translation start (Fig. 1). This
recently described short DNA element contributes to the suppression of
neuron-specific genes in non-neuronal tissues (26).
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The Structure and Chromosomal Localization of Human GLRA3
Gene--
A human genomic PAC library (27) was screened using the
radiolabeled 3-kb insert of cDNA clone p7. Based on the cDNA
sequences, ten positive PAC clones were characterized further by PCR
and Southern hybridization. Four clones proved to be overlapping and were used for the determination of the GLRA3 gene structure
by sequencing the exon-intron boundaries and flanking intronic
sequences. The coding sequence was found to be distributed over ten
exons (exons 1-9; Fig. 1 and Table I),
while exon 10 was completely located in the 3'-UTR. The sequences of
the exon-intron boundaries (Table I) largely match the consensus
sequences determined for mammalian splice sites (28). A comparison
showed that the exon-intron anatomy of GLRA3 resembled those
of the human and murine 1,
2, and
4 genes (8, 12, 29).
Positions of exon-intron boundaries proved to be highly conserved, and
gene structure homology was highest with GLRA1 sharing the
same lengths for exons 1-8. The alternatively spliced stretch of 45 bp
constitutes a separate novel exon, referred to as exon 8A. The putative
splice site located in the 3'-UTR of the
3 transcripts was analyzed
by sequencing the corresponding genomic region contained in PAC clone
ZP5.1. As the genomic sequences obtained were completely identical to the 3'-UTR within clone p7, the sequence starting from nucleotide 1648 in clone p7 is thought to represent the unspliced intron 9. To exclude
that the persistence of intron 9, as found in clone p7, was due to a
splice artifact, the expressed sequence tags (EST) data base of the
GenBankTM was searched using the 3'-UTR sequence of this
clone. Indeed, two independent cDNA clones (AA283885, AA488804)
were identified that contained the identical 3'-UTRs covering the exon
9/intron 9 transition sequence.
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GLRA3 Allelic Variants and the Human Hypertonic Motor Disorder, Hyperekplexia-- The human neurological disorder, hyperekplexia, is caused by mutations of the GLRA1 gene in a large number of cases. In the majority of cases, however, no association could be found with allelic variants of GLRA1 (12). We therefore investigated the role of GLRA3 as a candidate gene of hereditary hyperekplexia. DNA samples from 14 patients previously excluded to carry GLRA1 coding mutations, were subjected to SSCP screening. In analogy to GLRA1 allelic variants, where all amino acid substitutions identified are restricted to TM2 and its flanking polypeptide segments, we focused on the corresponding exons 6, 7, and 8 (12, 30-32). In 4 out of 14 patients, an SSCP polymorphism was identified in amplimers from exon 7. Direct sequencing revealed that this polymorphism was due to a silent C to T exchange in the wobble position of codon T292. Polymorphism frequencies did not significantly differ between a sample of normal probands and affected individuals (data not shown).
Functional Expression of GlyR 3 Variants in HEK 293 Cells--
The physiological properties of glycine receptor channels
encoded by both
3 splice variants were analyzed by patch-clamp recording from transfected HEK 293 cells. Upon expression of the GlyR
3L and
3K cDNA constructs, superfusion with glycine elicited current responses characterized by maximal membrane currents
(Imax) with mean amplitudes of 3988 ± 1169 pA and 3690 ± 737 pA, respectively (Fig.
4A,
Table II). Half-maximal responses
(EC50) were observed at glycine concentrations of 54 ± 12 µM (n = 7) and 64 ± 14 µM (n = 5) for GlyR
3L and GlyR
3K,
respectively (Fig. 4B, Table IIc).
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DISCUSSION |
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Here, we present the structure and chromosomal localization of the
human GlyR 3 subunit gene (GLRA3), and its splice
variants GlyR
3L and GlyR
3K. As revealed by functional
expression, these variants give rise to glycine-gated chloride channels
differing in desensitization kinetics.
Molecular cloning led to the identification of the human GlyR 3
subunit that was found to exist in two splice variants differing in
sizes of the polypeptides encoded. The amino acid sequences of both
variants were highly homologous to the previously characterized rodent
polypeptide (25), indicating that the gene family of glycine receptor
subunits is conserved during phylogeny (6, 8, 25). While subunit
variant
3L represented the homologue of the rat
3 transcript
previously described (25), a 45-bp segment was deleted from the novel
transcript
3K. When compared with the GLRA3 gene
structure, it became apparent that this alternative segment reflected a
distinct exon, termed exon 8A, which codes for 15 amino acids situated
within the putative cytoplasmic loop connecting TM3 and TM4 of the
mature polypeptide.
