Figure 1.
P2X7 mediates rapid ATP-dependent DAPI uptake.
(A) NRK cell lysates (30 µg/lane) stably expressing P2X1, P2X4, P2X7 or vector alone (mock) were resolved by SDS-PAGE, transferred to PVDF and membranes probed with the indicated antibodies. (B) NRK cells stably expressing P2X7 were treated with the indicated concentrations ATP for 15 min and DAPI uptake analyzed by flow cytometry. (C) NRK cells stably expressing either P2X7 or vector alone (mock) were incubated with the indicated concentrations of ATP for the indicated times, and DAPI uptake measured by flow cytometry. The geometric mean fluorescence intensity (GMFI) is shown. (D, E) NRK cells stably expressing P2X1, P2X4, P2X7 or vector alone (mock) were treated with ATP for 15 min and uptake of DAPI (D), Etd, YoPro1, and PI (E) measured by flow cytometry. Fold fluorescent increase was determined by dividing the GMFI of cells in the presence of ATP by the GMFI in the same cells without addition of ATP (GMFIATP/GMFIno-ATP). (F) NRK cells stably expressing P2X1, P2X4, P2X7 or vector alone (mock) were treated with ATP for 15 min in the absence (NT) or presence of 100 µM A74003 (A74) or 10 µM A438079 (A43) and uptake of DAPI was measured by flow cytometry. The data shown are representative of more than three independent experiments and two tailed Student's t-tests (unpaired) compared to mock yielded p values <0.05 (*), <0.01 (**), or <0.001(***).
Figure 2.
The C-termini of P2X1 and P2X4 do not substitute for the C-terminus of P2X7.
(A) Schematic representation of chimeric constructs generated by swapping the C-termini of P2X receptors. P2X7C1 is P2X7 where the C-terminus swapped for P2X1, while P2X7C4 is P2X7 where the C-terminus has been swapped for P2X4. (B) HEK293 cell lysates (30 µg/lane) stably expressing vector alone (mock), P2X1, P2X4, P2X7, P2X7C1 or P2X7C4 were resolved by SDS-PAGE, transferred to PVDF and membranes probed with the indicated antibodies. (C, D) HEK293 cells stably expressing vector alone (mock), P2X7, P2X7C1 or P2X7C4 were either (C) surface stained with the anti-P2X7 mAb HANO43 or (D) treated with 3 mM ATP for 30 min in the presence of DAPI, Etd and PI and analyzed by flow cytometry. The data shown are representative of more than three independent experiments and two tailed Student's t-tests (unpaired) compared to wild type P2X7 yielded p values <0.001 (###), while comparisons to mock yielded p values <0.01 (**), or <0.001 (***).
Figure 3.
The TM2 of P2X7 confers surface expression and channel activity.
(A) Schematic representation of the chimeric constructs used. All constructs are P2X7 with either the entire TM2 replaced with that of P2X1 (TM21), that of P2X4 (TM24) or the first or second half of TM2 replaced with that of P2X1 (TM21N and TM21C, respectively). A sequence alignment of P2X1, P2X4 and P2X7 are shown with *, :and. indicating identical, conserved and similar residues, respectively. (B, C) HEK293 cells stably expressing the vector alone (mock), P2X7 or the chimeric constructs were either (B) surface stained with anti-P2X7 mAb HANO43 or (C) treated with 3 mM ATP for 30 min in the presence of DAPI, Etd, YoPro1 and PI and analyzed by flow cytometry. The data shown are representative of more than three independent experiments and two tailed Student's t-tests (unpaired) compared to mock yielded p values of <0.05 (*), <0.01 (**), or <0.001(***) while comparisons to wild type P2X7 yielded p values <0.05 (#).
Figure 4.
Point mutations of P2X7 TM2 domain alter dye permeability.
The indicated cell lines were transduced with either vector alone (mock), wild type P2X7 or P2X7 expressing the indicated mutations and either (A) surface stained with anti-P2X7 mAb HANO43, or treated with 3 mM ATP for 30 min in the presence of (B) DAPI, (C) PI, (D) Etd, or (E) YoPro1 and analyzed by flow cytometry. GMFI was determined for live transduced cells. The data shown are representative of more than three independent experiments and two tailed Student's t-tests (unpaired) compared to wild type P2X7 yielded p values <0.05 (#), <0.01 (##), or <0.001(##), and compared to mock yielded p values of <0.05 (*), <0.01 (**) or <0.001(***).
Figure 5.
Point mutations of P2X7 TM2 alter cell viability.
NRK cells stably transduced with the indicated constructs were imaged at 2 frames/minute for 45 minutes in the presence of DAPI (blue) and PI (red) following the addition of 3 mM ATP. Arrowheads indicate blebs. Images shown are representative of at least 10 fields of cells from at least two independent experiments. Scale bar = 20 µm.