A Novel Class of GABAA Receptor Subunit in Tissues of the Reproductive System*

(Received for publication, April 24, 1997)

Eva Hedblom and Ewen F. Kirkness Dagger

From The Institute for Genomic Research, Rockville, Maryland 20850

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

A novel subunit of the gamma -aminobutyrate, type A (GABAA) receptor family has been identified in human and rat tissues. The subunit displays 30-40% amino acid identity with known family members and represents a distinct subunit class (termed pi ). Transcripts of the pi  subunit were detected in several human tissues and were particularly abundant in the uterus. The pi  subunit protein can assemble with known GABAA receptor subunits and confer unique ligand binding properties to the recombinant receptors in which it combines. Most notably, the presence of the pi  subunit alters the sensitivity of recombinant receptors to the endogenous steroid, pregnanolone. Identification of the pi  subunit indicates a new target for pharmacological manipulation of GABAA receptors that are located outside of the central nervous system.


INTRODUCTION

In mammalian brain, synaptic inhibition of neuronal activity is mediated mainly by gamma -aminobutyric acid (GABA)1 at GABAA receptors. In mammals, the 14 known subtypes of GABAA receptor subunits have been categorized within five structural classes (alpha 1-6, beta 1-3, gamma 1-3, delta , epsilon ). These subunits are thought to assemble in different pentameric complexes, with most functional receptors containing alpha /beta /(gamma , delta , or epsilon ) subunit combinations (1-3). A sixth class of subunit (rho ) form homomeric GABA receptors that do not appear to coexist with GABAA receptor subunits and correspond pharmacologically to a related (GABAC) receptor subtype (4, 5).

In addition to their location on central neurons and astroglia, functional GABAA receptors have been detected on peripheral neurons and non-neuronal cells. The non-neuronal cells include endocrine cells of the pituitary pars intermedia, adrenal medulla, islets of Langerhans, and placenta (6-9). The receptors have also been located on smooth muscle cells of the urinary bladder and uterus (10, 11).

The precise function of GABAA receptors in non-neuronal cells is presently ill-defined. Clearly, their location on endocrine cells suggests a role in the regulation of hormone secretion. However, their function in the uterus appears to be related directly to tissue contractility. Within the uterus, GABA, and its metabolic enzymes are found at high concentrations (12). The GABAA receptors of this tissue have been proposed to regulate uterine motility by inhibiting contractions (13). They may also mediate the relaxing effects of 5alpha ,3alpha -pregnanolone, an endogenous steroid that is capable of activating GABAA receptors directly (13, 14).

Here, we describe a novel class of GABAA receptor subunit that is expressed at relatively high levels in several peripheral tissues, including the uterus. This subunit can combine with known GABAA receptor subunits and alter the sensitivity of recombinant receptors to modulatory agents such as pregnanolone.


EXPERIMENTAL PROCEDURES

Materials

[35S]tert-Butyl bicyclophosphorothionate (TBPS), [3H] muscimol, [3H]Ro15-1788, and [3H]flunitrazepam were purchased from DuPont. Pentobarbital and 5alpha -pregnan-3alpha -ol-20-one were from Sigma. The HEK-293 cell line (ATCC CRL 1573) was obtained from American Type Culture Collection. The PANC cell line was provided by Dr. E. Jaffe (Johns Hopkins University). Human placenta and uterus were obtained from National Disease Research Interchange. Samples of poly(A)+ RNA from other tissues were purchased from Clontech.

Cloning of the Human pi  Subunit cDNA

A consensus sequence of amino acid residues that are conserved between all known vertebrate GABAA receptor subunits (3) was searched against the human cDNA data base (15), using the TBLASTN algorithm (16). The search uncovered an expressed sequence tag (EST) that encodes part of a subunit homologue that has not been described previously. This EST was derived from a partial cDNA clone of a pancreatic carcinoma mRNA (nucleotides 859-3300; Fig. 1). Flanking 5' sequence (nucleotides 1-984; Fig. 1) was obtained by anchored PCR (17), using a pancreatic carcinoma cDNA library as template. A fragment of the amplified cDNA (nucleotides 2-230; Fig. 1) was then used to screen approximately 5 × 105 plaques of the cDNA library, using standard procedures (17). Two independent clones that contained full-length inserts of 3.3 kb were isolated. A fragment of one insert (nucleotides 76-1503; Fig. 1) was transferred to the pCDM8 vector (Invitrogen) and re-sequenced over its entire length prior to expression studies.


