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Identification of P2X2/P2X4/P2X6 heterotrimeric receptors using atomic force microscopy (AFM) imaging
Author(s) -
Antonio Ligia S.,
Stewart Andrew P.,
Varanda Wamberto A.,
Edwardson J. Michael
Publication year - 2014
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/j.febslet.2014.04.048
Subject(s) - atomic force microscopy , heterotrimeric g protein , identification (biology) , chemistry , receptor , nanotechnology , biophysics , materials science , biology , biochemistry , g protein , ecology
Seven P2X purinergic receptor subunits have been identified: P2X1–P2X7. The overlapping expression of P2X2, P2X4 and P2X6 subunits has been shown in different cell types, and functional analysis of P2X receptors in Leydig cells suggests that the three subunits might interact. Here, His 6 ‐tagged P2X2, HA‐tagged P2X4 and FLAG‐tagged P2X6 subunits were co‐expressed in tsA 201 cells. After sequential co‐immunoprecipitation using anti‐HA and anti‐FLAG beads, all three subunits were present, demonstrating their interaction. Atomic force microscopy (AFM) imaging revealed receptors that were specifically decorated by both an anti‐His 6 antibody and an anti‐HA Fab fragment, indicating the presence of a P2X2/4/6 heterotrimer. To our knowledge, this is the first report of a P2X receptor containing three different subunits.