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Experimental Physiology 92.1 pp 139-145
DOI: 10.1113/expphysiol.2006.921304
© The Physiological Society 2007
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Genomic Physiology

Alternative splicing of P2X6 receptors in developing mouse brain and during in vitro neuronal differentiation

 Rogério L. da Silva1, Rodrigo R. Resende1 and Henning Ulrich1

1 Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, Caixa Postal 26077, CEP 05513-970, São Paulo, Brazil


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Adenosine triphosphate acts as a fast excitatory neurotransmitter by binding to and activating seven structurally related subtypes of purinergic P2X receptors, which act as ligand-gated ion channels. Besides its role in neurotransmission, ATP also has trophic functions during development of the neuronal system. P2X receptor expression, mainly of P2X4 and P2X6 subtypes, has been detected in adult brain and also during neuronal development. We have used the mouse teratocarcinoma P19 cell line as an in vitro model to study P2X6 receptor expression during early neuronal differentiation. We have detected a full-length and an alternatively spliced form of the mouse P2X6 receptor gene in P19 cells using reverse transcriptase-polymerase chain reaction. The alternatively spliced form was already present at the stage of pluripotent undifferentiated P19 cells, and was predominant compared to the full-length form during the whole course of neuronal differentiation of P19 cells. Alternative splicing of P2X6 receptor subunits was also confirmed during postnatal development of mouse brain. During postnatal development, however, the full-length form was predominant compared to the spliced form. Alternative splicing is suggested to regulate P2X6 receptor function during neuronal differentiation.

(Received 2 March 2006; accepted after revision 1 September 2006; first published online 7 September 2006)
Corresponding author H. Ulrich: Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, Caixa Postal 26077, CEP 05513-970, São Paulo, Brazil. Email: henning{at}iq.usp.br


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
P2X purinergic receptors are ligand-gated ion channels that respond to the binding of extracellular ATP, which acts as a neurotransmitter when released from presynaptic nerve terminals, causing fast excitatory postsynaptic potentials both in neurones and in smooth muscles. Up to now, seven subunits of structurally related receptors (P2X1–P2X7) have been cloned (North & Surprenant, 2000). All subunits share the same general structure, with intracellular N- and C-termini, two membrane-spanning domains and an extracellular loop containing 10 conserved cysteine residues, which are thought to form disulphide bonds (Ennion & Evans, 2002). P2X1-P2X7 subtypes have characteristic properties regarding their responses to agonists (e.g. {alpha},ß-methylene ATP, {alpha}ß-meATP), antagonists (e.g. suramin) and rates of receptor desensitization. They are able to form homomeric or heteromeric functional ion channels, possibly of a trimeric nature (Aschrafi et al. 2004; Barrera et al. 2005).

P2X6 and P2X4 subtypes are expressed during neuronal development (Cheung et al. 2005) and are the most predominant P2X receptors in rat brain. The P2X6 receptor was first isolated from a cDNA library obtained from rat superior cervical ganglion (Collo et al. 1996). According to various studies (Collo et al. 1996; Torres et al. 1999; Barrera et al. 2005), it does not form homomeric receptors, but it co-assembles with P2X2 and P2X4 subunits to form heteromeric ATP receptors (Le et al. 1998; King et al. 2000). Jones et al. (2004), however, reported the cloning and further expression of functional recombinant P2X6 homomeric receptors in human embryonic kidney (HEK) 293 cells. The co-assemblage of the P2X6 with either P2X2 or P2X4 subunits gives rise to functional ATP receptors with pharmacological properties different from those of homomeric P2X2 or P2X4 receptors.

The occurrence of alternative splicing in mRNAs coding for ion channels results in structural changes in the protein, thereby altering crucial properties of the channel, such as channel inactivation, steady-state kinetics, voltage dependency, desensitization time and ligand binding (Stamm et al. 2005). Alternative splicing has been described for some of P2X subunits (Dhulipala et al. 1998; Hardy et al. 2000). For instance, the P2X4 purinergic receptor and its splice variant, P2X4a, are expressed simultaneously in mouse brain, and both isoforms may co-assemble to form heteromeric functional channels (Townsend-Nicholson et al. 1999). The P2X2 purinergic receptor gene codes for at least three splice isoforms in rat brain (Simon et al. 1997).

