Characterization of Ca2+ influx through recombinant P2X receptor in C6BU-1 cells.

Abstract

1. The effects of exogenous adenosine 5'-triphosphate (ATP) and alpha,beta-methylene ATP (alpha,beta meATP) on C6BU-1 cells transfected with P2X2 and P2X3 subtypes, separately or together (P2X2+3), were investigated using fura-2 fluorescence recording and whole-cell patch clamp recording methods. 2. Untransfected C6BU-1 cells showed no intracellular Ca2+ ([Ca2+]i) increase in response to depolarizing stimulation with high K+ or stimulation with ATP. There was no current induced by ATP under voltage clamp conditions in untransfected C6BU-1 cells. ATP caused Ca2+ influx only from extracellular sources in C6BU-1 cells transfected with the P2X subtypes, suggesting that the C6BU-1 cell line is suitable for the characterization of Ca2+ influx through the P2X subtypes. 3. In C6BU-1 cells transfected with the P2X2 subtype, ATP (more than 10 microM) but not alpha,beta meATP (up to 100 microM) evoked a rise in [Ca2+]i. 4. In the cells transfected with the P2X3 subtype, current responses under voltage clamp conditions were observed at ATP concentrations higher than 0.1 microM of alpha,beta meATP were required. This discrepancy in the concentration dependence of the agonist responses with respect to the [Ca2+]i rise and the current response was seen only with the P2X3 subtype. In addition, the agonist-induced rise in [Ca2+]i was observed only after the first application because of desensitization of this subtype. 5. In C6BU-1 cells co-transfected with P2X2 and P2X3, ATP at 1 microM evoked a [Ca2+]i rise. This responsiveness was higher than that of the other subtype combinations tested. The efficiency of expression was improved by co-transfection with P2X2 and P2X3, when compared to transfection with the P2X3 subtype alone. The desensitization of the P2X2+3 was apparently slower than that of the P2X3 subtype alone. Therefore, this combination could respond to the repeated application of agonists each time with a [Ca2+]i rise. 6. These results suggest that the P2X2 and P2X3 subtypes assemble a heteromultimer and that this heterogeneous expression acquires more effective Ca2+ dynamics than that by homogeneously expressed P2X2 or P2X3.