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Molecular shape, architecture, and size of P2X4 receptors determined using fluorescence resonance energy transfer and electron microscopy.

Young MT, Fisher JA, Fountain SJ, Ford RC, North RA, Khakh BS - J. Biol. Chem. (2008)

Bottom Line: Single particle analysis of purified P2X(4) receptors was used to determine the three-dimensional structure at a resolution of 21A; the orientation of the particle with respect to the membrane was assigned by labeling the intracellular C termini with 1.8-nm gold particles and the carbohydrate-rich ectodomain with lectin.We found that human P2X(4) is a globular torpedo-like molecule with an approximate volume of 270 nm(3) and a compact propeller-shaped ectodomain.Thus, our data provide the first views of the architecture, shape, and size of single P2X receptors, furthering our understanding of this important family of ligand-gated ion channels.

View Article: PubMed Central - PubMed

Affiliation: Manchester Interdisciplinary Biocentre, University of Manchester, Manchester M1 7DN, United Kingdom.

ABSTRACT
P2X receptors are ATP-gated nonselective cation channels with important physiological roles. However, their structures are poorly understood. Here, we analyzed the architecture of P2X receptors using fluorescence resonance energy transfer (FRET) microscopy and direct structure determination using electron microscopy. FRET efficiency measurements indicated that the distance between the C-terminal tails of P2X(4) receptors was 5.6 nm. Single particle analysis of purified P2X(4) receptors was used to determine the three-dimensional structure at a resolution of 21A; the orientation of the particle with respect to the membrane was assigned by labeling the intracellular C termini with 1.8-nm gold particles and the carbohydrate-rich ectodomain with lectin. We found that human P2X(4) is a globular torpedo-like molecule with an approximate volume of 270 nm(3) and a compact propeller-shaped ectodomain. In this structure, the distance between the centers of the gold particles was 6.1 nm, which closely matches FRET data. Thus, our data provide the first views of the architecture, shape, and size of single P2X receptors, furthering our understanding of this important family of ligand-gated ion channels.

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FRET e for all homomeric P2X receptors for near membrane regions of interest and relationship of FRET e and interfluorophore distance. A-H, in each case, linear plots of YFP photodestruction and CFP photorecovery are shown for the indicated constructs. In each case, FRET e is given by the y axis intercept of the straight line fits. The FRET e value from a linear fit to the mean data for CFP/YFP represents FRET noise. In the case of tagged P2X6 receptors, only whole-cell regions were examined because these do not express in the plasma membrane, but are instead retained within intracellular membranes (see “Results”). I, the left panel shows a plot of the Förster equation relating FRET e (y axis) and interfluorophore distance (x axis), with FRET e data for all homomeric P2X receptors superimposed. The boxed region is expanded in the right panel. J, the left panel shows the measured distance between the C-terminal tails of P2X4 monomers within the trimer (5.6 nm). The right panel summarizes the trend that was observed in the P2X receptor data, viz. that receptors with short C termini display lower FRET e values (thus, greater interfluorophore distance), and those with longer tails display higher FRET e values (thus, greater interfluorophore distance). In interpreting these data, it is important to remember that the orientation factor κ2 is taken as 2/3 and assumes random tumbling as discussed by us and others (19, 30). aa, amino acids.
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fig3: FRET e for all homomeric P2X receptors for near membrane regions of interest and relationship of FRET e and interfluorophore distance. A-H, in each case, linear plots of YFP photodestruction and CFP photorecovery are shown for the indicated constructs. In each case, FRET e is given by the y axis intercept of the straight line fits. The FRET e value from a linear fit to the mean data for CFP/YFP represents FRET noise. In the case of tagged P2X6 receptors, only whole-cell regions were examined because these do not express in the plasma membrane, but are instead retained within intracellular membranes (see “Results”). I, the left panel shows a plot of the Förster equation relating FRET e (y axis) and interfluorophore distance (x axis), with FRET e data for all homomeric P2X receptors superimposed. The boxed region is expanded in the right panel. J, the left panel shows the measured distance between the C-terminal tails of P2X4 monomers within the trimer (5.6 nm). The right panel summarizes the trend that was observed in the P2X receptor data, viz. that receptors with short C termini display lower FRET e values (thus, greater interfluorophore distance), and those with longer tails display higher FRET e values (thus, greater interfluorophore distance). In interpreting these data, it is important to remember that the orientation factor κ2 is taken as 2/3 and assumes random tumbling as discussed by us and others (19, 30). aa, amino acids.

