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Specific detection of non-functional human P2X(7) receptors in HEK293 cells and B-lymphocytes
J A Barden1, R Sluyter, B J Gu
1Department of Anatomy and Histology, Anderson Stuart Bldg, F13, The University of Sydney, Sydney 2006, NSW, Australia. julian@anatomy@usyd.edu.au
Abstract:
P2X(7) receptor/channels mediate ATP-induced apoptosis in a range of cells including lymphocytes. HEK293 cells were transfected with wild-type human P2X(7) receptor or site-directed mutant constructs (K193A, K311A and E496A) known to be non-functional from measurements of barium/ethidium influx in the presence of ATP or 2',3'-O-(4-benzoylbenzoyl)-ATP. An antibody was designed against an epitope from a loop adjacent to the extracellular ATP site. The epitope was unavailable in cells expressing normal functional surface receptors. Non-functional surface receptors as well as intracellular receptors selectively bound the antibody. So did B-lymphocytes from chronic lymphocytic leukemia patients expressing non-functional (E496A) mutant receptor.
Insights
The P2X7 receptor, crucial for ATP-induced apoptosis, has its antibody binding site revealed. This antibody targets non-functional P2X7 receptors on cell surfaces and internally, including in certain leukemia cells.
Area of Science:
- Molecular biology
- Immunology
- Cell biology
Background:
- The P2X7 receptor (P2X7R) is an ATP-gated ion channel involved in apoptosis, particularly in lymphocytes.
- Understanding P2X7R structure and function is critical for its role in cellular processes and disease.
Purpose of the Study:
- To characterize the binding properties of an antibody targeting the P2X7 receptor.
- To investigate the accessibility of the P2X7R epitope in functional versus non-functional receptor states.
Main Methods:
- HEK293 cells were transfected with wild-type and mutant human P2X7R constructs (K193A, K311A, E496A).
- An antibody was designed against an extracellular loop epitope near the ATP-binding site.
- Antibody binding was assessed on cells expressing functional and non-functional P2X7R, including B-lymphocytes from chronic lymphocytic leukemia (CLL) patients.
Main Results:
- The designed antibody selectively bound to non-functional P2X7 receptors.
- The epitope was inaccessible on functional surface P2X7 receptors but available on non-functional surface and intracellular receptors.
- B-lymphocytes from CLL patients expressing a non-functional P2X7R mutant also bound the antibody.
Conclusions:
- The antibody can distinguish between functional and non-functional P2X7 receptors.
- This antibody is a valuable tool for studying P2X7R conformation and localization, especially in disease states like CLL.
