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Biochim Biophys Acta
2001 May 02;15121:111-24. doi: 10.1016/s0005-2736(01)00313-3.
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Molecular cloning and functional expression of Xenopus laevis oocyte ATP-activated P2X4 channels.
Juranka PF
,
Haghighi AP
,
Gaertner T
,
Cooper E
,
Morris CE
.
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All cells contain mechanosensitive ion channels, yet the molecular identities of most are unknown. The purpose of our study was to determine what encodes the Xenopus oocyte's mechanosensitive cation channel. Based on the idea that homologues to known channels might contribute to the stretch channels, we screened a Xenopus oocyte cDNA library with cation channel probes. Whereas other screens were negative, P2X probes identified six isoforms of the P2X4 subtype of ATP-gated channels. From RNase protection assays and RT-PCR, we demonstrated that Xenopus oocytes express P2X4 mRNA. In expression studies, four isoforms produced functional ATP-gated ion channels; however, one, xP2X4c, had a conserved cysteine replaced by a tyrosine and failed to give rise to functional channels. By changing the tyrosine to a cysteine, we showed that this cysteine was crucial for function. We raised antibodies against a Xenopus P2X4 C-terminal peptide to investigate xP2X4 protein expression. This affinity purified anti-xP2X4 antibody recognized a 56 kDa glycosylated Xenopus P2X4 protein expressed in stably transfected HEK-293 cells and in P2X4 cDNA injected oocytes overexpressing the cloned P2X4 channels; however, it failed to recognize proteins in control, uninjected oocytes. This suggests that P2X4 channels and mechanosensitive cation channels are not linked. Instead, oocyte P2X4 mRNA may be part of the stored pool of stable maternal mRNA that remains untranslated until later developmental stages.
Fig. 1.
Primary structure of the Xenopus oocyte P2X4 channel. Nucleotide and deduced amino acid sequences of isoform xP2X4f. The two putative transmembrane domains are underlined. Between these, in the extracellular loop, the ten conserved cysteines are undermarked and the seven putative N-linked glycosylation sites (N-X-S/T) are marked with an asterisk. The polyadenylation signal in the 3â² UTR is in bold and underlined and the KpnI site used for subcloning all isoforms into pcDNAI/Amp is in italics and underlined.
Fig. 2.
Alignment of different classes of P2X4 amino acid sequences. The amino acid sequence alignment of mammalian, bird, amphibian and fish P2X4. The number of amino acids and the GenBank accession numbers for these sequences are: rat 388 (AAA99777), chicken 384 (CAB56283), Xenopus 391 (AF308153) and zebra fish, an incomplete deduced amino acid sequence made by translating two partial cDNA sequences (AW133550 and AW115797). Underlined amino acids represent the C-terminal peptides used to generate the Xenopus (BSYN291) and rat [18] antisera. As in Fig. 1 the two transmembrane domains are overlined. Asterisks below the alignments indicate identity in all sequences while a colon or period indicates decreasing degrees of amino acid conservation between the four sequences. The bolded residues (Q, E, E, H for the four organisms) are mentioned in Section 3 in relation to PPADS.
Fig. 3.
Detection of Xenopus P2X4 mRNA in oocytes and surrounding tissues by RNase protection assay. RNase protection assays performed on total cellular RNA (20 μg A and 4 μg B) extracted from defolliculated, devitellinated oocytes (lane 1), oocytes with intact follicle cell layer (lane 2), the transparent vascularized epithelial sac which surrounds each ovarian lobe minus the folliculated oocytes (lane 3) and the entire ovarian lobe with folliculated oocytes at various stages I–VI and the vascularized sac surrounding them (lane 4). 200 000 cpm of each riboprobe ((a) the 318 nucleotide Xenopus P2X4 riboprobe (asterisk in lane P; protects 256 nucleotides), (b) the 220 nucleotide Xenopus histone H4 riboprobe (asterisk in lane P; protects multiple sizes of RNA) was used in the reactions and the gels were exposed to X-ray film for 25 h (a) or 3 h (b). The riboprobes were used at 2000 cpm/lane as size markers (lane P, size in nucleotides as indicated).
Fig. 4.
Electrophysiology of P2X4 channels. (a) Functional expression of xP2X4 clones in Xenopus oocytes. (b) Coinjection of α4β2 nAChR and rat P2X4 or Xenopus P2X4 (xP2X4c) clones in Xenopus oocytes. (c,d) Comparison of rectification for rat P2X4 and Xenopus P2X4 (xP2X4f) ATP-gated ion channels; in d, the Xenopus current is scaled to the maximum inward rat current at â100 mV. (e) Block of Xenopus P2X4 (xP2X4f) ATP-gated channels by PPADS.
Fig. 5.
Detection of in vitro translated P2X4 protein by 35S-methionine incorporation and by Western blot. The cDNAs for Xenopus P2X4 (xP2X4f-pcDNAI/Amp), rat P2X4 (rP2X4-pcDNAI [14]) and a control plasmid coding for luciferase (Promega) were transcribed and translated in a coupled reticulocyte lysate system (TnT, Promega). The reaction was primed with 1 μg DNA and the 35S-methionine-labeled proteins (1 μl) were resolved on an 8% polyacrylamide gel by SDS-PAGE. The peptides were transferred to nitrocellulose and either exposed to X-ray film for 3 days (lanes 1, 2, 3) or probed with anti-X-P2X4 antibody (lanes 4, 5, 6) as described in Fig. 6. Luciferase (lanes 1, 4), Xenopus P2X4 (lanes 2, 5) and rat P2X4 (lanes 3, 6). The mass of the proteins was determined by comparison to prestained molecular weight marker proteins (New England Biolabs) shown on the left.
Fig. 6.
Detection of Xenopus and rat P2X4 protein by Western blot using (a) anti-Xenopus and (b) anti-rat P2X4 antisera. The Westerns were probed with affinity purified anti-C-terminal P2X4 peptide antisera (1.5 μg IgG/ml). Lanes 1â4 were cell membrane extracts from: 1, HEK-293, 15 μg; 2, HEK-293 stably transfected with Xenopus P2X4f, 15 μg; 3, Xenopus brain, 5 μg; 4, rat olfactory bulb, 5 μg; and lanes 5 and 6 were 10 μg of crude cell extracts of Xenopus oocytes which were either nuclear injected with xP2X4f isoform (lane 6) or uninjected (lane 5). Enhanced chemiluminescence with horseradish peroxidase-conjugated anti-rabbit secondary antibody was used to develop the Western. The mass of the proteins was determined by comparison to prestained molecular weight marker proteins (New England Biolabs) shown on the right.
