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Characterization of Ca2+ influx through recombinant P2X receptor in C6BU-1 cells
1Division of Pharmacology, National Institute of Health Sciences, Tokyo, Japan.
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.
Insights
Investigating adenosine triphosphate (ATP) receptors P2X2 and P2X3 in C6BU-1 cells revealed that co-expression of both subtypes (P2X2+3) enhances calcium signaling dynamics. This heteromultimer formation offers improved responsiveness and slower desensitization compared to individual subtypes.
Area of Science:
- Cellular and Molecular Neuroscience
- Ion Channel Physiology
- Receptor Pharmacology
Background:
- Adenosine 5'-triphosphate (ATP) is a key extracellular signaling molecule.
- P2X receptors, particularly subtypes P2X2 and P2X3, are ligand-gated ion channels activated by ATP.
- Understanding the functional properties of P2X receptor subtypes is crucial for elucidating cellular signaling pathways.
Purpose of the Study:
- To investigate the functional effects of exogenous adenosine 5'-triphosphate (ATP) and alpha,beta-methylene ATP (alpha,beta meATP) on C6BU-1 cells expressing P2X2 and P2X3 receptor subtypes.
- To characterize the calcium (Ca2+) influx and current responses mediated by individual and co-expressed P2X2 and P2X3 subtypes.
- To determine if co-expression of P2X2 and P2X3 leads to altered functional properties, such as responsiveness and desensitization, compared to their individual expression.
Main Methods:
- Utilized fura-2 fluorescence recording to measure intracellular Ca2+ ([Ca2+]i) changes.
- Employed whole-cell patch clamp recording to assess ion channel currents.
- Expressed P2X2 and P2X3 receptor subtypes, individually and together (P2X2+3), in the C6BU-1 cell line.
Main Results:
- Untransfected C6BU-1 cells did not exhibit Ca2+ influx or current responses to ATP stimulation.
- P2X2 subtype expression in C6BU-1 cells showed ATP-evoked [Ca2+]i rise at >10 microM ATP, while alpha,beta meATP was ineffective.
- P2X3 subtype expression demonstrated ATP-evoked currents at >0.1 microM ATP and [Ca2+]i rise at >10 microM ATP, with significant desensitization observed.
- Co-expression of P2X2 and P2X3 (P2X2+3) in C6BU-1 cells resulted in a robust [Ca2+]i rise at 1 microM ATP, higher than individual subtypes, with slower desensitization and improved expression efficiency.
Conclusions:
- The C6BU-1 cell line is a suitable model for studying Ca2+ influx through P2X subtypes.
- P2X2 and P2X3 subtypes likely assemble as a heteromultimer (P2X2+3).
- Heterogeneous expression of P2X2 and P2X3 results in enhanced Ca2+ dynamics, including increased responsiveness and slower desensitization, compared to homogeneous expression of either subtype alone.