Allsopp RC et al. (2013), P2X receptor chimeras highlight roles of the am...

XB-ART-48126

J Biol Chem 2013 Jul 19;28829:21412-21421. doi: 10.1074/jbc.M113.464651.

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P2X receptor chimeras highlight roles of the amino terminus to partial agonist efficacy, the carboxyl terminus to recovery from desensitization, and independent regulation of channel transitions.

Allsopp RC , Farmer LK , Fryatt AG , Evans RJ .

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P2X receptor subtypes can be distinguished by their sensitivity to ATP analogues and selective antagonists. We have used chimeras between human P2X1 and P2X2 receptors to address the contribution of the extracellular ligand binding loop, transmembrane segments (TM1 and TM2), and intracellular amino and carboxyl termini to the action of partial agonists (higher potency and efficacy of BzATP and Ap5A at P2X1 receptors) and antagonists. Sensitivity to the antagonists NF449, suramin, and PPADS was conferred by the nature of the extracellular loop (e.g. nanomolar for NF449 at P2X1 and P2X2-1EXT and micromolar at P2X2 and P2X1-2EXT). In contrast, the effectiveness of partial agonists was similar to P2X1 levels for both of the loop transfers, suggesting that interactions with the rest of the receptor played an important role. Swapping TM2 had reciprocal effects on partial agonist efficacy. However, TM1 swaps increased partial agonist efficacy at both chimeras, and this was similar for swaps of both TM1 and 2. Changing the amino terminus had no effect on agonist potency but increased partial agonist efficacy at P2X2-1N and decreased it at P2X1-2N chimeras, demonstrating that potency and efficacy can be independently regulated. Chimeras and point mutations also identified residues in the carboxyl terminus that regulated recovery from channel desensitization. These results show that interactions among the intracellular, transmembrane, and extracellular portions of the receptor regulate channel properties and suggest that transitions to channel opening, the behavior of the open channel, and recovery from the desensitized state can be controlled independently.

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Species referenced: Xenopus laevis
Genes referenced: p2rx1 p2rx2

