Characterization of Ca2+ 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 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 μM) but not α,βmeATP (up to 100 μM) 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 μM of α,βmeATP 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 μM 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 homogenously expressed P2X₂ or P2X₃