Mentions: Structure Determination by Electron Microscopy and Single Particle Analysis—Protein samples (20 μg/ml) were adsorbed onto glow-discharged carbon-coated copper grids and negatively stained with 2% (w/v) uranyl acetate. Transmission electron microscopy images were recorded on Kodak SO-163 film at a calibrated magnification of ×43,500 using a Tecnai 10 electron microscope operating at 100 kV. Images were digitized using a UMAX 2000 Powerlook scanner at 1200 pixels/inch, providing a specimen level increment of 4.87 Å/pixel. EMAN software (29) was used to manually select, filter, and process 6826 particles in 48 × 48 pixel boxes. Using the command “startcsym,” we compared the best match of the two-dimensional reference-free class averages with symmetrized classes using C2, C3, C4, C5, and C6 symmetry (see Fig. 3E). C3 symmetry provided the best fit to the data, and so a start model was generated from a manually selected set of reference-free class averages with C3 symmetry applied using the command “start-Any”; this model was refined by 12 rounds of iterative projection matching using the “refine” command. Fourier shell correlation analysis of the final iteration using comparison of structures generated from even- or odd-numbered particles (“eotest”; see Fig. 3D) indicated a final resolution of ∼21 Å. Structures were displayed at volume shells corresponding to at least 3 S.D. values above the mean density.


Molecular shape, architecture, and size of P2X4 receptors determined using fluorescence resonance energy transfer and electron microscopy.

Young MT, Fisher JA, Fountain SJ, Ford RC, North RA, Khakh BS - J. Biol. Chem. (2008)

FRET e for all homomeric P2X receptors for near membrane regions of interest and relationship of FRET e and interfluorophore distance. A-H, in each case, linear plots of YFP photodestruction and CFP photorecovery are shown for the indicated constructs. In each case, FRET e is given by the y axis intercept of the straight line fits. The FRET e value from a linear fit to the mean data for CFP/YFP represents FRET noise. In the case of tagged P2X6 receptors, only whole-cell regions were examined because these do not express in the plasma membrane, but are instead retained within intracellular membranes (see “Results”). I, the left panel shows a plot of the Förster equation relating FRET e (y axis) and interfluorophore distance (x axis), with FRET e data for all homomeric P2X receptors superimposed. The boxed region is expanded in the right panel. J, the left panel shows the measured distance between the C-terminal tails of P2X4 monomers within the trimer (5.6 nm). The right panel summarizes the trend that was observed in the P2X receptor data, viz. that receptors with short C termini display lower FRET e values (thus, greater interfluorophore distance), and those with longer tails display higher FRET e values (thus, greater interfluorophore distance). In interpreting these data, it is important to remember that the orientation factor κ2 is taken as 2/3 and assumes random tumbling as discussed by us and others (19, 30). aa, amino acids.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2533801&req=5