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	FIGURE 1. Partial agonist sensitivity at P2X1 and P2X2 extracellular loop chimeras. a, schematic representation of the P2X1 receptor (black), P2X2 receptor (gray), and receptor chimeras P2X1-2EXT and P2X2-1EXT generated by replacement of the entire extracellular loop. b, representative currents mediated by 3-s application (indicated by bar) of 100 μm ATP (black), α,β-meATP (red), AP5A (green), or BzATP (blue) to P2X1, P2X2, and chimeric receptors expressed in Xenopus oocytes. c, histogram summary showing the average peak current mediated by partial agonist application plotted as a percentage of maximum 100 μm ATP response. d, concentration-response curves (normalized to maximum ATP response) for ATP, α,β-meATP, BzATP, and AP5A at the P2X1, P2X1-2EXT, P2X2-1EXT, and P2X2 receptors. Dotted line represents concentration-response curves published previously (19, 41). , p < 0.05; **, p < 0.001 (n = 4–8). Error bars, S.E.
	FIGURE 2. Antagonist sensitivity tracks with the extracellular loop. a, schematic showing P2X1 (black), P2X2 (gray), and chimeras P2X1-2EXT and P2X2-1EXT. Representative current traces mediated by a 3-s application (indicated by bar) of an EC90 concentration of ATP (open circles) and ATP plus suramin (filled circles) were recorded from Xenopus oocytes. b, histogram summary showing percentage of control current when suramin was presuperfused and co-applied with ATP. Error bars, S.E. c, representative current traces in response to an EC90 concentration of ATP (open triangles) and ATP plus NF449 (filled triangles). d, inhibition curves showing the effects of NF449 on WT and chimeric receptors. , p < 0.05; *, p < 0.01 (n = 3–6).
	FIGURE 3. PPADS sensitivity tracked with the extracellular loop. a, schematic showing P2X1 (black) and P2X2 (gray) and chimeras P2X1-2EXT and P2X2-1EXT. Representative current traces mediated by a 3-s application (indicated by bar) of an EC90 concentration of ATP (open squares) and ATP plus PPADS (filled squares) were recorded from Xenopus oocytes. b, histogram showing percentage current change when PPADS was co-applied with ATP. , p < 0.05; , p < 0.01; **, p < 0.001 (n = 3–5). Error bars, S.E.
	FIGURE 4. Partial agonist sensitivity at P2X1 and P2X2 transmembrane domain chimeras. a, c, and e, schematic representations of TM reciprocal chimeras and representative currents mediated by 3-s application (indicated by bar) of 100 μm ATP (black open circles), AP5A (gray hatched circles), or BzATP (black filled circles). a, TM1 chimeras. c, TM2 chimeras. e, TM1 + 2 chimeras. b, d, and f, histogram summaries showing the average peak currents mediated by partial agonist application plotted as a percentage of maximum 100 μm ATP response. , p < 0.05; , p < 0.01; **, p < 0.001 (n = 4–8). Error bars, S.E.
	FIGURE 5. Partial agonist sensitivity at P2X1 and P2X2 intracellular domain chimeras. a, c, e, and g, schematic representations of amino- and carboxyl-terminal reciprocal chimeras and representative currents mediated by 3-s application (indicated by bar) of 100 μm ATP (black open circles), AP5A (gray hatched circles), or BzATP (black filled circles). a, amino-terminal chimeras. c, amino-terminal chimera subdivisions. e, carboxyl-terminal chimers. g, amino- and carboxyl-terminal chimeras. b, d, f, and h, histogram summaries showing the average peak current mediated by partial agonist application plotted as a percentage of maximum 100 μm ATP response. , p < 0.05; , p < 0.01; **, p < 0.001 (n = 4–8). Error bars, S.E.
	FIGURE 6. Carboxyl terminus contribution to recovery from desensitization. a, representative currents mediated by 3-s application (indicated by bar) of 100 μm ATP (black traces) and subsequent repeat application (time interval as indicated) resulting in approximately 50% recovery from desensitization compared with stable, reproducible, maximum ATP response. b, amino acid sequence line up showing the initial residues of the carboxyl-terminal section of P2X1 (top) and P2X2 (bottom). Mutations are based on the nonconserved amino acid residues within the Cβ region (shown in gray). c, histogram summary showing the time required in between successive, maximal, reproducible recordings (3-s 100 μm ATP applications) to achieve 50% recovery from desensitization. d, scatter plot showing the time to 50% recovery from desensitization after 3-s 100 μm ATP application versus time to 50% decay as measured over a 3-s or 20-s continuous 100 μm ATP application. , p < 0.05; **, p < 0.001 (n = 5–17). Error bars, S.E.
	FIGURE 7. Efficacy of partial agonists (compared with ATP) for WT and chimeric receptors shows no correlation with the time course of ATP-mediated currents. Efficacy of partial agonists AP5A (a) and BzATP (b) as a percentage of maximum ATP evoked response (100 μm ATP) versus the receptor time course to desensitization is shown. Time course is expressed as the time to 50% desensitization over 3-s ATP application, or percentage current remaining at the end of prolonged 20-s ATP application, for fast/slow desensitizing receptors, respectively (n = 5–17).
	FIGURE 8. Summary of effects of chimeras on partial agonist potency and efficacy. Replacement of different regions of the P2X1 receptor with those from the P2X2 receptor, and vice versa modified partial agonist action. Residues in the extracellular loop determine antagonist sensitivity. The TM regions are involved in control of both agonist sensitivity and efficacy. The amino terminus can regulate agonist efficacy independently of sensitivity, and the carboxyl terminus is involved in recovery of the receptor from the desensitized state.

References [+] :

Allsopp, Cysteine scanning mutagenesis (residues Glu52-Gly96) of the human P2X1 receptor for ATP: mapping agonist binding and channel gating. 2011, Pubmed, Xenbase