Fig. 1. Sequences of the human and rat pi  subunits. The human cDNA sequence contains an open reading frame that is flanked by in-frame stop codons (***) and contains a polyadenylation signal (underlined) at bases 3237-3242. The deduced amino acid sequence of the open reading frame is written below the corresponding codons, from the first in-frame methionine residue. Amino acid residues of the rat pi  subunit that differ from the human sequence are indicated below the human peptide sequence. The four putative transmembrane domains (M1-M4) are highlighted by lines under the corresponding peptide sequences. The peptide sequences contain 72 of the 78 amino acid residues that are conserved between all known vertebrate GABAA receptor subunits (bold type). The six differences are indicated by diamonds.
[View Larger Version of this Image (86K GIF file)]

Cloning of the Rat pi  Subunit cDNA

Initially, two fragments of the rat pi  subunit gene were amplified by PCR from total genomic DNA using degenerate primers that were derived from the human pi  subunit protein sequence. For fragment 1, the primers were 5'-GGWAATGATGTKGARTTYACYTGG-3' and 5'-CGATCTKGTKACTAAKGTRAARTA-3' (nucleotides 709-732 and 799-822; Fig. 1). For fragment 2, they were 5'-CACTAGRTTRGTYTTRCARTTTGA-3' and 5'-GCAGGTTCTTGCAGGGACTGAATC-3' (nucleotides 843-866 and 961-984; Fig. 1). Amplification at 95 °C for 45 s, 55 °C for 60 s, 72 °C for 2 min was performed for 30 cycles using the GenAmp system (Perkin-Elmer). Sequences of the cloned fragments were then used to design primers for amplification of two overlapping fragments of the rat pi  subunit cDNA. Total RNA (5 µg) was isolated from rat uterus (18), and reverse-transcribed using 200 units of Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc.) at 42 °C for 60 min. The primer was a 15-mer poly(dT) oligonucleotide. Samples (5%) of the reaction products were then subjected to 35 cycles of PCR as described above. For the 5'-region of the cDNA, the primers were 5'-gcggaattcCAGAGCCTCAACAACTACCTG-3' (nucleotides 94-114; Fig. 1) and 5'-gcggaattcCCAAAAGGAGACCCAGGATAA-3' (nucleotides 820-840 of GenBankTM accession number U95368[GenBank]). For the 3'-region, the primers were 5'-gcggaattcGATTCGGTACGTGGACTCGAA-3' (nucleotides 635-654 of U95368[GenBank]) and 5'-gcggaattcGTTGAAGACCTATGGCATGCA-3' (nucleotides 1497-1516; Fig. 1). The two amplified cDNA fragments (858 and 772 base pairs) were purified and sequenced directly. The protein-coding region of the cDNA sequence was then amplified from total uterus cDNA using the XL-PCR system (Perkin-Elmer). The primers were 5'-ccggccactaGTGAATCAGCTCCTTCAATATGAGCTACA-3' (nucleotides 27-55 of GenBankTM number U95368[GenBank]) and 5'-ccggccactagTCAAAAATACATGTAGTATGCCCAGTAA-3' (nucleotides 1341-1368 of GenBankTM number U95368[GenBank]). The reaction products were ligated into the pCDM8 vector, and individual clones were sequenced over their entire lengths to ensure that no mutations had been introduced.

Northern Blot Analysis

Poly(A)+ RNA was prepared from placenta, uterus, and PANC cells using the Oligotex system (Qiagen). Approximately 2-µg samples of poly(A)+ RNA were electrophoresed on 1.2% formaldehyde-agarose gels, transferred to nylon membranes, and hybridized with a 32P-labeled fragment of the 3'-untranslated pi  subunit cDNA (nucleotides 1501-2340; Fig. 1). The blots were washed at 60 °C in 0.1 × SSC, 0.1% SDS prior to exposure. The blots were stripped of probe by boiling in 0.5% SDS, and re-hybridized with a 32P-labeled fragment of the cDNA that encodes human glyceraldehyde phosphate dehydrogenase (nucleotides 789-1140; Ref. 19).