Both transcripts exhibited further divergency. The 3'-UTR of transcript
3K represented a continuous copy of the corresponding genomic
region. In contrast, a diverging sequence was contained in the 3'-UTR
of
3L, indicative of a further splice event. The difference in
3
transcript sizes (2.4 versus 9 kb) as observed by Northern
analysis is most likely explained by the persistence of large intronic
sequences within the 3'-UTR of the
3K mRNA. This analysis also
showed that the inclusion of exon 8A is linked to the excision of these
large 3' sequences (>6 kb) from the
3L transcript, generating an
inverted repeat of 31 bp. A sequence motif overlapping the boundary
between exons 6 and 7 is invertedly repeated by a stretch of
nucleotides contained within the segment of the 3'-UTR unique for
variant
3L. As inverted repeats are capable to form stem structures
of RNA loops, these
3 transcript variants are likely to differ
substantially in mRNA secondary structures. Thus, inclusion of 3'
intronic sequences may lead to an altered mRNA folding, suggesting
the formation of variant-specific mRNA secondary structures. The
functional importance of these mRNA structures is not understood. A
search for specific RNA consensus sequences revealed, however, that a
cluster of three pentanucleotides occurred in the 5'-UTR, reminiscent
of the consensus sequence (UCAU(N)0-2)3
recognized by the RNA binding protein Nova-1 (33). Binding of Nova-1
has been demonstrated for primary transcripts encoding the human GlyR
2 subunit (33), suggesting that the motif analyzed here may indeed
serve a similar function. Taken together, it may be tempting to
speculate that splicing regulates the formation of long stem-loop RNA
structures, thus exposing determinants for RNA-protein interaction.
The GlyR 3 variants generated by alternative splicing differ within
the cytoplasmic loop between TM3 and TM4. A similar heterogeneity exists for the splice variants of the GlyR
1 subunit (9) and the
5-HT3 receptor (34), where usage of an alternate acceptor splice site for exon 9 results in the insertion of eight and six amino
acids, respectively, within a homologous position. These structural
characteristics of variants
3K and
3L coincide with distinct
desensitization behaviors of the receptor channels formed upon
recombinant expression, thus defining the cytoplasmic loop as an
important determinant of channel inactivation kinetics. Further
analysis reveals the alternative insert to carry a consensus target
sequence for casein kinase II-dependent phosphorylation of
serine 370 (35). Indeed, protein phosphorylation may significantly affect channel desensitization kinetics, as studied in detail with the
nicotinic acetylcholine receptor (36, 37). In the
and
subunits
of this receptor, those phosphorylation sites associated with this
effect are also situated within the cytoplasmic loop between TM3 and
TM4. In addition, phosphorylation of the GlyR
1 large cytoplasmic
loop by protein kinases A and C affects the sizes of glycine-evoked
membrane currents in opposing ways (38, 39). In contrast to the
functional expression of GlyR
3 splice variants in HEK 293 cells, no
physiological differences were apparent upon heterologous expression of
the GlyR
1 and 5-HT3 receptor splice variants in
Xenopus laevis oocytes (9, 34). It should be noted, however,
that the phosphorylation background is high in Xenopus
laevis oocytes due to a high basal level of protein kinase A
activity, thus potentially masking functional differences (37). It
remains to be shown whether the differences in GlyR
3
desensitization kinetics can be attributed to changes in
phosphorylation status or to alterations in receptor architecture due
to inclusion of the additional peptide sequence.
Analysis of the GLRA3 gene structure revealed a high degree of homology to the previously characterized glycine receptor subunit genes in mouse and man (8, 12, 19, 29). The alternatively spliced exon 8A, however, represents a structural element unique among glycine receptor genes. Mapping of the GLRA3 gene locus revealed its chromosomal localization in the vicinity of the GLRB gene, which was assigned to the human chromosomal band 4q31.3 (19, 40). This chromosomal region is linked by synteny homology to a region on mouse chromosome 8, where the murine Glra3 gene is situated (7). While no obvious correlations to currently known disease loci exist, the human gene GLRA3 nevertheless remains a major candidate gene for hypertonic and convulsive disorders.
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ACKNOWLEDGEMENTS |
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We thank G. Hebel-Klebsch, A. Heister, and R. Fäcke-Kühnhäuser for technical assistance, H. Betz, N. Milani, and C. Kling for support, and H.-G. Breitinger and T. Bonk for a critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by Bundesministerium für Bildung und Forschung, Deutsche Forschungsgemeinschaft (SFB 539) and Deutsche Krebshilfe (10-1124-Li1).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF0177715-AF017724, AF018157, and U93917.
** To whom correspondence should be addressed. Tel.: 49-913185-4190; Fax: 49-9131-85-2485; E-mail: C.-M.Becker{at}biochem.uni-erlangen.de.
1 The abbreviations used are: GlyR, glycine receptor; TM1-TM4, transmembrane spanning regions; GLRA, designation for human glycine receptor genes; Glra, designation for murine glycine receptor genes; PCR, polymerase chain reaction; FISH, fluorescence in situ hybridization; SSCP, single strand conformation polymorphism; UTR, untranslated region; PAC, P1 artificial chromosome; kb, kilobase(s); contig, group of overlapping clones; FITC, fluorescein isothiocyanate; DAPI, 4,6-diamidino-2-phenylindol-dihydrochloride.
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REFERENCES |
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