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Figure 1. Overview of P2XR structure and close-up view of the orthosteric binding site. X-ray crystal structure of the ATP-bound human P2X3R (PDB accession no. 5SVK), with individual subunits color coded and black bars representing approximate boundaries of the cell membrane. Inset shows conserved hydrophilic side chains and backbone carbonyls suggested to interact with ATP (rP2X2R numbering), as indicated by dotted lines.
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Figure 2. The role of Lys69 side chain and backbone carbonyl in ATP recognition. (A) Closeup of the ATP-binding pocket with dotted lines indicating proposed interactions between ATP and Lys69. (B and C) Example recordings (B) and ATP-elicited concentrationâresponse data (C) for WT and mutant P2X2Rs in response to increasing concentrations of ATP. Bars in B, x, 15 s; y, µA; data in C displayed as mean ± SD (n = 10â15). (D) Schematic showing the reduced H-bond acceptor propensity as a result of amide-to-ester substitutions. (E) ATP-elicited concentrationâresponse data for WT and Val70Vah and Val96Vah mutants (the latter serving as a control for Vah incorporation). Inset represents the mean peak current amplitude (± SD) elicited by 3 mM ATP at oocytes injected with Val70TAG mRNA and uncharged (âVah) or charged (+Vah) tRNAs (n = 6â12; ****, P < 0.0001; Studentâs t test).
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Figure 3. Differential effects of charge-retaining and -neutralizing mutations at other conserved basic side chains lining the orthosteric binding site. (AâD) ATP-elicited concentrationâresponse data for WT, and Lys/Arg/Gln mutants at positions Lys71, Lys188, Lys308, and Arg290 (mean ± SD; n = 7â24).
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Figure 4. Role of side chain- and backbone-mediated H-bonds in CTP recognition. (A) Closeup of the ATP-binding pocket with dotted lines indicating proposed interactions between CTP and Lys69 (upper panel) and Thr184 (lower panel). (BâD) CTP-elicited concentrationâresponse data for WT and Val70Vah (B), WT, Thr184Ser, and Thr184Val (C), and WT, Ile185Val, and Ile185Vah (D; mean ± SD; n = 6â8). Insets show the mean peak current amplitude (±SD) elicited by 10 mM CTP at oocytes injected with Val70TAG (B) or Ile185TAG (D) mRNA and uncharged (âVah) or charged (+Vah) tRNAs, respectively (n = 6â7; ****, P < 0.0001; Studentâs t test).
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Figure 5. The role of Thr184 side chain and backbone carbonyl in ATP recognition. (A) Closeup of the ATP-binding pocket with dotted lines indicating proposed interactions between ATP and Thr184. (B and C) Example recordings (B) and ATP-elicited concentrationâresponse data (C) for WT and mutant P2X2Rs in response to increasing concentrations of ATP. Bars in B, x, 15 s; y, µA; data in C displayed as mean ± SD (n = 14â25). (D) ATP-elicited concentrationâresponse data for WT, Ile185Vah, Ile185Val, and Val96Vah mutants (the latter serving as a control for Vah incorporation). Inset represents the mean peak current amplitude (±SD) elicited by 10 mM of ATP in oocytes injected with Ile185TAG mRNA and uncharged (âVah) or charged (+Vah) tRNAs (n = 7â10; ****, P < 0.0001, Studentâs t test).
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Figure 6. Probing the effects of base modifications on ligand recognition. (A) Chemical structure of ATP (upper structure) and CTP (lower structure). (B) Peak current recorded in response to the highest tested concentration of base-modified nucleoside triphosphate analogues (chemical base structures shown in frames), compared with that elicited by 1 mM of ATP. Bars, x, 12 s; y, 2 µA. (C) ATP-elicited concentrationâresponse data for base-modified analogues, normalized to current elicited by 1 mM of ATP (mean ± SD; n = 5â15). Arrow indicates data for ITP. Note that the cost of 6-ClârTP, ITP, isoGTP, and GTP prevented us from using higher concentrations than those stated in the text (data for GTP could not be reliably fit).
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Figure 7. Probing the effects of sugar modification on ligand recognition. (A) Scheme of possible sugar pucker conformations: C3â²-endo ribose conformation with C3â²-hydroxyl group puckered in the endo face (upper panel) and C2â²-endo ribose conformation (C2â²-hydroxyl group puckered in the endo face; lower panel). (B) Peak current recorded in response to the highest tested concentration of sugar-modified nucleoside triphosphate analogues (chemical base structures shown in frames) compared with that elicited by 1 mM of ATP. Bars, x, 12 s; y, 2 µA. (C) ATP-elicited concentrationâresponse data for sugar-modified analogues, normalized to current elicited by 1 mM of ATP (mean ± SD; n = 4â9).
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Figure 8. UNA-ATP does not elicit macroscopic current responses. Left: Currents elicited by increasing concentrations of UNA-ATP (orange lines) compared with application of 1 mM ATP to the same cell. Bars, x, 10 s; y, 1 µA. Right: Chemical structure of UNA-ATP; note the lack of a covalent bond between the C2′ and C3′ ring positions, which makes the analogue highly flexible.
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Figure 9. Single channel current amplitude and flicker are independent of agonist structure. (A–G) Left panels: Example of all-point histograms fitted by a sum of Gaussian distributions; c represents the closed channel; O1, full-conductance open state; O2 and O3, full-conductance open state of two, three, or more channels open simultaneously; and S1, subconductance state. The histograms are obtained from the current analysis of the patch shown on the right (note leak current was not subtracted for histogram visualization). Right panels: Examples of single channel currents from excised outside-out patches expressing rP2X2-3T receptors. Channel openings (downward deflections) were elicited by application of different agonists for 10 s (indicated by light blue line). Bars, x, 1 s; y, 5 pA; insets, x, 0.1 s; y, 5 pA. (H) Mean ± SD of unitary current amplitudes (left) and SDopen (right; n = 4–20; two to five sweeps were averaged for each patch). Concentrations used: ATP (1 µM), CTP (10 µM), GTP (100 µM), UTP (10 µM), LNA-ATP (1 µM), C2′-F-dATP (10 µM), and Ara-ATP (1 µM).
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Figure 10. Cartoon summary of the key findings. Note that this study focused on conserved side chains only. Other factors are likely to also contribute to ligand recognition and discrimination.
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