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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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Purification and 21 Å structure of P2X4 receptor trimers. A, Coomassie Blue-stained 8% (w/v) PFO-polyacrylamide gel of partially purified P2X4 after nickel chromatography. The molecular masses of monomer (55 kDa), dimer (110 kDa), and trimer (165 kDa) bands are indicated (1, 2, and 3 respectively). B, silver-stained SDS-polyacrylamide gel of purified human P2X4 trimers following gel excision and electroelution. A high purity was obtained; SDS-stable dimeric and trimeric forms of protein are also visible. C, silver-stained PFO-polyacrylamide gel of purified trimers demonstrating that the trimeric form was stable after purification (indicated by the arrow). D, particle field from an electron micrograph of negatively stained (2% (w/v) uranyl acetate) single particles of human P2X4-His10. Single particles are indicated by 48 × 48 pixel square boxes (top/bottom views) and 48 pixel radius circles (side views). E, symmetry analysis of two-dimensional reference-free class averages using the command startcsym at C2, C3, C4, C5, and C6 symmetry. The best fitting class average is displayed above the corresponding symmetrized view (sym); C3 symmetry produces the best fit with the class average. F, Fourier shell correlation (FSC) analysis of structures of wild-type human P2X4 generated from even- and odd-numbered particles using the command eotest. The resolution of the structure given by the Fourier shell correlation value of 0.5 is indicated and corresponds to ∼21 Å. G, class averages generated by single particle analysis of the wild-type particle data set. Each column represents a back-projection from the final C3-symmetrized three-dimensional volume paired with its corresponding unsymmetrized class average. A selected range of views from top/bottom (top left column) to side (bottom right column) are displayed. Box size is 234 × 234 Å. H, structure of human P2X4 at 21 Å resolution displayed at two volume thresholds (5.6σ, light blue inner core; and 3σ, dark blue outer shell) above the mean density. Scale bar = 50 Å.
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fig5: Purification and 21 Å structure of P2X4 receptor trimers. A, Coomassie Blue-stained 8% (w/v) PFO-polyacrylamide gel of partially purified P2X4 after nickel chromatography. The molecular masses of monomer (55 kDa), dimer (110 kDa), and trimer (165 kDa) bands are indicated (1, 2, and 3 respectively). B, silver-stained SDS-polyacrylamide gel of purified human P2X4 trimers following gel excision and electroelution. A high purity was obtained; SDS-stable dimeric and trimeric forms of protein are also visible. C, silver-stained PFO-polyacrylamide gel of purified trimers demonstrating that the trimeric form was stable after purification (indicated by the arrow). D, particle field from an electron micrograph of negatively stained (2% (w/v) uranyl acetate) single particles of human P2X4-His10. Single particles are indicated by 48 × 48 pixel square boxes (top/bottom views) and 48 pixel radius circles (side views). E, symmetry analysis of two-dimensional reference-free class averages using the command startcsym at C2, C3, C4, C5, and C6 symmetry. The best fitting class average is displayed above the corresponding symmetrized view (sym); C3 symmetry produces the best fit with the class average. F, Fourier shell correlation (FSC) analysis of structures of wild-type human P2X4 generated from even- and odd-numbered particles using the command eotest. The resolution of the structure given by the Fourier shell correlation value of 0.5 is indicated and corresponds to ∼21 Å. G, class averages generated by single particle analysis of the wild-type particle data set. Each column represents a back-projection from the final C3-symmetrized three-dimensional volume paired with its corresponding unsymmetrized class average. A selected range of views from top/bottom (top left column) to side (bottom right column) are displayed. Box size is 234 × 234 Å. H, structure of human P2X4 at 21 Å resolution displayed at two volume thresholds (5.6σ, light blue inner core; and 3σ, dark blue outer shell) above the mean density. Scale bar = 50 Å.

Mentions: Following nickel affinity chromatography, the majority of P2X4 eluted in the 500 mm imidazole fraction as a diffuse 55-kDa band. The size of the band was in agreement with that observed by Western blotting, and purity was estimated to be ∼50%. To purify P2X4 to homogeneity, we used nondenaturing PFO-PAGE (28), followed by electroelution. When separated by PFO-PAGE, 50% pure P2X4 presented as three bands corresponding to a monomer (55 kDa), a faint dimer (110 kDa), and a trimer (165 kDa) (Fig. 5A). No higher order oligomers were observed. We assumed that the observed monomers and dimers corresponded to immature nonassembled proteins recovered from internal membranes. Trimers were selectively purified by excising bands from preparative nonstained PFO-polyacrylamide gels and eluting protein under an electric field. SDS-PAGE analysis of eluted protein demonstrated the high purity obtained using this purification method (Fig. 5B); the only bands visible on the silver-stained gel corresponded to P2X4. (SDS-stable dimeric and trimeric forms were also observed.) In addition, PFO-PAGE of purified trimers was used to demonstrate that the trimeric form was stable after purification (Fig. 5C, arrow). After diafiltration into 0.1% (w/v) DDM, 40 μl of P2X4 was recovered at an approximate concentration of 1 mg/ml. Yield was estimated by comparing the band intensity of pure P2X4 on Coomassie Blue-stained SDS-polyacrylamide gels with known quantities of bovine serum albumin. In summary, 40 μg of pure P2X4 trimer was recovered from 2.5 × 108 cells (equivalent to a confluent monolayer of 5000 cm2 in area).


