Heteromeric channels with different phenotypes are generated when coexpressing two P2X2 receptor isoforms

• Published in: Biochemical and biophysical research communications Vol. 477; no. 1; pp. 54 - 61
• Main Authors: Jaramillo-Polanco, Josue, Liñán-Rico, Andrómeda, Espinosa-Luna, Rosa, Jiménez-Bremont, Juan F., Montaño, Luis M., Miranda-Morales, Marcela, Barajas-López, Carlos
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 12.08.2016
• Subjects: adenosine triphosphate; Adenosine Triphosphate - metabolism; agonists; Animals; binding sites; Cells, Cultured; Heterologous expression; Ion Channels - metabolism; Kinetics; mutants; oocytes; Oocytes - metabolism; P2X receptor activation; P2X receptors; P2X2 splice variants; patch-clamp technique; Patch-Clamp Techniques; phenotype; PPADS binding; Protein Isoforms - chemistry; Protein Isoforms - drug effects; Protein Isoforms - metabolism; Pyridoxal Phosphate - analogs & derivatives; Pyridoxal Phosphate - pharmacology; receptors; Receptors, Purinergic P2X2 - chemistry; Receptors, Purinergic P2X2 - drug effects; Receptors, Purinergic P2X2 - metabolism; stoichiometry; Xenopus laevis; P2X receptor activation; P2X2 splice variants; P2X receptors; PPADS binding; Heterologous expression
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2016.06.020

To investigate if channels with different stoichiometry are formed from P2X2 receptor isoforms during their heterologous co-expression. The two-electrode voltage-clamp technique was used to measured ATP induced currents in Xenopus laevis oocytes. We used a mutant (P2X2-2bm) because its ATP sensitivity is lower than P2X2-2b receptors, which highlights the differences with its splice variant P2X2-1a.Currents through homomeric channels had significantly different Hill coefficients. P2XR are trimeric proteins with three agonist binding sites; therefore, only two homomeric and two heteromeric stoichiometries are possible when both P2X2 isoforms are coexpressed, the heteromeric channels might be formed by: i) 2(P2X2-1a)+1(P2X2-2bm); or ii) 1(P2X2-1a)+2(P2X2-2bm). Because P2X2 channels open when two binding sites are occupied, these stoichiometries are expected to have different ATP sensitivities. Thus, co-expressing both P2X2 isoforms, two oocyte populations were distinguished based on their sensitivity to ATP and Hill coefficients. For the first population (P2X2-1a like), the ATP EC50 and the Hill coefficient were not different than those of homomeric P2X2-1a channels similarly, for the second population (P2X2-2bm like), these variables were also not different than for those of homomeric P2X2-2bm channels. Various findings indicate that homomeric channel expression is not responsible for such differences. Our observations indicate that two heteromeric channels can be assembled from two P2X2 receptor isoforms. Our data support a current model, according to which, ATP activation of two subunits can open P2X2 channel. However, PPADS appears to bind to all three subunits in order to inhibit ATP effects on P2X2 receptors.