Molecular cloning and functional expression of a novel rat heart P2X purinoceptor

Abstract

Here we describe a novel purinergic receptor, the P2X5 receptor, cloned from rat heart. The full-length cDNA encodes a protein 455 amino acids long which shares an overall identity of 40-47% with other members of the P2X purinergic receptor family. P2X5 mRNA transcripts are found predominantly in rat heart but are also present in brain, spinal cord and adrenal gland. Functional expression of the recombinant receptor in HEK-293 cells shows a current that resembles mostly the P2X2 phenotype: the ATP-activated current reveals little agonist desensitization, is not activated by a,~meATP and is completely blocked by suramin and PPADS.

Multiple P2 Receptors Contribute to a Transient Increase in Intracellular Ca2+ Concentration in Atp‐Stimulated Rat Brown Adipocytes

“…Among seven ionotropic P2X receptor subtypes tested, the expression of P2X 2 and P2X 6 receptor subtype genes were undetectable in these rat brown adipocytes. Amplifications of P2X 1 , P2X 3 , P2X 4 , P2X 5 and P2X 7 receptor subtype genes resulted in single bands around the predicted sizes of 435, 473, 432 and 433 bp, respectively, and these PCR products were found to show 100 % homology for the sequences reported previously (Valera et al 1994;Chen et al 1995;Wang et al 1996;Garcia-Guzman et al 1996;Surprenant et al 1996). During amplification of the P2X 5 receptor gene, the predicted size of the signal appeared to be very weak and the signal from the lower molecular weight product was found to be the major band.…”