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)

Purification and 21 Å structure of P2X4 receptor trimers. A, Coomassie Blue-stained 8% (w/v) PFO-polyacrylamide gel of partially purified P2X4 after nickel chromatography. The molecular masses of monomer (55 kDa), dimer (110 kDa), and trimer (165 kDa) bands are indicated (1, 2, and 3 respectively). B, silver-stained SDS-polyacrylamide gel of purified human P2X4 trimers following gel excision and electroelution. A high purity was obtained; SDS-stable dimeric and trimeric forms of protein are also visible. C, silver-stained PFO-polyacrylamide gel of purified trimers demonstrating that the trimeric form was stable after purification (indicated by the arrow). D, particle field from an electron micrograph of negatively stained (2% (w/v) uranyl acetate) single particles of human P2X4-His10. Single particles are indicated by 48 × 48 pixel square boxes (top/bottom views) and 48 pixel radius circles (side views). E, symmetry analysis of two-dimensional reference-free class averages using the command startcsym at C2, C3, C4, C5, and C6 symmetry. The best fitting class average is displayed above the corresponding symmetrized view (sym); C3 symmetry produces the best fit with the class average. F, Fourier shell correlation (FSC) analysis of structures of wild-type human P2X4 generated from even- and odd-numbered particles using the command eotest. The resolution of the structure given by the Fourier shell correlation value of 0.5 is indicated and corresponds to ∼21 Å. G, class averages generated by single particle analysis of the wild-type particle data set. Each column represents a back-projection from the final C3-symmetrized three-dimensional volume paired with its corresponding unsymmetrized class average. A selected range of views from top/bottom (top left column) to side (bottom right column) are displayed. Box size is 234 × 234 Å. H, structure of human P2X4 at 21 Å resolution displayed at two volume thresholds (5.6σ, light blue inner core; and 3σ, dark blue outer shell) above the mean density. Scale bar = 50 Å.
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fig5: Purification and 21 Å structure of P2X4 receptor trimers. A, Coomassie Blue-stained 8% (w/v) PFO-polyacrylamide gel of partially purified P2X4 after nickel chromatography. The molecular masses of monomer (55 kDa), dimer (110 kDa), and trimer (165 kDa) bands are indicated (1, 2, and 3 respectively). B, silver-stained SDS-polyacrylamide gel of purified human P2X4 trimers following gel excision and electroelution. A high purity was obtained; SDS-stable dimeric and trimeric forms of protein are also visible. C, silver-stained PFO-polyacrylamide gel of purified trimers demonstrating that the trimeric form was stable after purification (indicated by the arrow). D, particle field from an electron micrograph of negatively stained (2% (w/v) uranyl acetate) single particles of human P2X4-His10. Single particles are indicated by 48 × 48 pixel square boxes (top/bottom views) and 48 pixel radius circles (side views). E, symmetry analysis of two-dimensional reference-free class averages using the command startcsym at C2, C3, C4, C5, and C6 symmetry. The best fitting class average is displayed above the corresponding symmetrized view (sym); C3 symmetry produces the best fit with the class average. F, Fourier shell correlation (FSC) analysis of structures of wild-type human P2X4 generated from even- and odd-numbered particles using the command eotest. The resolution of the structure given by the Fourier shell correlation value of 0.5 is indicated and corresponds to ∼21 Å. G, class averages generated by single particle analysis of the wild-type particle data set. Each column represents a back-projection from the final C3-symmetrized three-dimensional volume paired with its corresponding unsymmetrized class average. A selected range of views from top/bottom (top left column) to side (bottom right column) are displayed. Box size is 234 × 234 Å. H, structure of human P2X4 at 21 Å resolution displayed at two volume thresholds (5.6σ, light blue inner core; and 3σ, dark blue outer shell) above the mean density. Scale bar = 50 Å.
Mentions: Following nickel affinity chromatography, the majority of P2X4 eluted in the 500 mm imidazole fraction as a diffuse 55-kDa band. The size of the band was in agreement with that observed by Western blotting, and purity was estimated to be ∼50%. To purify P2X4 to homogeneity, we used nondenaturing PFO-PAGE (28), followed by electroelution. When separated by PFO-PAGE, 50% pure P2X4 presented as three bands corresponding to a monomer (55 kDa), a faint dimer (110 kDa), and a trimer (165 kDa) (Fig. 5A). No higher order oligomers were observed. We assumed that the observed monomers and dimers corresponded to immature nonassembled proteins recovered from internal membranes. Trimers were selectively purified by excising bands from preparative nonstained PFO-polyacrylamide gels and eluting protein under an electric field. SDS-PAGE analysis of eluted protein demonstrated the high purity obtained using this purification method (Fig. 5B); the only bands visible on the silver-stained gel corresponded to P2X4. (SDS-stable dimeric and trimeric forms were also observed.) In addition, PFO-PAGE of purified trimers was used to demonstrate that the trimeric form was stable after purification (Fig. 5C, arrow). After diafiltration into 0.1% (w/v) DDM, 40 μl of P2X4 was recovered at an approximate concentration of 1 mg/ml. Yield was estimated by comparing the band intensity of pure P2X4 on Coomassie Blue-stained SDS-polyacrylamide gels with known quantities of bovine serum albumin. In summary, 40 μg of pure P2X4 trimer was recovered from 2.5 × 108 cells (equivalent to a confluent monolayer of 5000 cm2 in area).

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