Allsopp RC and Evans RJ (2015), Contribution of the Juxtatransmembrane Intracel...

XB-ART-50574

J Biol Chem 2015 Jun 05;29023:14556-66. doi: 10.1074/jbc.M115.642033.

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Contribution of the Juxtatransmembrane Intracellular Regions to the Time Course and Permeation of ATP-gated P2X7 Receptor Ion Channels.

Allsopp RC , Evans RJ .

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P2X7 receptors are ATP-gated ion channels that contribute to inflammation and cell death. They have the novel property of showing marked facilitation to repeated applications of agonist, and the intrinsic channel pore dilates to allow the passage of fluorescent dyes. A 60-s application of ATP to hP2X7 receptors expressed in Xenopus oocytes gave rise to a current that had a biphasic time course with initial and secondary slowly developing components. A second application of ATP evoked a response with a more rapid time to peak. This facilitation was reversed to initial levels following a 10-min agonist-free interval. A chimeric approach showed that replacement of the pre-TM1 amino-terminal region with the corresponding P2X2 receptor section (P2X7-2Nβ) gave responses that quickly reached a steady state and did not show facilitation. Subsequent point mutations of variant residues identified Asn-16 and Ser-23 as important contributors to the time course/facilitation. The P2X7 receptor is unique in having an intracellular carboxyl-terminal cysteine-rich region (Ccys). Deletion of this region removed the secondary slowly developing current, and, when expressed in HEK293 cells, ethidium bromide uptake was only ∼5% that of WT levels, indicating reduced large pore formation. Dye uptake was also reduced for the P2X7-2Nβ chimera. Surprisingly, combination of the chimera and the Ccys deletion (P2X7-2NβdelCcys) restored the current rise time and ethidium uptake to WT levels. These findings suggest that there is a coevolved interaction between the juxtatransmembrane amino and carboxyl termini in the regulation of P2X7 receptor gating.

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Species referenced: Xenopus laevis
Genes referenced: p2rx1 p2rx2 p2rx7