Transient Transfection of Subunit cDNAs

Human embryonic kidney cells (HEK-293) were maintained in Dulbecco's modified Eagle's medium, supplemented with calf serum (10%), penicillin (50 units/ml), and streptomycin (50 µg/ml). Cells were plated at ~20% confluence, 24 h prior to transfection. Transfection of subunit cDNAs was performed with a calcium phosphate-DNA precipitate in HEPES buffer (17). Following incubation with the precipitate for 24 h, the cells were washed and cultured for a further 48 h before harvesting. All subunit cDNAs were derived from rat and were cloned in pCDM8. Transfection efficiencies were standardized by cotransfecting a beta -galactosidase cDNA, as described previously (20).

Radioligand Binding Assays

A crude membrane fraction was prepared from transfected cells as described previously (3). The [35S]TBPS binding activity of membranes (50-100 ng of protein) was assayed by incubation for 120 min at 25 °C in 100 µl of a solution containing Tris-HCl (20 mM), NaCl (1 M), pH 7.5. Assays included [35S]TBPS at 1-40 nM (for Scatchard analysis) or 5 nM (all other experiments). Nonspecific binding was determined in the presence of picrotoxin (100 µM) and was equal to the binding of mock-transfected cell membranes (<0.03 pmol/mg of protein). Following incubation, 5 ml of ice-cold assay buffer was added, and the membranes filtered through Whatman GF/C filters under vacuum. The filters were washed twice with 5 ml of the same buffer prior to scintillation counting. The binding of [3H]muscimol (20 nM) was assayed by incubation of membranes for 60 min at 0 °C in 100 µl of TEN (10 mM Tris-HCl, 1 mM EDTA, 100 mM NaCl, ph 7.5). Nonspecific binding was determined in the presence of GABA (1 mM). The binding of [3H]Ro15-1788 and [3H]flunitrazepam was assayed by incubation of membranes for 60 min at 0 °C in 100 µl of TEN, NaCl (0.1 M). Nonspecific binding was determined in the presence of flunitrazepam (10 µM). Assays included [3H]Ro15-1788 at 0.5-6 nM (for Scatchard analysis) or 2 nM (all other experiments). Bound ligand was detected by filtration as described above. All binding data are mean values ± S.D. of three experiments, using membranes from at least two independent transfections.


RESULTS AND DISCUSSION

A novel class of GABAA receptor subunit was identified by searching a data base of ESTs with a peptide consensus sequence of known family members. The sequence of this EST was used to isolate a 3.3-kb cDNA from a pancreatic carcinoma cDNA library. The cDNA contains a large open reading frame that is flanked by stop codons and encodes a polypeptide of 440-amino acid residues (Fig. 1). The polypeptide, termed the pi  subunit, has all of the hallmarks of a ligand-gated anion channel subunit. The N-terminal half of the protein exhibits five sites for potential N-linked glycosylation, and two cysteine residues separated by 13 amino acids (residues 160 and 174). The C-terminal half of the protein contains four hydrophobic regions that are potential transmembrane domains. The first of these contains a conserved proline residue, while the second contains an octamer sequence (residues 280-287) that is similar, but not identical, to that found in most GABAA and glycine receptor subunits. Within this octamer, the pi  subunit contains a serine residue at position 284, where threonine is found in all other GABAA receptor subunits. This minor difference in protein sequence may explain why the pi  subunit cDNA has not been uncovered previously by the cross-hybridization approaches that have relied almost exclusively on conservation of this motif.

