Characterization of Ca 2+ influx through recombinant P2X receptor in C6BU‐1 cells

1 The effects of exogenous adenosine 5′‐triphosphate (ATP) and α,β‐methylene ATP (α,βmeATP) on C6BU‐1 cells transfected with P2X 2 and P2X 3 subtypes, separately or together (P2X 2+3 ), were investigated using fura‐2 fluorescence recording and whole‐cell patch clamp recording methods. 2 Untransfected C6BU‐1 cells showed no intracellular Ca 2+ ([Ca 2+ ] 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 Ca 2+ 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 Ca 2+ influx through the P2X subtypes. 3 In C6BU‐1 cells transfected with the P2X 2 subtype, ATP (more than 10 μ m ) but not α,βmeATP (up to 100 μ m ) evoked a rise in [Ca 2+ ] i . 4 In the cells transfected with the P2X 3 subtype, current responses under voltage clamp conditions were observed at ATP concentrations higher than 0.1 μ m of α,βmeATP were required. This discrepancy in the concentration dependence of the agonist responses with respect to the [Ca 2+ ] i rise and the current response was seen only with the P2X 3 subtype. In addition, the agonist‐induced rise in [Ca 2+ ] i was observed only after the first application because of desensitization of this subtype. 5 In C6BU‐1 cells co‐transfected with P2X 2 and P2X 3 , ATP at 1 μ m evoked a [Ca 2+ ] i rise. This responsiveness was higher than that of the other subtype combinations tested. The efficiency of expression was improved by co‐transfection with P2X 2 and P2X 3 , when compared to transfection with the P2X 3 subtype alone. The desensitization of the P2X 2+3 was apparently slower than that of the P2X 3 subtype alone. Therefore, this combination could respond to the repeated application of agonists each time with a [Ca 2+ ] i rise. 6 These results suggest that the P2X 2 and P2X 3 subtypes assemble a heteromultimer and that this heterogeneous expression acquires more effective Ca 2+ dynamics than that by homogenously expressed P2X 2 or P2X 3British Journal of Pharmacology (1998) 124 , 1484–1490; doi: 10.1038/sj.bjp.0701963