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	FIGURE 1. The pre-TM1 region regulates hP2X7 facilitation. A, representative traces showing the speeding/facilitation of WT P2X7 receptor responses to prolonged (60 s, bar) repeat applications of 1 mm ATP at 3-min intervals (black, red, and blue trace consecutively). B, increasing the time interval between repeat ATP applications 2 and 3 (black trace to red trace, 3-min interval; red trace to blue trace, 10-min interval) returns the receptor response back to its naïve state. C, schematics and representative traces showing the effect of replacing the entire N terminus of the hP2X7 receptor (black) with that of hP2X2 receptor (magenta) or just the 16 pre-TM1 amino acids (P2X7–2Nβ). Traces represent first (black) and second (red) receptor responses (60-s application at 3-min intervals) to 1 mm ATP. ex, the extracellular region of the receptor. D, histogram summary showing the 10–50% rise times (seconds) of the first (black) versus second (red) receptor response to EC90 ATP (1 mm ATP at P2X7 and 100 μm ATP at P2X7–2N, P2X7–2Nβ, and P2X2) at 3-min intervals. Inset, concentration response to ATP for P2X7 and P2X7–2Nβ receptors. E, histogram summary showing the peak amplitude (microamperes) of the response to the first ATP application. Inset, representative Western blot of the equivalent levels of surface expression of P2X7 and P2X7–2Nβ receptors. Data are mean ± S.E. (n = 7–25). , p < 0.05; ***, p < 0.0001.
	FIGURE 2. Contribution of variant pre-TM1 residues to P2X7 receptor current facilitation. A, amino acid sequence lineup showing the pre-TM1 residues of the hP2X7 receptor (top row) and the hP2X2 receptor (bottom row). Non-conserved amino acids are shown in green. B, representative traces (the first and second responses are shown in black and red, respectively) demonstrating the effect of pre-TM1 substitution of non-conserved amino acid residues between the P2X7 and P2X2 receptor in response to ATP (1 mm, 60-s agonist addition at 3-min intervals). C and D, histogram summaries showing the peak current amplitude (microamperes and 10–50% rise time (seconds) of the first and second responses to ATP. Statistical significance shown in black is relative to the P2X7 WT and, in red, for between the first and second response at a particular receptor. Data are mean ± S.E. (n = 4–25). , p < 0.01; , p < 0.001; ***, p < 0.0001.
	FIGURE 3. Contribution of the pre-TM1 region to the time course of P2X1, P2X2, and P2X7 receptor currents. A, normalized traces demonstrating the effect on the time course of pre-TM1 β-region substitution of P2X1 into P2X7, P2X7 into P2X2, and P2X7 into P2X1 receptors, respectively. In each case, ATP was applied for the duration indicated by the gray bars (1 mm for P2X7 and P2X7–1Nβ and 100 μm for P2X1 and P2X2 WT receptors and chimeras). B—D, histograms showing the peak current amplitude (microamperes), 10–50% rise time (seconds), and percent current remaining at the end of the ATP pulse (EC90 concentration of ATP applied for the duration as indicated on the adjacent traces). Western blots show the surface expression of the respective WT and chimeric receptors. For P2X2 and P2X7 receptors, the lanes are from the same blot and exposure, and the white line between them indicates that they have been reordered from that loaded on the gel. Data are mean ± S.E. (n = 4–25). , p < 0.05; , p < 0.01; ***, p < 0.0001.
	FIGURE 4. Contribution of the hP2X7 receptor C-terminal cysteine-rich region (amino acid residues 362–379) to the time course. A, peak normalized traces showing the effect on the time-course of cysteine-rich region deletion of the WT P2X7 receptor, insertion into the P2X2 receptor, and insertion into the P2X1 receptor. ATP application was as indicated by the gray bars (1 mm for P2X7 and P2X7–1Nβ and 100 μm for P2X1 and P2X2 WT receptors and chimeras). For the P2X7-delCcys, the trace in yellow shows the relative amplitude of the response compared with the P2X7 receptor to show the similar time course to the initial rise time of the P2X7 receptor response. B—D, histograms showing the peak current amplitude (microamperes), 10–50% rise time (seconds), and percent current remaining at the end of the ATP pulse. Western blots show equivalent surface expression of the respective WT and chimeric receptors (the WT control for P2X7 is the same as in Fig. 3, the original blot compared P2X7 WT with a range of P2X7 chimeras). Data are mean ± S.E. (n = 4–25). , p < 0.05; , p < 0.01; , p < 0.001; **, p < 0.0001 (n = 4–25).
	FIGURE 5. Functional interaction between the pre-TM1 β-region and C-terminal cysteine-rich region and its effect on P2X7 receptor time course. A, peak normalized traces showing the effect of deletion of the cysteine-rich region deletion from the P2X7 receptor (P2XYdelCcys, yellow) compared with the WT P2X7 receptor (black) and the P2X7–2Nβ chimera (red). ATP application is indicated by the gray bars (1 mm for 60 s). B, peak normalized traces showing reversion to slow receptor facilitation at the P2X7–2Nβ delCcys chimera (blue) with both 60-s (i) and 120-s (ii) applications of 1 mm ATP highlighting the faster desensitization of the chimeric receptor. C–E, histograms showing the peak amplitude (microamperes), rise time (seconds), and time to 50% decay (seconds) at the end of the ATP pulse for the P2X7 receptor versus the P2X7–2Nβ delCcys chimera. Western blots show equivalent levels of surface expression of P2X7 and the P2X7–2Nβ delCcys chimera. Data are mean ± S.E. (n = 4–25). *, p < 0.001 **, p < 0.0001.
	FIGURE 6. Sucrose buffer enhances ethidium bromide dye uptake through the P2X7 receptor pore. A and B, representative FlexStation responses showing agonist-induced dye uptake through the rat (A) and human (B) P2X7 receptor when preloaded for 30 min with ethidium bromide (20 μm) under different buffer conditions (sucrose dye uptake buffer versus normal extracellular solution low divalent buffer). Agonist addition (300 μm BzATP) at 240 s is indicated by the arrow. C, histogram summary showing the total RFU (relative fluorescence units) change under the different buffer loading conditions. D, histogram summary showing the -fold increase in dye uptake in sucrose dye uptake buffer compared with low divalent normal extracellular solution buffer. Data are mean ± S.E. ****, p < 0.0001 (n = 24).
	FIGURE 7. Interaction of the intracellular pre-TM1 β region and C-terminal cysteine-rich region of the P2X7 receptor regulates pore formation and dye uptake. A, FlexStation responses demonstrating agonist-induced dye uptake through the pore of the P2X7 receptor, reduced dye uptake though the N terminus chimeric receptor, and C terminus deletion receptor (P2X7–2Nβ and P2X7 delCcys, respectively) and “P2X7-like” dye uptake through the P2X7–2Nβ delCcys mutant receptor. Agonist addition (300 μm BzATP) at 240 s is indicated by the arrow. S.E. is only shown in one direction to not obscure the mean values. Western blots show equivalent levels of surface expression for the P2X7 receptor WT and mutants. B, P2X7–2Nβ receptor response normalized to the peak P2X7 receptor response identifying an increased initial rate of dye uptake. C, P2X7–2Nβ delCcys receptor response normalized to the peak P2X7 receptor response demonstrating the relative dye uptake speeds through these two receptors. D, histogram summary showing the relative dye uptake for each receptor. E, histogram summary demonstrating the amount of dye uptake (as a percentage of the peak maximum) 1 min after BzATP addition (300 μm BzATP addition made at 240 s). Data are mean ± S.E. , p < 0.01; **, p < 0.0001 (n = 7–21).
	FIGURE 8. Effects of mutations in the P2X7 receptor pre-TM1 β-region on pore formation and dye uptake. A, FlexStation responses demonstrating agonist-induced dye uptake through the pore of the P2X7 receptor and reduced dye uptake though the N terminus mutants N16P, Y26L, S23N, and NRRL. Agonist addition (300 μm BzATP) at 240 s is indicated by the arrow. Standard errors are only shown in one direction to not obscure the mean values. B, histogram summary showing the relative peak dye uptake for each receptor. Western blots show equivalent levels of surface receptor expression for P2X7 receptor WT and mutants. C, histogram summary demonstrating the amount of dye uptake (as a percentage of the peak maximum) 1 min after BzATP addition (300 μm BzATP addition made at 240 s). Data are mean ± S.E. , p < 0.01; , p < 0.001; ***, p < 0.0001 (n = 24).