Alternative splicing is present in all tissues. Brain cells, however, possess the most prevalent tissue-specific splicing (Stamm et al. 2005). Alternative splicing plays a key role in brain development. For instance, murine neocortical development is altered when splice variants of acetylcholinesterase gene are silenced by antisense oligonucleotide suppression (Dori et al. 2005).

We have used P19 cells as an in vitro model of early differentiation (McBurney et al. 1982) to study splice variants of the P2X6 receptor. P19 cells are murine embryonal carcinoma cells that undergo in vitro neuronal differentiation when treated with retinoic acid (reviewed by Bain et al. 1994). Following induction of neuronal differentiation, the mRNA levels of several splicing factors are modulated, suggesting an association with the occurrence of alternative splicing during this period (Shinozaki et al. 1999).

Here we describe the occurrence of a splice variant of the P2X6 purinergic receptor, which is predominant during neuronal differentiation of P19 embryonal carcinoma cells and is less apparent during postnatal development and in adult mouse brain.


   Methods
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 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
If not otherwise indicated, reagents were purchased from Sigma-Aldrich Corp. (St Louis, MO, USA). Rats (Rattus norvegicus, Wistar strain), and adult and postnatal mice (days 0, 8 and 15 after birth; Mus musculus, Balb C strain) were obtained from the Animal Facility, Instituto de Química, University of São Paulo. Animals were kept and killed by decapitation or cervical dislocation in compliance with the Ethical Standards of the Institute of Chemistry, University of São Paulo, Brazil.

Cell culture and neuronal differentiation in vitro

P19 mouse embryonal carcinoma (EC) cells were cultured and differentiated into neurones as previously described (Tárnok & Ulrich, 2001; Martins et al. 2005). Briefly, P19 cell cultures were maintained in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Cultilab, Campinas, Brazil), 100 i.u. ml–1 penicillin, 100 µg ml–1 streptomycin, 2 mM L-glutamine and 2 mM sodium pyruvate. For induction of neuronal differentiation, 5 x 105 P19 cells ml–1 in defined medium containing DMEM supplemented with 2 mM glutamine, 2 mM sodium pyruvate, 2.4 mg ml–1 sodium bicarbonate, 5 µg ml–1 insulin, 30 µg ml–1 human apo-transferrin, 20 µM ethanolamine, 30 nM sodium selenite, 100 i.u. ml–1 penicillin, 100 µg ml–1 streptomycin, and 10 mM HEPES, pH 7.2, were treated with 1 µM ‘all-trans’ retinoic acid (RA) and plated into bacterial dishes previously coated with 0.5% agarose, to avoid adhesion of the cell culture to plastic surfaces. After 2 days of culture in suspension in the presence of RA, P19 cells formed embryonic body stages (EBs). These EBs were collected from suspension cultures and replated in adherent culture flasks in DMEM medium with 10% FBS for 48 h. The serum-containing medium was replaced with defined medium, on day 4, followed by a further 4 days of culture until neuronal maturation was completed as determined by neurone-specific protein expression (neurofilament-200 and ß-3-tubulin) according to Martins et al. (2005). Glial cells were eliminated from differentiating neurone cultures by addition of cytosine-arabinoside (5 µg ml–1) 2 days before cell collection.

