Figure 1.
Expression and Purification of P2X4R
(A) Overexpression of P2X4R in human 1321N1 astrocytoma cells. In Western blotting, P2X4R protein was only detected in cells transfected with the P2X4R gene.
(B) Purification of P2X4R protein. The molecular weight of the purified P2X4R protein band was detected at about 150 kDa in native-PAGE (left) and 50 kDa in SDS-PAGE (right). After electrophoresis, the gels were stained with silver staining.
Figure 2.
AFM Observations of P2X4Rs on PDL-Coated Mica
(A) P2X4Rs attach stably to PDL-coated mica. P2X4Rs on PDL-coated mica exhibited stable attachment and the majority did not shift position during AFM observation. Scale bar, 10 nm.
(B) AFM images of P2X4Rs at (i) low resolution, (ii) high resolution, and (iii) single particle level. (i) At low resolution, the P2X4Rs were relatively homogenous. Slight differences were observed after ATP treatment (1 mM, 30 min), but they are not very clear at this resolution. Scale bar, 20 nm. (ii, iii) At high resolution and at a single particle level, there were significant structural differences between the control P2X4Rs and the P2X4Rs after ATP addition. Each single particle was selected based on our criteria (Materials and Methods, Figure S1C). In the control, the P2X4Rs were nearly circular, ellipsoid, or triangular with obtuse angles. After ATP addition, the P2X4Rs had a tripartite morphology. PDL-polymers were also observed (arrows). Scale bar, 10 nm.
(C) Percentage of trimeric P2X4R was significantly increased after ATP (1 mM, 30 min). ***, p < 0.001.
(D) Averaged images of P2X4Rs. (i) Nonsymmetrized averaging of P2X4Rs in the control (left) and after ATP addition (right). (ii) Symmetrized averaging of P2X4Rs in the control (left) and after ATP addition (right). 3-fold symmetrized images were obtained after symmetrized averaging. Scale bar, 5 nm.
(E) Three lobes are individual subunits in one P2X4R trimer. The distance between lobes was significantly less than that between trimers. ***, p < 0.001.
(F) P2X4R trimer shifts position as one unit. (i) When a P2X4R trimer moves during AFM, the three lobes were not dissociated but moved as a trimer. Scale bar, 20 nm. (ii) Enlarged images of single P2X4R trimer in a rectangle at 5 s, nonsymmetrized and symmetrized averaging images of ten particles in the same scan area. Scale bar, 10 nm. AFM observation was performed in AFM imaging buffer A.
Figure 3.
Fast-Scanning AFM Observations of the ATP-Induced Structural Changes in the P2X4Rs
Time-lapse imaging of ATP-induced structural changes of P2X4R. Before activation, P2X4R was in circular shape and exhibited some fluctuation (−2.5 s to ≈0.0 s). Caged ATP (200 μM) was uncaged at 0 s. After uncaging, the P2X4R structure changed to a trimer structure within 0.5 s. Then, P2X4R exhibited a further structural change and adopted a pore dilation-like conformation. Ten P2X4R particles were averaged for each frame. Scale bar, 10 nm. AFM observation was performed in AFM imaging buffer B.
Figure 4.
AFM Observation and Functional Analysis of Membrane-Inserted P2X4Rs
(A) Diagrams illustrating domain structures in (i) one P2XR subunit and (ii) P2XR trimer. The ECD of a P2XR subunit is assumed to have a six-stranded antiparallel β-pleated sheet structure. Three characteristic ECDs are assumed to exist in a P2XR trimer.
(B) Hypothetical view of P2X4Rs reconstituted in a lipid bilayer. If P2X4R were inserted upwardly, ATP-induced structural changes and the Ca2+ flow of P2X4R would be observed.
(C) AFM images of membrane-inserted P2X4Rs. In the control, the P2X4Rs were homogenous and largely circular, ellipsoid, or triangular with obtuse angles (upper panels). After ATP addition (200 μM, 1 min), the P2X4Rs exhibited tripartite morphologies (lower panels).
(D) Averaged images of membrane-inserted P2X4Rs. (i) Nonsymmetrized averaging of P2X4Rs in the control (left) and after ATP addition (right). (ii) Symmetrized averaging of P2X4Rs in the control (left) and after ATP addition (right). Scale bar, 10 nm. AFM observation was performed in AFM imaging buffer B.
(E) Ca2+ and dye-uptake imaging of P2X4Rs. (i) Green fluorescence derived from fluo3/Ca2+ after ATP (100 μM, at 0 s) addition was detected only in the hole made in the plastic plate. (ii, iii) Simultaneous recording of ATP-induce Ca2+ permeability and EtBr uptake of P2X4R. Under 2 mM Ca2+ conditions, the green fluorescence intensity immediately increased after ATP addition (see also Video S2). There was no significant increase in red fluorescence (DNA/EtBr) intensity. Each trace is the mean ± SEM of five independent experiments. Scale bar, 100 μm.
Figure 5.
Pore Dilation-Like Structural Changes Are Related to the Dye-Uptake of P2X4R
(A) Pore dilation-like structural change of P2X4R on mica without coating. P2X4R was a trimer immediately after activation (0.5 s, 200 μM ATP, middle) and had a pore dilation-like structure 5 s after ATP binding (right).
(B) Pore dilation-like structural change of P2X4R on PDL-coated mica. After 30 min of ATP (1 mM) treatment, P2X4R exhibited a pore dilation-like structure (right) but it had a tripartite topology without a pore dilation-like structure after 15 min of ATP treatment (middle). The AFM observation was performed in AFM imaging buffer B. Scale bar, 10 nm.
(C) With 0 mM Ca2+, the red fluorescence (DNA/EtBr) intensity gradually increased after ATP addition (see also Video S3). Each trace is the mean ± SEM of five independent experiments. Scale bar, 100 μm.
(D) AFM images of activated P2X4R (5 s uncaging) under (i) 0 mM and (ii) 2 mM Ca2+ conditions. With Ca2+, P2X4R did not exhibit a pore dilation-like structure. Scale bar, 10 nm.
Figure 6.
Model of Structural Changes of ECDs in P2X4R and Corresponding Pore States Based on AFM and Functional Analysis Data
In the control, the ECDs are close to each other and so individual ECDs (or subunits) cannot be observed with AFM. Immediately after ATP binding, three ECDs are disengaged and the TMD pore is Ca2+ permeable.
(A) In the absence of Ca2+, the distances between the ECDs are further increased and the TMD pore becomes EtBr permeable (right).
(B) With 2 mM Ca2+, P2X4R does not exhibit any further structural changes after the disengagement of the ECDs and exhibits no permeability changes.