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Identification of a site that modifies desensitization of P2X2 receptors
1Department of Biology, University of Michigan, Natural Science Building, Ann Arbor, Michigan, 48109-1048, USA.
Abstract:
The time course of desensitization of P2X2 receptors expressed in Xenopus oocytes and HEK 293 cells was examined. We found that there was virtually no desensitization in response to 5 microM ATP, but that responses to 50 microM ATP desensitized with a highly variable time course. The time constant of desensitization varied 250fold among oocytes (from 4 to over 1000 seconds) and 10 fold among HEK cells (from 4 to 40 seconds). Mutation of D349, which lies at the cytoplasmic end of the second transmembrane domain (to either A, N, or R), resulted in a dramatic acceleration of the median rate of desensitization, and eliminated variation in the rate of desensitization. In contrast, mutation of four other charged residues in putative transmembrane domains had no effect on desensitization. These results suggest that P2X2 receptor desensitization is strongly modulated by the intracellular environment, and that D349 is essential for this modulation.
Insights
P2X2 receptor desensitization to ATP is highly variable and influenced by the intracellular environment. Mutation of residue D349 significantly speeds up and stabilizes this desensitization process.
Area of Science:
- Neuroscience
- Molecular Biology
- Cellular Physiology
Background:
- P2X2 receptors are ligand-gated ion channels activated by extracellular ATP.
- Receptor desensitization is a critical process regulating cellular responses to stimuli.
- Understanding P2X2 receptor desensitization mechanisms is key to deciphering purinergic signaling.
Purpose of the Study:
- To investigate the time course and variability of P2X2 receptor desensitization.
- To identify key residues involved in modulating P2X2 receptor desensitization.
- To elucidate the role of the intracellular environment in P2X2 receptor desensitization.
Main Methods:
- Expressing P2X2 receptors in Xenopus oocytes and HEK 293 cells.
- Applying varying concentrations of ATP (5 µM and 50 µM) to elicit responses.
- Performing site-directed mutagenesis on specific charged residues within transmembrane domains.
- Analyzing the kinetics and variability of receptor desensitization.
Main Results:
- P2X2 receptors showed minimal desensitization at 5 µM ATP but highly variable desensitization at 50 µM ATP.
- Desensitization time constants varied significantly across oocytes and HEK cells.
- Mutation of aspartate at position 349 (D349) drastically accelerated and normalized desensitization rates.
- Mutations at other charged transmembrane residues did not affect desensitization.
Conclusions:
- P2X2 receptor desensitization is strongly modulated by the intracellular environment.
- Residue D349 is crucial for the intracellular modulation of P2X2 receptor desensitization.
- The findings provide insights into the molecular mechanisms governing P2X2 receptor channel gating and regulation.