Specific detection of non-functional human P2X(7) receptors in HEK293 cells and B-lymphocytes.
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.
Barden
J A
JA
Department of Anatomy and Histology, Anderson Stuart Bldg, F13, The University of Sydney, Sydney 2006, NSW, Australia. julian@anatomy@usyd.edu.au
Sluyter
R
R
Gu
B J
BJ
Wiley
J S
JS
D000255:Adenosine Triphosphate / Q000494:pharmacology; D001402:B-Lymphocytes / Q000378:metabolism*; D002460:Cell Line; D004996:Ethidium / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000196:embryology / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D018048:Receptors, Purinergic P2 / Q000235:genetics / Q000378:metabolism*; D058486:Receptors, Purinergic P2X7
C546468:P2RX7 protein, human; D018048:Receptors, Purinergic P2; D058486:Receptors, Purinergic P2X7; D000255:Adenosine Triphosphate; D004996:Ethidium