References [+] :

Adinolfi, Trophic activity of a naturally occurring truncated isoform of the P2X7 receptor. 2010, Pubmed

	FIGURE 1. The pre-TM1 region regulates hP2X7 facilitation. A, representative traces showing the speeding/facilitation of WT P2X7 receptor responses to prolonged (60 s, bar) repeat applications of 1 mm ATP at 3-min intervals (black, red, and blue trace consecutively). B, increasing the time interval between repeat ATP applications 2 and 3 (black trace to red trace, 3-min interval; red trace to blue trace, 10-min interval) returns the receptor response back to its naïve state. C, schematics and representative traces showing the effect of replacing the entire N terminus of the hP2X7 receptor (black) with that of hP2X2 receptor (magenta) or just the 16 pre-TM1 amino acids (P2X7–2Nβ). Traces represent first (black) and second (red) receptor responses (60-s application at 3-min intervals) to 1 mm ATP. ex, the extracellular region of the receptor. D, histogram summary showing the 10–50% rise times (seconds) of the first (black) versus second (red) receptor response to EC90 ATP (1 mm ATP at P2X7 and 100 μm ATP at P2X7–2N, P2X7–2Nβ, and P2X2) at 3-min intervals. Inset, concentration response to ATP for P2X7 and P2X7–2Nβ receptors. E, histogram summary showing the peak amplitude (microamperes) of the response to the first ATP application. Inset, representative Western blot of the equivalent levels of surface expression of P2X7 and P2X7–2Nβ receptors. Data are mean ± S.E. (n = 7–25). , p < 0.05; ***, p < 0.0001.
	FIGURE 2. Contribution of variant pre-TM1 residues to P2X7 receptor current facilitation. A, amino acid sequence lineup showing the pre-TM1 residues of the hP2X7 receptor (top row) and the hP2X2 receptor (bottom row). Non-conserved amino acids are shown in green. B, representative traces (the first and second responses are shown in black and red, respectively) demonstrating the effect of pre-TM1 substitution of non-conserved amino acid residues between the P2X7 and P2X2 receptor in response to ATP (1 mm, 60-s agonist addition at 3-min intervals). C and D, histogram summaries showing the peak current amplitude (microamperes and 10–50% rise time (seconds) of the first and second responses to ATP. Statistical significance shown in black is relative to the P2X7 WT and, in red, for between the first and second response at a particular receptor. Data are mean ± S.E. (n = 4–25). , p < 0.01; , p < 0.001; ***, p < 0.0001.
	FIGURE 3. Contribution of the pre-TM1 region to the time course of P2X1, P2X2, and P2X7 receptor currents. A, normalized traces demonstrating the effect on the time course of pre-TM1 β-region substitution of P2X1 into P2X7, P2X7 into P2X2, and P2X7 into P2X1 receptors, respectively. In each case, ATP was applied for the duration indicated by the gray bars (1 mm for P2X7 and P2X7–1Nβ and 100 μm for P2X1 and P2X2 WT receptors and chimeras). B—D, histograms showing the peak current amplitude (microamperes), 10–50% rise time (seconds), and percent current remaining at the end of the ATP pulse (EC90 concentration of ATP applied for the duration as indicated on the adjacent traces). Western blots show the surface expression of the respective WT and chimeric receptors. For P2X2 and P2X7 receptors, the lanes are from the same blot and exposure, and the white line between them indicates that they have been reordered from that loaded on the gel. Data are mean ± S.E. (n = 4–25). , p < 0.05; , p < 0.01; ***, p < 0.0001.
	FIGURE 4. Contribution of the hP2X7 receptor C-terminal cysteine-rich region (amino acid residues 362–379) to the time course. A, peak normalized traces showing the effect on the time-course of cysteine-rich region deletion of the WT P2X7 receptor, insertion into the P2X2 receptor, and insertion into the P2X1 receptor. ATP application was as indicated by the gray bars (1 mm for P2X7 and P2X7–1Nβ and 100 μm for P2X1 and P2X2 WT receptors and chimeras). For the P2X7-delCcys, the trace in yellow shows the relative amplitude of the response compared with the P2X7 receptor to show the similar time course to the initial rise time of the P2X7 receptor response. B—D, histograms showing the peak current amplitude (microamperes), 10–50% rise time (seconds), and percent current remaining at the end of the ATP pulse. Western blots show equivalent surface expression of the respective WT and chimeric receptors (the WT control for P2X7 is the same as in Fig. 3, the original blot compared P2X7 WT with a range of P2X7 chimeras). Data are mean ± S.E. (n = 4–25). , p < 0.05; , p < 0.01; , p < 0.001; **, p < 0.0001 (n = 4–25).