Reverse transcription and polymerase chain reaction

Total RNA was isolated using TRIzol (Invitrogen, Carlsbad, CA, USA) from undifferentiated P19 cells and from P19 cells at various time points of neuronal differentiation. Total brain tissue was obtained from adult rats and from adult and postnatal mice (days 0, 8 and 15 after birth), followed by total RNA extraction using the TRIzol reagent. Integrity of the isolated RNA was analysed on a 1% ethidium bromide-stained agarose gel. Three micrograms of total RNA were used for cDNA synthesis in a total volume of 20 µl in the presence of 50 mM Tris-HCl, pH 8.3, 3 mM MgCl2, 10 mM dithiothreitol, 0.5 mM dNTPs and 50 ng random primers with 200 U of RevertAidTM H Minus M-MuLV-reverse transcriptase (Fermentas Inc., Hanover, MD, USA) for 45 min at 42°C. For polymerase chain reaction (PCR), 5 µl of the reverse transcription (RT) reaction was used as a template. Reactions were performed in the presence of 2.5 mM MgCl2, 0.2 mM dNTPs, 20 µM each reverse and forward primers and 0.5 U of Taq polymerase (Invitrogen). Primer sequences (Integrated DNA Technologies, Coralville, IA, USA) for amplification of mouse and rat P2X6 receptor (GenBank accession no. X92070. [GenBank] As cDNA sequences for mouse P2X6 receptors were not available at this time, therefore sequences published for rats were used) and ß-actin (GenBank accession no. NM-007393) cDNAs were: forward 5'-CGATTCACTCTCCAGTCCG-3', reverse 5'-GGTCCTCCAGTAGAAACCG-3'; and forward 5'-AGGAAGAGGATGCGGCAGTGG-3', reverse 5'-CGAGGCCCAGAGCAAGAGAG-3', respectively. Reactions were cycled 35 times (94°C for 60 s, 62°C for 60 s and 72°C for 60 s) plus a final extension of 10 min at 72°C. The PCR products were electrophoresed on a 2% agarose gel, and visualized by ethidium bromide staining, followed by UV illumination of the gel. The 317 and 427 bp PCR products were excised and gel-purified.

Cloning and sequencing

Each fragment was inserted into pGEM T-Easy Vectors (Promega, Madison, WI, USA), which then were transfected into competent E. coli cells through calcium treatment and heat shock. Following overnight bacterial growth, plasmids containing the inserted fragments were recovered from 10 ml of bacterial culture using the Wizard Plus SV Miniprep System (Promega). For DNA sequencing, 200 ng of the purified cDNAs were amplified in the presence of M13 forward primers, using the Big Dye Terminator v3.0 DNA Sequencing Kit (Applied Biosystems, Foster City, CA, USA). Sequences were determined on an ABI Prism 3100 Genetic Analyser (Applied Biosystems). Confirmation of sequence identities was obtained using the BLAST software (http://www.ncbi.nlm.nih.gov/BLAST), and sequence alignments were performed using the ClustalW software (http://www.ebi.ac.uk/clustalw). Semi-quantitative RT-PCR analysis was performed as described elsewhere (Martins et al. 2005). Relative mRNA transcription levels were determined by densitometric analysis of ethidium bromide-stained bands of PCR products in agarose gels using the TINA software (Covance, Harrogate, UK). ß-Actin mRNA transcription was used as internal control for normalizing P2X6 receptor subunit transcription levels.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Following reverse transcription and PCR amplification of mouse and rat cDNA in the presence of primers specific for the P2X6 receptor, a fragment of a size of 427 bp was expected. The presence of this fragment was confirmed when mouse or rat brain mRNA, or mRNA from different stages of in vitro differentiation of P19 cells was used as a template for the reactions (Fig. 1). However, a second smaller fragment of approximately 320 bp was also visualized, when the template for RT-PCR amplification had been RNA from mouse brain or RNAs from undifferentiated mouse P19 embryonal carcinoma cells or P19 cultures undergoing neuronal differentiation.


Figure 1
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  		Figure 1.  Detection of an alternative splice variant of the mouse P2X6 receptor by RT PCR
Reverse transcription followed by PCR amplification of mouse and rat P2X6 receptor cDNA was performed as detailed in the Methods, followed by detection of PCR products in an ethidium bromide-stained agarose gel. D0–D12 (day 0–day 12) represent RT-PCR amplification of P2X6-specific cDNA from P19 cells of various days of neuronal differentiation. MB, mouse brain; RB, rat brain; AC, ß-actin cDNA used as positive control for PCR reactions.

 
The fragments were DNA-sequenced and aligned with the published sequence for P2X6 cDNA of rats (Collo et al. 1996), since the mouse sequence of this receptor was not available in GenBank (Fig. 2). The assigned GenBank accession numbers for the sequences submitted as expressed sequence tags (ESTs) coding for the mouse P2X6 receptor (427 bp) and its splice variant (317 bp) are DY543045 [GenBank] and DY543046 [GenBank] , respectively. The longer fragment exactly matched the size of the published sequence. A sequence that could be similar to exon 8 of the rat P2X6 receptor gene (nucleotides 6242–6351), encoding for a segment on the extracellular loop, was missing in the shorter fragment. Further comparison revealed that in the splice variant, 36 amino acids are missing in addition to there being a shift in the reading frame beginning from the site of exclusion of the exon 8-like region by alternative splicing.


