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P2 purinoceptor-activated inward currents in follicular oocytes of Xenopus laevis
B F King1, S Wang, G Burnstock
1Department of Anatomy and Developmental Biology, University College London, UK. ucgabfk@ucl.ac.uk or b.king@ucl.ac.uk
Abstract:
1. ATP, UTP and 2MeSATP (10 microM) evoked fast, transient (2-20 s duration) and slow, sustained (60-120 s duration) inward currents in 118 of 285 follicular Xenopus oocytes but failed to activate inward currents in defolliculated Xenopus oocytes (n = 136). 2. The potency order for nucleotides was (at 10 microM): UTP = 2MeSATP > 2ClATP = ATP > ADP > alpha, beta-methylene-ATP > adenosine > ITP, with beta, gamma-methylene-ATP, CTP and GTP inactive. This potency order is atypical for any single P2 purinoceptor subtype. 3. EC50 values (for fast and slow inward currents) were (microM): ATP, 5.6 +/- 2.1 and 6.7 +/- 3.0; UTP, 1.0 +/- 0.3 and 0.9 +/- 0.3; 2MeSATP, 0.6 +/- 0.2 and 9.2 +/- 7.4 (n = 3). Suramin inhibited fast (IC50: 65 +/- 20 microM; n = 7) and slow (IC50: 57 +/- 10 microM; n = 7) inward currents evoked by ATP. Suramin also inhibited slow inward currents produced by UTP but potentiated those caused by 2MeSATP. 4. Fast and slow inward currents evoked in response to ATP and UTP reversed at -12 +/- 3 mV (n = 3), a value close to Erev for Na(+)-permeable ion channels present in oocytes. These same currents were reduced significantly when extracellular Na+ was substituted with choline and Erev changed to -30 +/- 5 mV (n = 3). Inward currents evoked by ATP and UTP were inhibited in a dose-dependent manner by amiloride (10-100 microM). 5. Fast and slow inward currents caused by 2MeSATP reversed at -29 +/- 3 mV (n = 3), a value close to Erev for Cl- channels in oocytes. Fast and slow responses to 2MeSATP were inhibited in a dose-dependent manner by the Cl- channel blocker NPPB (10-100 microM) and were only reduced slightly by amiloride (100 microM). 6. We conclude that two P2 purinoceptor subtypes are present on follicular Xenopus oocytes. 2MeSATP activates fast and slow Cl- currents (Erev = -30 mV) via a suramin-insensitive P2Y purinoceptor, while UTP and ATP activate fast and slow Na(+)-dependent currents (Erev = -10 mV) via a suramin-sensitive P2U purinoceptor.
Insights
Follicular Xenopus oocytes express two P2 purinoceptor subtypes. One activates chloride currents via a suramin-insensitive P2Y receptor, while the other activates sodium currents via a suramin-sensitive P2U receptor.
Area of Science:
- Molecular Biology
- Cell Physiology
- Pharmacology
Background:
- Purinergic signaling involves P2 receptors, which are activated by extracellular nucleotides.
- Xenopus oocytes are a common model system for studying ion channel function and receptor pharmacology.
Purpose of the Study:
- To investigate the subtypes of P2 purinoceptors present on follicular Xenopus oocytes.
- To characterize the ion selectivity and pharmacological properties of nucleotide-evoked currents.
Main Methods:
- Electrophysiological recordings (two-electrode voltage clamp) were performed on follicular and defolliculated Xenopus oocytes.
- Nucleotides (ATP, UTP, 2MeSATP) were applied to evoke inward currents.
- Pharmacological agents (suramin, amiloride, NPPB) and ionic substitutions (choline for Na+) were used to characterize receptor subtypes and ion channel properties.
Main Results:
- Follicular oocytes, but not defolliculated ones, responded to ATP, UTP, and 2MeSATP with fast and slow inward currents.
- 2MeSATP activated chloride currents (reversed at -30 mV) via a suramin-insensitive receptor, distinct from ATP and UTP which activated Na+-dependent currents (reversed at -10 mV) via a suramin-sensitive receptor.
- The potency order of nucleotides and differential sensitivity to suramin suggest the presence of at least two distinct P2 purinoceptor subtypes.
Conclusions:
- Follicular Xenopus oocytes express two P2 purinoceptor subtypes: a suramin-insensitive P2Y receptor activated by 2MeSATP, and a suramin-sensitive P2U receptor activated by ATP and UTP.
- These receptors mediate distinct ion currents, primarily Cl- and Na+ respectively, highlighting differential signaling pathways in oocytes.