	FIGURE 5. Functional interaction between the pre-TM1 β-region and C-terminal cysteine-rich region and its effect on P2X7 receptor time course. A, peak normalized traces showing the effect of deletion of the cysteine-rich region deletion from the P2X7 receptor (P2XYdelCcys, yellow) compared with the WT P2X7 receptor (black) and the P2X7–2Nβ chimera (red). ATP application is indicated by the gray bars (1 mm for 60 s). B, peak normalized traces showing reversion to slow receptor facilitation at the P2X7–2Nβ delCcys chimera (blue) with both 60-s (i) and 120-s (ii) applications of 1 mm ATP highlighting the faster desensitization of the chimeric receptor. C–E, histograms showing the peak amplitude (microamperes), rise time (seconds), and time to 50% decay (seconds) at the end of the ATP pulse for the P2X7 receptor versus the P2X7–2Nβ delCcys chimera. Western blots show equivalent levels of surface expression of P2X7 and the P2X7–2Nβ delCcys chimera. Data are mean ± S.E. (n = 4–25). *, p < 0.001 **, p < 0.0001.
	FIGURE 6. Sucrose buffer enhances ethidium bromide dye uptake through the P2X7 receptor pore. A and B, representative FlexStation responses showing agonist-induced dye uptake through the rat (A) and human (B) P2X7 receptor when preloaded for 30 min with ethidium bromide (20 μm) under different buffer conditions (sucrose dye uptake buffer versus normal extracellular solution low divalent buffer). Agonist addition (300 μm BzATP) at 240 s is indicated by the arrow. C, histogram summary showing the total RFU (relative fluorescence units) change under the different buffer loading conditions. D, histogram summary showing the -fold increase in dye uptake in sucrose dye uptake buffer compared with low divalent normal extracellular solution buffer. Data are mean ± S.E. ****, p < 0.0001 (n = 24).
	FIGURE 7. Interaction of the intracellular pre-TM1 β region and C-terminal cysteine-rich region of the P2X7 receptor regulates pore formation and dye uptake. A, FlexStation responses demonstrating agonist-induced dye uptake through the pore of the P2X7 receptor, reduced dye uptake though the N terminus chimeric receptor, and C terminus deletion receptor (P2X7–2Nβ and P2X7 delCcys, respectively) and “P2X7-like” dye uptake through the P2X7–2Nβ delCcys mutant receptor. Agonist addition (300 μm BzATP) at 240 s is indicated by the arrow. S.E. is only shown in one direction to not obscure the mean values. Western blots show equivalent levels of surface expression for the P2X7 receptor WT and mutants. B, P2X7–2Nβ receptor response normalized to the peak P2X7 receptor response identifying an increased initial rate of dye uptake. C, P2X7–2Nβ delCcys receptor response normalized to the peak P2X7 receptor response demonstrating the relative dye uptake speeds through these two receptors. D, histogram summary showing the relative dye uptake for each receptor. E, histogram summary demonstrating the amount of dye uptake (as a percentage of the peak maximum) 1 min after BzATP addition (300 μm BzATP addition made at 240 s). Data are mean ± S.E. , p < 0.01; **, p < 0.0001 (n = 7–21).
	FIGURE 8. Effects of mutations in the P2X7 receptor pre-TM1 β-region on pore formation and dye uptake. A, FlexStation responses demonstrating agonist-induced dye uptake through the pore of the P2X7 receptor and reduced dye uptake though the N terminus mutants N16P, Y26L, S23N, and NRRL. Agonist addition (300 μm BzATP) at 240 s is indicated by the arrow. Standard errors are only shown in one direction to not obscure the mean values. B, histogram summary showing the relative peak dye uptake for each receptor. Western blots show equivalent levels of surface receptor expression for P2X7 receptor WT and mutants. C, histogram summary demonstrating the amount of dye uptake (as a percentage of the peak maximum) 1 min after BzATP addition (300 μm BzATP addition made at 240 s). Data are mean ± S.E. , p < 0.01; , p < 0.001; ***, p < 0.0001 (n = 24).