Fryatt AG , Dayl S , Cullis PM , Schmid R , Evans RJ .

PG/11/64/28772 British Heart Foundation

	Figure 1. MD simulations of ATP binding at WT subunits and one incorporating the K68A binding site mutation.(A–C) 2-D rmsd plots of ATP in P2X1R wild type and P2X1R (K68A) binding sites. 1000 Snapshots were extracted from the 100 ns molecular dynamics trajectories (one snapshot every 10 ps) and used to calculate all-against-all rmsd values for ATP. The rmsd value (in Å) for each calculation is indicated by the colour gradient. ATP bound in a wild type P2X1R binding site (left and centre panel) shows very little variation. In contrast ATP bound to a P2X1R K68A binding site (right panel) shows generally more conformational variability. In the trajectory visualised a conformational transition appears after around 80 ns. (D) Ribose pucker phase angle frequency for ATP in P2X1R wild type and K68A binding sites. ATP in the wild type site (black) is almost exclusively in the C2′ endo conformation while in the K68A binding site (orange) C′3 endo conformations are also present (peak at ~270 degree). Values are reported according to the Cremer convention. (E) Distance between the gamma phosphate phosphor atom and the C′2 of the ribose. Plotted are the relative frequencies of γ-P – C′2 distances for ATP in a wild type P2X1R binding site (black) and ATP bound to a P2X1R K68A binding site (orange). ATP in the wild type binding site remains in the U shaped conformation, while in K68A binding site additional conformations are accessible indicated by the peaks at ~6.5 Å and ~8 Å. (F) Binding conformations for ATP are shown for two snapshots before and after the conformational transition in an overlay corresponding to trajectories at 0.8 ns and 6.8 ns in the right panel of (C).
	Figure 2. Fluorescence changes can be rescued in ATP insensitive subunits by co-expression of WT subunits.(A) Example VCF traces from K190C showing rapidly desensitizing current and decrease in fluorescence with ATP (100 μM). Insert shows expanded timescale during initial ATP application, with fluorescence change normalized to peak current. (B) Example of VCF traces from oocytes expressing wild type hP2X1R (upper panel) and double mutant P2X1R K190C-K68A (lower panel). Both show no change on fluorescence with 100 μM ATP application (30 s indicated by bar) and no current was evoked from K190C-K68A. (C) Schematic showing a heteromeric receptor with two WT and one K190C-K68A mutant subunit (grey), inter-subunit binding sites are shown in green and labelling of the mutant subunit by cysteine reactive MTS-TAMRA (in red). (D) The ATP evoked current and fluorescence changes were recovered by co-expressing K190C-K68A with WT P2X1R, although the fluorescence change was significantly smaller than those seen for K190C. Insert shows expanded timescale during initial ATP application, with fluorescence change normalized to peak current. (E). There was no difference in the %Fmax at Imax between K190C and K190C-K68A/WT (F). (G) The recovery of the fluorescence change following agonist wash out was significantly faster for K190C-K68A/WT compared to K190C (traces normalized to peak decrease in fluorescence), with time from 90% to 50% fluorescence recovery of 0.7 ± 0.3 s and 38.8 ± 4.7 s respectively. Data are shown as mean ± SEM, n ≥ 6, ****p < 0.0001.
	Figure 3. The hydroxyl group at the 2′, but not 3′, position on the ribose ring of ATP is important for agonist action at P2X1Rs.(A) Agonist evoked currents (3 s application indicated by bar) at P2X1Rs. Representative traces from a single oocyte are shown for 3′-deoxy ATP and 2′-deoxy ATP (a response to a maximal concentration of ATP 100 μM from the same oocyte is shown for comparison). (B) Concentration response curves for agonist evoked currents, ATP (open circles), 3′-deoxy ATP (purple diamonds) and 2′-deoxy ATP (green squares). Due to cost a maximal concentration of 10 μM 3′-deoxy ATP was used. Data are shown as mean ± SEM, n = 4.