“…Single strand cDNA was synthesized from 5 mg of total RNA with 20 units of RAV-2 reverse transcriptase (Takara Biochemicals, Kyoto, Japan) using random primers. A portion of cDNA was amplified by the polymerase chain reaction (PCR) method using rat P2X 1 -, P2X 2 , P2X 3 -, P2X 4 -, P2X 5 -, P2X 6 -, P2X 7 -, P2Y 1 -, P2Y 2 -, P2Y 4 -, P2Y 6 -and human P2Y 11 -specific primers synthesized according to the nucleotide sequences of rat receptor genes P2X 1 (GenBank accession number X80477) (Valera et al 1994), P2X 2 (GenBank accession number U14414) (Brake et al 1994), P2X 3 (GenBank accession number X90651) (Chen et al 1995), P2X 4 (GenBank accession number U47031) (Wang et al 1996), P2X 5 (GenBank accession number X97328) (Garcia-Guzman et al 1996), P2X 6 (GenBank accession number X92070) (Collo et al 1995), P2X 7 (GenBank accession number X95882) (Surprenant et al 1996), P2Y 1 (GenBank accession number U22830) (Tokuyama et al 1995), P2Y 2 (GenBank accession number L46865) (Chen et al 1996), P2Y 4 (GenBank accession number Y14705) (Bogdanov et al 1998), P2Y 6 (GenBank accession number D63665) (Chang et al 1995) and P2Y 11 (GenBank accession number AF030335) (Communi et al 1997), respectively: P2X 1 sense, 5‚-CCC-AGAAGGTGGCATATGCCAG-3‚ (nt 569-590); P2X 1 antisense, 5‚-CAGTCCAGATCACACTTCCAGTC-3‚ (nt 1003-981); P2X 2 sense, 5‚-ATCCTCATCAAGAACAGCATCCAC-3‚ (nt 589-612); P2X 2 antisense, 5‚-TTGGCAAACCTGAAGTTGTAGCCT-3‚ (nt 914-891); P2X 3 sense, 5‚-CTGTGAGATCCAGGGCTGGTG-3‚ (nt 619-639); P2X 3 antisense, 5‚-GGATGCCAAAAGCCT-TCAGGAG-3‚ (nt 1091-1070); P2X 4 sense, 5‚-AACATGATT-GTCACCGTGAACCAG-3‚ (nt 340-363); P2X 4 antisense, 5‚-TGCCATCACCTGGAAGCTATGTC-3‚ (nt 771-749); P2X 5 sense, 5‚-GACACTTCCCTGCAGAGTGCTG-3‚ (nt 225-246); P2X 5 antisense, 5‚-AAGGGGATCCGTTGGCATGGAC-3‚ (nt 593-572); P2X 6 sense, 5‚-GTAACCCAGGTTAAGGAACTG-GAG-3‚ (nt 239-262); P2X 6 antisense, 5‚-GTATCTAAG-GCATTGGTTCTGGAG-3‚ (nt 622-645); P2X 7 sense, 5‚-TCT-TCGACACGGCCGACTACAC-3‚ (nt 381-402); P2X 7 antisense, 5‚-AGCCGGAAGATGGGACACTGAG-3‚ (nt 813-792); P2Y 1 sense, 5‚-TCCGATGTGCCCTGATCAAGAC-3‚ (nt 738-759); P2Y 1 antisense, 5‚-ATGAGCCATACCAGCACACTGAC-3‚ (nt 1152-1130); P2Y 2 sense, 5‚-CGCTTCAACGAGGACTTCAAG-TA-3‚ (nt 559-581); P2Y 2 antisense, 5‚-CCCCAGCTCAGG-GAGTGCAGA-3‚ (nt 917-897); P2Y 4 sense, 5‚-GTTCTG-GAGATGGTGATTGTAGG-3‚ (nt 1407-1429); P2Y 4 antisense, 5‚-CAGTATCCCAGGTATCGGTGCA-3‚ (nt 1758-1737); P2Y 6 sense, 5‚-CTACCGTGAGGATTTCAAGCGAC-3‚ (nt 496-518); P2Y 6 antisense, 5‚-CTGGAAGCTAATGCAGGTGAGGA-3‚ (nt 808-786); P2Y 11 sense, 5‚-TGGAGCGCTTCCTCTTCACCTG-3‚ (nt 302-323); P2Y 11 antisense, 5‚-AGTCATGCCTGGGCT-GCGTAG-3‚ (nt 708-688). The PCR reaction included 0.4 ml of template cDNA, 2.5 units of KOD dash polymerase (Toyobo Biochemicals, Osaka, Japan), 1 mM KCl, 6 mM (NH 4 ) 2 SO 4 , 0.1 % Triton X-100, 10 mg ml _1 BSA, 0.2 mM each of deoxynucleotide triphosphates and 4 pmol primers in 20 ml of 120 mM Tris-HCl buffer (pH 8.0).…”

Mouse Leydig cells express multiple P2X receptor subunits

“…Seven P2X receptor subunits (P2X 1-7 ) were cloned and characterized in vertebrates, and each of them can form two type of assemblies, homomeric, P2X 1 [50], P2X 2 [3], P2X 3 [8], P2X 4 [2], P2X 5 [18], P2X 7 [46], and P2X 6 [23], or heterotrimeric P2X 2/3 [31], P2X 1/5 [48], P2X 4/6 [30], P2X 2/6 [27], P2X 1/2 [5], P2X 1/4 [38], and P2X 4/7 [19]. Each subunit is composed by two transmem-brane regions (TM1 and TM2) forming the channel pore, a large extracellular domain, which contains the ATP binding site and both N and C termini located intracellularly [14,48,22].…”

Identification and Characterization of Adenosine 5′-Tetraphosphate in Human Myocardial Tissue

“…Additionally the P 2X5 receptor was cloned in rat heart (27). In accordance with pertinent literature, the P 2X2 receptor and the P 2X5 receptor are not activated by ␣,␤-meATP (27,28). The P 2X4 receptor cannot be inhibited by PPADS (29).…”

“…Only mRNAs for P 2X1 , P 2X2 , and P 2X4 have been detected in coronary vasculature thus far (26). Additionally the P 2X5 receptor was cloned in rat heart (27). In accordance with pertinent literature, the P 2X2 receptor and the P 2X5 receptor are not activated by ␣,␤-meATP (27,28).…”