	FIGURE 1. Partial agonist sensitivity at P2X1 and P2X2 extracellular loop chimeras. a, schematic representation of the P2X1 receptor (black), P2X2 receptor (gray), and receptor chimeras P2X1-2EXT and P2X2-1EXT generated by replacement of the entire extracellular loop. b, representative currents mediated by 3-s application (indicated by bar) of 100 μm ATP (black), α,β-meATP (red), AP5A (green), or BzATP (blue) to P2X1, P2X2, and chimeric receptors expressed in Xenopus oocytes. c, histogram summary showing the average peak current mediated by partial agonist application plotted as a percentage of maximum 100 μm ATP response. d, concentration-response curves (normalized to maximum ATP response) for ATP, α,β-meATP, BzATP, and AP5A at the P2X1, P2X1-2EXT, P2X2-1EXT, and P2X2 receptors. Dotted line represents concentration-response curves published previously (19, 41). , p < 0.05; **, p < 0.001 (n = 4–8). Error bars, S.E.
	FIGURE 2. Antagonist sensitivity tracks with the extracellular loop. a, schematic showing P2X1 (black), P2X2 (gray), and chimeras P2X1-2EXT and P2X2-1EXT. Representative current traces mediated by a 3-s application (indicated by bar) of an EC90 concentration of ATP (open circles) and ATP plus suramin (filled circles) were recorded from Xenopus oocytes. b, histogram summary showing percentage of control current when suramin was presuperfused and co-applied with ATP. Error bars, S.E. c, representative current traces in response to an EC90 concentration of ATP (open triangles) and ATP plus NF449 (filled triangles). d, inhibition curves showing the effects of NF449 on WT and chimeric receptors. , p < 0.05; *, p < 0.01 (n = 3–6).
	FIGURE 3. PPADS sensitivity tracked with the extracellular loop. a, schematic showing P2X1 (black) and P2X2 (gray) and chimeras P2X1-2EXT and P2X2-1EXT. Representative current traces mediated by a 3-s application (indicated by bar) of an EC90 concentration of ATP (open squares) and ATP plus PPADS (filled squares) were recorded from Xenopus oocytes. b, histogram showing percentage current change when PPADS was co-applied with ATP. , p < 0.05; , p < 0.01; **, p < 0.001 (n = 3–5). Error bars, S.E.
	FIGURE 4. Partial agonist sensitivity at P2X1 and P2X2 transmembrane domain chimeras. a, c, and e, schematic representations of TM reciprocal chimeras and representative currents mediated by 3-s application (indicated by bar) of 100 μm ATP (black open circles), AP5A (gray hatched circles), or BzATP (black filled circles). a, TM1 chimeras. c, TM2 chimeras. e, TM1 + 2 chimeras. b, d, and f, histogram summaries showing the average peak currents mediated by partial agonist application plotted as a percentage of maximum 100 μm ATP response. , p < 0.05; , p < 0.01; **, p < 0.001 (n = 4–8). Error bars, S.E.
	FIGURE 5. Partial agonist sensitivity at P2X1 and P2X2 intracellular domain chimeras. a, c, e, and g, schematic representations of amino- and carboxyl-terminal reciprocal chimeras and representative currents mediated by 3-s application (indicated by bar) of 100 μm ATP (black open circles), AP5A (gray hatched circles), or BzATP (black filled circles). a, amino-terminal chimeras. c, amino-terminal chimera subdivisions. e, carboxyl-terminal chimers. g, amino- and carboxyl-terminal chimeras. b, d, f, and h, histogram summaries showing the average peak current mediated by partial agonist application plotted as a percentage of maximum 100 μm ATP response. , p < 0.05; , p < 0.01; **, p < 0.001 (n = 4–8). Error bars, S.E.
	FIGURE 6. Carboxyl terminus contribution to recovery from desensitization. a, representative currents mediated by 3-s application (indicated by bar) of 100 μm ATP (black traces) and subsequent repeat application (time interval as indicated) resulting in approximately 50% recovery from desensitization compared with stable, reproducible, maximum ATP response. b, amino acid sequence line up showing the initial residues of the carboxyl-terminal section of P2X1 (top) and P2X2 (bottom). Mutations are based on the nonconserved amino acid residues within the Cβ region (shown in gray). c, histogram summary showing the time required in between successive, maximal, reproducible recordings (3-s 100 μm ATP applications) to achieve 50% recovery from desensitization. d, scatter plot showing the time to 50% recovery from desensitization after 3-s 100 μm ATP application versus time to 50% decay as measured over a 3-s or 20-s continuous 100 μm ATP application. , p < 0.05; **, p < 0.001 (n = 5–17). Error bars, S.E.
	FIGURE 7. Efficacy of partial agonists (compared with ATP) for WT and chimeric receptors shows no correlation with the time course of ATP-mediated currents. Efficacy of partial agonists AP5A (a) and BzATP (b) as a percentage of maximum ATP evoked response (100 μm ATP) versus the receptor time course to desensitization is shown. Time course is expressed as the time to 50% desensitization over 3-s ATP application, or percentage current remaining at the end of prolonged 20-s ATP application, for fast/slow desensitizing receptors, respectively (n = 5–17).
	FIGURE 8. Summary of effects of chimeras on partial agonist potency and efficacy. Replacement of different regions of the P2X1 receptor with those from the P2X2 receptor, and vice versa modified partial agonist action. Residues in the extracellular loop determine antagonist sensitivity. The TM regions are involved in control of both agonist sensitivity and efficacy. The amino terminus can regulate agonist efficacy independently of sensitivity, and the carboxyl terminus is involved in recovery of the receptor from the desensitized state.