	Figure 4. VCF reports changes in fluorescence induced by deoxyadenosine 5′-triphosphates.(A) Example of VCF traces from oocytes expressing P2X1R K190C (10 μM ATP indicated by bar, black trace = current recording, red trace = fluorescence). Inward currents and decreases in fluorescence were also seen with the application of (B) 2′-deoxy ATP and (C) 3′-deoxy ATP (both 10 μM). The fluorescence changes were significantly smaller than those seen with ATP while 2′-deoxy ATP evoked a significantly reduced current (D) The proportion of the fluorescence change at peak current (%Fmax at Imax, (E)) was also significantly greater that ATP with the application of 2′-deoxyATP. (F) The recovery of the fluorescence change following agonist wash out was significantly faster for both 2′-dATP and 3′-deoxy ATP compared to ATP, with time from 90% to 50% fluorescence recovery of 1.0 ± 0.5 s, 2.5 ± 0.9 s and 30.4 ± 2.0s respectively. Data are shown as mean ± SEM, n ≥ 5, **p < 0.01, ****p < 0.0001.
	Figure 5. Conformations of ATP, 3′-deoxy ATP and 2′-deoxy ATP in the P2X1R binding site.(A,B) Nucleotide conformations in the MD simulations extracted from a total of six traces (three binding sites, two replicates) are monitored via their ribose pucker phase angle (A) and the distance between the gamma phosphate phosphor atom and the C′2 of the ribose (B) Plotted are the relative frequencies of the respective angle (3.6 degrees window) and distance (0.1 à window) for ATP (black), 3′deoxy-ATP (purple) and 2′-deoxy-ATP (green). The second peak in the ribose pucker phase angle distribution and the broad shoulder in the distribution of 2′-deoxy ATP γ–phosphate C′2-ribose distances indicates at least two conformational states. (C) Overlay of ATP (black), 3′-deoxy-ATP (purple), and the non U shaped 2′-deoxy ATP (green) conformation, the P2X1R is shown as a cartoon, the nucleotides are shown as stick models.
	Figure 6. VCF profile is ATP analogue dependent.(A) Example of VCF traces from oocytes expressing P2X1R K190C (100 μM ATP indicated by bar). Inward currents and decreases in fluorescence were seen with the application of (B) Ap5A and (C) αβmeATP (100 μM). The fluorescence changes induced by αβmeATP were significantly smaller than those seen with ATP while both Ap5A and αβmeATP generated significantly reduced currents (D). The proportion of the fluorescence change at peak current (%Fmax at Imax, (E)) was significantly greater with the application of Ap5A while significantly reduced with αβmeATP application. (F) The recovery of the fluorescence change following agonist wash out was significantly faster for both Ap5A and αβmeATP compared to ATP, with time from 90% to 50% fluorescence recovery of 1.4 ± 0.4 s, 6.5 ± 1.4 s and 38.8 ± 4.7 s respectively. Data are shown as mean ± SEM, n ≥ 4, *p < 0.05, **p < 0.01, ****p < 0.0001.
	Figure 7. Differential recovery of fluorescence and channel activity from activation by ATP analogues.(A) Example of VCF traces from oocytes expressing P2X1R K190C to two 30 second ATP applications (100 μM indicated by bar) with 30 second washout between. A sustained fluorescence decrease was observed during the first agonist application that partially recovered with washout. The subsequent application induced a smaller peak current with a decrease in fluorescence to a similar level as previous. (B) Replacing ATP with Ap5A (100 μM) in the above protocol generated an attenuated current during the second application with a similar decrease in fluorescence as the initial application. The response seen with αβmeATP (C) was similar to that seen with ATP. Measuring the average %Fmax at Imax during the first and second applications (D) showed no change in the proportion of the fluorescence change with application of Ap5A, while both ATP and αβmeATP showed more rapid change in fluorescence with the subsequent agonist trial. (E) Graph showing the average evoked current during the second agonist application as a percentage of the first, with Ap5A showing a significantly reduced current compared to ATP or αβmeATP. Data are shown as mean ± SEM, n ≥ 3, ****p < 0.0001.

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