Figure 2
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  		Figure 2.  Alignment of the complete and splice variant nucleotide sequence of the mouse P2X6 receptor with the published sequence from rat (GenBank accession no. X92070)
Lower case letters correspond to exon 8 of the rat gene present in the complete isoform (GeneID: 25041) that is missing in the product of alternative splicing. The stop codon is underlined.

 
Full-length products were expressed 1.7 times more than the splice variant when total RNA from mouse brain was used for RT-PCR amplification. In cDNA from P19 cells, the splice variant was expressed 1.5 times more than the full-length product. The percentage of the expression of the product of alternative splicing compared to the full-length cDNA was unchanged throughout the course of neuronal differentiation. However, overall transcription of the splice variant increased during neuronal differentiation of P19 cells.

In brain tissue obtained from postnatal mice, the P2X6 receptor coding cDNA increased between days 0 and 15 (Fig. 3). The splice variant was less present when compared to differentiating P19 cells. An increase in transcription rate of the splice isoform during postnatal development, such as observed during the neuronal differentiation of P19 cells, was not found.


Figure 3
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  		Figure 3.  Gene expression of the P2X6 receptor subunit and its splice variant during mouse postnatal development
A, agarose gel showing complete and alternative spliced isoforms of P2X6 receptor cDNA fragments from postnatal days (P) 0, 8 and 15. ß-Actin (Ac) was used as a control for RNA integrity and as a standard for quantification of relative transcription of P2X6 receptor coding mRNA. P0, P8 and P15 represent P2X6 receptor mRNA transcription. AcP0, AcP8 and AcP15 represent determination of ß-actin mRNA transcription in RNA samples used for detection of P2X6 receptor gene expression. B, the data shown are representative of three independent experiments. *, Increases of transcription rates of mRNAs coding for the full-length P2X6 receptor were statistically significant as determined by ANOVA analysis.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
P2X purinergic receptors, especially P2X4 and P2X6 subunits, are widely expressed in the central nervous system (Collo et al. 1996). The co-assemblage of the seven described subunits in trimeric functional ion channels gives rise to a wide range of responses to agonists and antagonists in cells expressing these receptors. When alternative splicing is considered, this range of responses is increased.

Alternative splicing has already been described for P2X1, P2X2, P2X4, P2X5 and P2X7 receptors. The splice variant of the P2X1 receptor lacks part of the second transmembrane domain, together with 28 amino acids in the intracellular carboxy-terminal region (Hardy et al. 2000). Three isoforms resulting from alternative splicing were identified for the P2X2 subunit (Simon et al. 1997). One of them (P2X2b) has a deletion of 69 amino acids towards the carboxyl terminus, but proved to be functional when expressed in Xenopus oocytes or HEK 293 cells. The other two isoforms have mutations in the first membrane-spanning domain, and it is not known whether they are able to form functional channels. In the human P2X4 subunit, the first transmembrane region is eliminated in the described splice variant (Dhulipala et al. 1998), thus originating a non-functional receptor, whereas in mouse the protein originated from alternative splicing lacks 27 amino acids in the extracellular domain (Townsend-Nicholson et al. 1999). This variant hardly forms functional homomeric receptors, but appears to interact with the full-length form of P2X4, thus forming a functional channel with altered properties. The variant form of P2X5 looses a cassette containing 24 amino acids, originating a subunit which lacks the first transmembranic domain (Le et al. 1997). Eight variations of the P2X7 receptors were isolated (Cheewatrakoolpong et al. 2005). Two of them lacked the first transmembrane domain, and at least one of these was shown to originate an inactive P2X7 receptor. Three isoforms contain an intron located between exons 10 and 11, causing the introduction of a new stop codon and shortening the protein to a sequence of 171 amino acids. This shortened variant seemed to be unable to adopt the regular pore structure that is formed by the full-length form.

