Molecular mechanisms of human P2X3 receptor channel activation and modulation by divalent cation bound ATP
- National Institute of Neurological Disorders and Stroke, National Institutes of Health, United States
- Fudan University, China
- National Heart, Lung and Blood Institute, National Institutes of Health, United States
Figures
Figure 1 with 1 supplement
X-ray structures of hP2X3 receptors with divalent ions bound..
(A) Overall structure of hP2X3 MFCslow with ATP and Mg2+ bound (PDB ID 6AH5). Agonist binding pockets are formed at each of the three interfaces between adjacent subunits (blue, orange, gray). (B) …
Figure 1—figure supplement 1
Conformational changes associated with ATP binding in the presence and absence of Mg2+.
(A) Overall structure of hP2X3 MFCslow with ATP and Mg2+ bound (PDB ID 6AH5). The cytoplasmic domain is omitted for clarity. (B) Comparison of the structures of hP2X3 MFCslow bound to ATP only (PDB …
Figure 2 with 3 supplements
Computational study of the mode of ATP and Mg2+ recognition by P2X3.
(A) Occurrence of different modes of Mg2+-ATP and Ca2+-ATP interaction in non-redundant high-resolution crystal structures available in the Protein Data Bank. Each mode is indicated by the phosphate …
Figure 2—figure supplement 1
Simulated ATP dissociation from P2X3 in the presence and in the absence of Mg2+.
(A) Solute-tempering simulations of the P2X3-ATP-Mg2+ complex. The plots quantify the distance between ATP and either its binding site in P2X3 or Mg2+, as a function of simulation time. Each plot …
Figure 2—figure supplement 2
Change in the mode of Mg2+ binding to P2X3 following ATP dissociation.
(A–D) Distance between Mg2+ and the carboxyl groups of D158, E156, E111 and E109, in the ATP-bound state (black) and following ATP dissociation (red). The data are shown as histograms calculated …
Figure 2—figure supplement 3
Cooperativity between Mg2+ and P2X3 slows down ATP dissociation.
Histograms of the ATP-receptor distance, for either P2X3 ATP (black) or P2X3-ATP-Mg2+ (red), calculated from all the solute-tempering simulations carried out in each case. The peaks at ~ 10 Å …
Figure 3 with 3 supplements
Mg2+ and Ca2+ slow deactivation of hP2X3 receptors.
(A) Superimposed fast-desensitizing hP2X3 wt (orange) and slow-desensitizing hP2X3 slow (black) macroscopic receptor channel currents activated by extracellular application of a saturating …
Figure 3—figure supplement 1
Mg2+ slows deactivation of hP2X3 slow receptor channel currents at acid pH.
Macroscopic hP2X3 slow receptor channel currents activated by 100 μM ATP in the presence of EDTA (10 mM; left) or MgCl2 (right) at pH 5. Similar results were obtained from four different cells and …
Figure 3—figure supplement 2
Mg2+ increases the apparent affinity for activation of hP2X3 slow receptor channels by ATP.
Concentration-response relations for ATP activation of hP2X3 slow receptor channels in the absence and presence of 0.5 mM MgCl2. In each case, currents were normalized to the maximal current in the …
Figure 3—figure supplement 3
Effects of Mg2+ on single mutations in the lower or upper modes within the divalent cation binding chamber.
(A) 100 μM ATP activated macroscopic ATP-activated currents for E156A hP2X3 slow recorded in the presence of 10 mM EDTA (10 mM; left) or MgCl2 (right). (B) Deactivation time constants obtained by …
Figure 4 with 1 supplement
Influence of Mg2+ and Ca2+ on desensitization of hP2X3 receptor channels.
(A) Macroscopic wt hP2X3 receptor channel currents activated by a saturating concentration of ATP (300 μM) under different conditions. (B) Summary of desensitization time constants obtained by …
Figure 4—figure supplement 1| A
Triple mutation in the Mg2+ binding chamber eliminates inhibition caused by Mg2+.
A) Mg2+ can inhibit wt hP2X3 receptor channels when applied during repetitive activation of the channel by ATP. ATP was applied every 2 min. In four cells, Mg2+ inhibited current activation by 93 ± …
Figure 5 with 3 supplements
The divalent cation binding chamber is critical for Mg2+-ATP activation.
(A) Concentration-response relations for ATP activation of wt hP2X3, E156A, D158A and the E156A/D158A double mutant in divalent-free solution. Each data point is the mean of four measurements (± S.E.…
Figure 5—figure supplement 1
The divalent cation binding chamber is critical for Mg-ATP activation of hP2X3 slow.
A) Concentration-response relations for ATP activation of hP2X3 slow and with the E109A/E156A/D158A triple mutant. Each data point is an average of four measurements. Smooth lines are fits of the …
Figure 5—figure supplement 2
The divalent cation binding chamber is critical for Mg-ATP activation of hP2X3.
A) Concentration-response relations for ATP activation of hP2X3 E109A, E109A/E156A, E109A/D158A and the E109A/E156A/D158A triple mutant in divalent-free solution. Each data point is an mean of four …
Figure 5—figure supplement 3
Mg2+-ATP can bind but not activate hP2X3 slow receptors when the acidic chamber is mutated.
A) hP2X3 slow currents activated by 0.4 μM ATP4− and two different concentrations of Mg2+-ATP. EDTA solution was applied to the cell between the ATP application. Resurgent current marked with an …
Figure 6
Schematic illustration of the mechanism of Mg2+-ATP/Ca2+-ATP activation of P2X3 receptor channels.
Mg2+ or Ca2+ binds to hP2X3 receptors in the upper mode under physiological conditions. When ATP divalent cation complex (M-ATP) enters the nucleotide binding pocket, a divalent cation will rapidly …
Videos
Video 1
Close-up of the binding site for ATP-Mg2+ in P2X3, in the ATP-bound state, in a representative simulation snapshot (same as Figure 2E and Figure 2—figure supplement 2E).
Residue numbers are indicated in Figure 2—figure supplement 3.
Video 2
Close-up of the binding site for ATP-Mg2+ in P2X3, in the ATP-dissociated state, in a representative simulation snapshot (same as Figure 2F and Figure 2—figure supplement 2F).
Residue numbers are indicated in Figure 2—figure supplement 2F.
Additional files
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Supplementary file 1
Data collection, phasing and refinement statistics.
- https://doi.org/10.7554/eLife.47060.021
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Transparent reporting form
- https://doi.org/10.7554/eLife.47060.022