The amino acid sequence of the pi  subunit is most closely related to GABAA receptor beta  subunits (37% amino acid identity), followed by the delta  subunit (35%) and rho  subunits (33%). It displays less similarity to other GABA and glycine receptor subunits. The maximum levels of sequence identity are similar to those found between the different classes of GABAA receptor subunits (20-40%; Refs. 1 and 2) and justifies its categorization within a distinct subunit class. Of the 78 amino acid residues that are conserved between all known GABAA receptor subunits (3), only six are substituted in the pi  subunit sequence (Fig. 1). A rat cDNA that encodes a homologue of the human pi  subunit was also isolated. The rat and human pi  subunits display a high degree of sequence conservation (93% amino acid identity; Fig. 1). This conservation includes the six amino acid residues that vary from all other known GABAA receptor subunits.

Expression of pi  subunit mRNA in human tissues was first assessed by searching EST data bases for additional examples of pi subunit cDNA fragments. The dbEST data base (21; release 32897) contains eight entries2 that were derived from six partial human pi  subunit cDNA clones. In common with the cDNA described here, four of these clones were derived from two independent pancreatic carcinomas. The remainder were isolated from a neuroepithelial cell line (NT2) and breast tissue.

A more systematic examination of expression patterns employed a collection cDNA libraries that were derived from most major human tissues (22). After 35 cycles of PCR, using approximately 1 × 106 phage, a detectable pi  subunit cDNA fragment was amplified from the cDNA libraries of uterus, prostate, ovaries, placenta, gall bladder, lung, small intestine, and two brain regions (hippocampus and temporal cortex; data not shown). When available, these tissues were examined by Northern analysis to obtain a more quantitative estimation of subunit mRNA expression levels. A hybridizing transcript of the predicted size (3.3 kb) could be detected in several tissues (Fig. 2). Although this transcript was barely detectable in small intestine, ovaries, and placenta, it was relatively enriched in lung, thymus, and prostate and was particularly abundant in the uterus. The pi  subunit transcript was also enriched in a cell line (PANC) that was derived from a human pancreatic adenocarcinoma.3 Notably, no hybridizing transcripts were detected in samples of whole brain or pancreas. It appears that the pi  subunit is expressed in only a minor subpopulation of cells in these tissues. Future in situ hybridization studies should help to identify these cell types.


Fig. 2. Distribution of pi  subunit mRNA in human tissues. A, hybridization of poly(A)+ RNA from heart (lane 1), brain (lane 2), placenta 1 (lane 3), lung (lane 4), liver (lane 5), skeletal muscle (lane 6), kidney (lane 7), pancreas (lane 8), spleen (lane 9), thymus (lane 10), prostate (lane 11), testis (lane 12), ovary (lane 13), small intestine (lane 14), colon (lane 15), leukocytes (lane 16), placenta 2 (lane 17), PANC cells (lane 18), and uterus (lane 19) with a 32P-labeled fragment of the 3'-untranslated pi  subunit cDNA. B, the same blot was reprobed with a fragment of the cDNA that encodes human glyceraldehyde phosphate dehydrogenase.
[View Larger Version of this Image (57K GIF file)]

The pharmacological properties that are conferred to GABAA receptors by the pi  subunit were examined after transient expression in HEK-293 cells. Cells that were transfected with only the pi subunit cDNA did not express binding sites for the GABAA receptor ligands, [3H]muscimol or [35S]TBPS (Table I). These cells also failed to elicit chloride currents in response to GABA or glycine (each 100 µM).4 The pi  subunit is therefore unlike the rho  class of GABA receptor subunits (2) or the alpha  class of glycine receptor subunits (23), which can each assemble homomeric chloride channels that are gated by these ligands. When cells were cotransfected with cDNAs encoding the pi  subunit, and either an alpha 1 subunit or a beta 1 subunit, there was also a failure to detect expression of any ligand binding activities. The pi  subunit therefore does not behave like a typical GABAA receptor alpha  or beta  subunit (2). As expected, transfection of cells with a combination of alpha , beta , and pi  subunits resulted in the expression of both [3H]muscimol and [35S]TBPS binding sites (Table I). However, the transfected cell membranes did not bind either [3H]Ro15-1788 or [3H]flunitrazepam. Therefore, the pi  subunit is also unlike the gamma class of subunits, which confers a sensitivity to benzodiazepines when expressed with alpha  and beta  subunits (2).