fig3: FRET e for all homomeric P2X receptors for near membrane regions of interest and relationship of FRET e and interfluorophore distance. A-H, in each case, linear plots of YFP photodestruction and CFP photorecovery are shown for the indicated constructs. In each case, FRET e is given by the y axis intercept of the straight line fits. The FRET e value from a linear fit to the mean data for CFP/YFP represents FRET noise. In the case of tagged P2X6 receptors, only whole-cell regions were examined because these do not express in the plasma membrane, but are instead retained within intracellular membranes (see “Results”). I, the left panel shows a plot of the Förster equation relating FRET e (y axis) and interfluorophore distance (x axis), with FRET e data for all homomeric P2X receptors superimposed. The boxed region is expanded in the right panel. J, the left panel shows the measured distance between the C-terminal tails of P2X4 monomers within the trimer (5.6 nm). The right panel summarizes the trend that was observed in the P2X receptor data, viz. that receptors with short C termini display lower FRET e values (thus, greater interfluorophore distance), and those with longer tails display higher FRET e values (thus, greater interfluorophore distance). In interpreting these data, it is important to remember that the orientation factor κ2 is taken as 2/3 and assumes random tumbling as discussed by us and others (19, 30). aa, amino acids.
Mentions: Structure Determination by Electron Microscopy and Single Particle Analysis—Protein samples (20 μg/ml) were adsorbed onto glow-discharged carbon-coated copper grids and negatively stained with 2% (w/v) uranyl acetate. Transmission electron microscopy images were recorded on Kodak SO-163 film at a calibrated magnification of ×43,500 using a Tecnai 10 electron microscope operating at 100 kV. Images were digitized using a UMAX 2000 Powerlook scanner at 1200 pixels/inch, providing a specimen level increment of 4.87 Å/pixel. EMAN software (29) was used to manually select, filter, and process 6826 particles in 48 × 48 pixel boxes. Using the command “startcsym,” we compared the best match of the two-dimensional reference-free class averages with symmetrized classes using C2, C3, C4, C5, and C6 symmetry (see Fig. 3E). C3 symmetry provided the best fit to the data, and so a start model was generated from a manually selected set of reference-free class averages with C3 symmetry applied using the command “start-Any”; this model was refined by 12 rounds of iterative projection matching using the “refine” command. Fourier shell correlation analysis of the final iteration using comparison of structures generated from even- or odd-numbered particles (“eotest”; see Fig. 3D) indicated a final resolution of ∼21 Å. Structures were displayed at volume shells corresponding to at least 3 S.D. values above the mean density.

Bottom Line: Single particle analysis of purified P2X(4) receptors was used to determine the three-dimensional structure at a resolution of 21A; the orientation of the particle with respect to the membrane was assigned by labeling the intracellular C termini with 1.8-nm gold particles and the carbohydrate-rich ectodomain with lectin.We found that human P2X(4) is a globular torpedo-like molecule with an approximate volume of 270 nm(3) and a compact propeller-shaped ectodomain.Thus, our data provide the first views of the architecture, shape, and size of single P2X receptors, furthering our understanding of this important family of ligand-gated ion channels.

View Article: PubMed Central - PubMed

Affiliation: Manchester Interdisciplinary Biocentre, University of Manchester, Manchester M1 7DN, United Kingdom.

ABSTRACT
P2X receptors are ATP-gated nonselective cation channels with important physiological roles. However, their structures are poorly understood. Here, we analyzed the architecture of P2X receptors using fluorescence resonance energy transfer (FRET) microscopy and direct structure determination using electron microscopy. FRET efficiency measurements indicated that the distance between the C-terminal tails of P2X(4) receptors was 5.6 nm. Single particle analysis of purified P2X(4) receptors was used to determine the three-dimensional structure at a resolution of 21A; the orientation of the particle with respect to the membrane was assigned by labeling the intracellular C termini with 1.8-nm gold particles and the carbohydrate-rich ectodomain with lectin. We found that human P2X(4) is a globular torpedo-like molecule with an approximate volume of 270 nm(3) and a compact propeller-shaped ectodomain. In this structure, the distance between the centers of the gold particles was 6.1 nm, which closely matches FRET data. Thus, our data provide the first views of the architecture, shape, and size of single P2X receptors, furthering our understanding of this important family of ligand-gated ion channels.

Show MeSH
Related in: MedlinePlus