We describe here the occurrence of a splice variant of the P2X6 purinergic receptor subunit during neuronal development of P19 cells, which is not present in adult rat brain. This splice variant was originally described in dystrophic muscle from an X-linked muscular dystrophy (mdx) mouse model (Jiang et al. 2005). This splice variant lacks an 110 bp fragment, similar in size to exon 8 from the rat gene for the P2X6 subunit. Since this exon has a length of 110 bp, its removal changes the reading frame when the mRNA is being translated. In rats, it would cause the appearance of a stop codon (TAA) two amino acids after the splicing region, but a punctual mutation in mRNA mouse for P2X6 changes it to a codon for glutamine (CAA) with a stop codon 22 amino acids ahead towards the carboxy-terminal region. No insertions or deletions capable of re-establishing the original reading frame were present before the stop codon. These data suggest that the second transmembrane domain is not formed in this protein, derived from alternative splicing, thereby generating a non-functional P2X6 receptor subunit.

The generation of a non-functional protein can be used as a form of regulation of the active gene product. A distinct mechanism for this is the use of alternative splicing to induce nonsense-mediated mRNA decay (NMD; Lareau et al. 2004). Gene expression can be regulated post-transcriptionally by the production of splice forms that will be degraded by NMD rather than translated into protein, a process termed regulated unproductive splicing and translation (RUST). Nonsense-mediated mRNA decay is an RNA surveillance function that recognizes mRNAs containing premature termination codons (PTC+ mRNAs) and targets the transcripts for destruction rather than translation into protein (Maquat, 2004). Nonsense and frame shift mutations, errors in pre-mRNA processing and alternative splicing are among the many sources of PTC+ mRNAs.

Our RT-PCR results demonstrate that the mRNA for the splice variant form of the P2X6 receptor is more frequently present in differentiating P19 cells than the full-length coding mRNA. Although the functional expression of homomeric P2X6 receptor ion channels has been reported (Jones et al. 2004), this receptor is more commonly found forming heteromeric receptors either with P2X2 or P2X4 subunits. The low expression of the full-length P2X6 subunit during neuronal differentiation of P19 cells indicates that relatively few functional heteromeric P2X2,6 or P2X4,6 purinergic receptors are being formed. During postnatal development and in adult mouse brain, the functional full-length subunit is predominately expressed, suggesting an increase in expression of functional P2X2,6 and P2X4,6 receptors.

Many mRNAs coding for neurone-specific proteins are generated by tissue-specific splicing in the nervous system, resulting in structural changes and altered activities of these proteins. In some cases, proteins derived from alternatively spliced mRNAs lose their biological activity or even fail to be expressed. Although there are eight possible splice variants of the N-methyl-D-aspartate (NMDA) subunit NR1, only one of them is expressed in motor neurones (Stegenga & Kalb, 2001). However, a further splice variant of the same receptor subunit was found in respiratory-related neurones (Paarmann et al. 2005).

Alternative splicing events are also frequent during neuronal differentiation. The neuronal differentiation of P19 cells, used as the in vitro model for early neuronal development in our study, has already been used as a tool for studying neural-specific alternative splicing. Chen and co-workers demonstrated alternative splicing of the glutamate receptor subunit B during neuronal differentiation of P19 cells (Chen et al. 2004).

In summary, we have identified a splice variant of the mouse P2X6 receptor which is present during neuronal differentiation of P19 embryonal carcinoma cells and is the less predominant isoform in developing and adult mouse brain. It may function as a post-transcriptional regulation mechanism for P2X6 receptor gene expression during neuronal differentiation in vitro and postnatal neuronal development.

The origin of the P19 cell line as tumour cells may account for differences between the transcription rates of the isoform of alternative splicing in differentiating murine P19 embryonal carcinoma cells and developing mouse brain.


   Footnotes
 
R. L. da Silva and R. R. Resende contributed equally to this work.


   References
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
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   Acknowledgements
 
H.U. is grateful for a grant (no. 2001/08827-4) from FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo) and financial support from CNPq (Conselho Nacional de Desenvolvimento Cientifico e Tecnológico), Brazil. R.L.S. and R.R.R. have been supported by fellowships from FAPESP and CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior), Brazil respectively. The Center for Applied Toxicology (CAT-CEPID) at the Butantan Institute, São Paulo, Brazil, is acknowledged for performing DNA sequencing.





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