Table I. Binding of radioligands to cell membranes after transient expression of GABAA receptor subunit combinations


Subunit combination Radioligand bindinga
[35S]-TBPS (5 nM) [3H]Muscimol (20 nM) [3H]Ro15-1788 (2 nM)

 alpha  -  -  -
    beta 1  -  -  -
    beta 3 +  -  -
        gamma 2  -  -  -
             pi  -  -  -
 alpha 1  beta 1 + +  -
 alpha 1  beta 3 + +  -
 alpha 1      gamma 2  -  -  -
 alpha 1           pi  -  -  -
    beta 1  -  -  -
    beta 3 +  -  -
    beta 1  -  -  -
    beta 3 +  -  -
         gamma 2  pi  -  -  -
 alpha 1  beta 1  gamma 2 + + +
 alpha 1  beta 3  gamma 2 + + +
 alpha 1  beta 1      pi + +  -
 alpha 1  beta 3      pi + +  -
     beta 1   gamma 2  pi  -  -  -
     beta 3  gamma 2  pi +  -  -
 alpha 1  beta 1  gamma 2  pi + + +
 alpha 1  beta 3  gamma 2  pi + + +

a Cell membranes displayed specific binding activities of either 0.20-3.00 pmol/mg protein (+) or 0.00-0.03 pmol/mg of protein (-).

In common with the delta  and epsilon  subunit classes, the absence or presence of binding sites for [35S]TBPS, [3H]muscimol, and [3H]Ro15-1788 failed to demonstrate that the pi  subunit can assemble with known GABAA receptor subunits. However, such an ability could be inferred from the observation that inclusion of the pi  subunit with alpha /beta /gamma subunit combinations caused a significant reduction of [3H]Ro15-1788 binding to transfected cell membranes. This was demonstrated clearly by transfecting different ratios of the subunit cDNAs (Table II). When the pi  subunit cDNA was transfected with an alpha /beta /gamma subunit combination in an equimolar ratio, the density of [3H]Ro15-1788 binding sites was reduced by 45%. A 5-fold increase in the molar ratio of transfected pi  subunit cDNA caused a much larger reduction (90%) of the Bmax value for [3H]Ro15-1788 binding. In contrast, a 5-fold increase in the molar ratio of transfected gamma 2 subunit cDNA, blocked the reduction caused by the presence of the pi  subunit, and returned the Bmax value to 84% of that obtained with alpha /beta /gamma subunits alone. Notably, these variable subunit combinations had little effect on the binding of [35S]TBPS and [3H]muscimol (Table II). It was concluded that expression of the pi  subunit with the alpha /beta /gamma subunit combination results in expression of alpha /beta /gamma /pi receptors that lack [3H]Ro15-1788 binding sites and/or a mixed population of alpha /beta /gamma and alpha /beta /pi receptors.

Table II. Binding of radioligands to cell membranes after transient expression of alpha 1, beta 1, gamma 2, and pi  subunit combinations


Molar ratio of transfected subunit cDNAs
Radioligand bindinga
[3H]Ro15-1788 bindingb
 alpha 1  beta 1  gamma 2  pi [35S]TBPS (5 nM) [3H]Muscimol (20 nM) [3H]Ro15-1788 (2 nM) Kd Bmax

1 1 1 7.7  (0.4) 19.6  (1.3) 21.6  (1.5) 1.5  (0.3) 37.2  (6.4)
1 1 1 7.3  (0.1) 19.0  (1.6) NDc NDc NDc
1 1 1 1 6.8  (1.1) 18.5  (3.8) 10.7  (0.8) 1.6  (0.4) 20.3  (4.3)
1 1 1 5 7.9  (1.0) 18.3  (1.6) 2.3  (0.4) 1.3  (0.3) 3.6  (0.1)
1 1 5 1 6.4  (0.3) 16.1  (2.8) 17.5  (1.3) 1.6  (0.2) 31.1  (4.0)

a Specific binding activities are expressed in units of fmol/unit beta -galactosidase and represent mean values (S.D.) of three independent experiments. For [35S]TBPS binding to the alpha 1/beta 1/gamma 2 subunit combination, the value represents 0.14 ± 0.02 pmol/mg of protein.
b The Kd and Bmax values are expressed in units of nM and fmol/unit beta -galactosidase, respectively. The Bmax value for the alpha 1/beta 1/gamma 2 subunit combination was 0.67 ± 0.02 pmol/mg of protein.
c Specific binding activity was not detected.

Clearly, transfection of cells with a combination of alpha , beta , gamma , and pi  subunit cDNAs can give rise to multiple receptor types, composed of different subunit combinations and stoichiometries. For this reason, a less complex system was chosen to perform an initial characterization of the pharmacological properties that are conferred to GABAA receptors by the pi  subunit. This system utilized the beta 3 subunit, which assembles homomeric chloride channels that bind [35S]TBPS with high affinity (24, 25). Cells were transfected with either the beta 3 cDNA alone or a combination of beta 3 and pi  cDNAs. The presence of the pi  subunit had no significant effects on the Kd and Bmax values for [35S]TBPS binding to transfected cell membranes. Also, displacement of [35S]TBPS binding by pentobarbital, yielded similar Ki values for the two membrane preparations (9 and 10 µM, respectively; Fig. 3). However, displacement of binding by pregnanolone demonstrated a significant difference between the recombinant receptors. The concentration of steroid that displaced binding from the beta 3 homomeric receptor by 50% (3.5 µM) had no significant effect on binding to membranes of beta 3/pi transfected cells (Fig. 3). The Ki values for the two membrane preparations were 2 and 18 µM, respectively. The same effect was observed with human beta 3 and pi  subunit cDNAs, expressed in either HeLa or HEK-293 cells (data not shown).


Fig. 3. Effects of the pi  subunit on modulation of [35S]TBPS binding by anesthetic agents. The binding of [35S]TBPS to the membranes of cells transfected with either beta 3 (open circle ) or beta 3/pi (bullet ) cDNAs was displaced with pentobarbital or 5alpha -pregnan-3alpha -ol-20-one. Scatchard analysis indicated a single affinity class of [35S]TBPS binding sites for each receptor population. The beta 3 cell membranes displayed Kd and Bmax values of 11.2 ± 1.9 nM and 45 ± 3.1 fmol/unit beta -galactosidase, respectively. The corresponding values for the beta 3/pi cell membranes were 12.5 ± 2.8 nM and 46.8 ± 9.6 fmol/unit beta -galactosidase.
[View Larger Version of this Image (12K GIF file)]

The physiological significance of this reduced sensitivity to pregnanolone is unknown at present. To address this question, it will be necessary to identify the subunits with which the pi subunit combines in vivo and re-examine the properties of relevant subunit combinations in recombinant systems. However, this effect of the pi  subunit provides further evidence that it can assemble with known GABAA receptor subunits and alter the sensitivity of receptors to modulatory agents. The actions of pregnanolone at GABAA receptors have been proposed to regulate uterine motility by inhibiting contractions (13, 14). A quiescence of uterine motility is essential for embryonic implantation and maintenance of pregnancy (26). The identification of a novel subunit class in the uterus is therefore likely to provide new insights to the role of GABAA receptors in this important process.


FOOTNOTES

*   This work was supported by National Institutes of Health Grant NS34702.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) U95367[GenBank] and U95368[GenBank].


Dagger    To whom correspondence should be addressed: The Institute for Genomic Research, 9712 Medical Center Dr., Rockville, MD 20850. Tel.: 301-838-3536; Fax: 301-838-0208; E-mail: ekirknes{at}tigr.org.
1   The abbreviations used are: GABA, gamma -aminobutyric acid; GABAA, gamma -aminobutyric acid, type A; EST, expressed sequence tag.
2   GenBankTM accession numbers H43243[GenBank], U54596[GenBank], U54597[GenBank], U54599[GenBank], AA101225, AA102670, AA120821, and AA120822.
3   E. Jaffe, personal communication.
4   T. G. Hales, personal communication.

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