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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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Functional properties and FRET for CFP- and YFP-tagged P2X4 receptors. A, average 100 μm ATP-evoked current traces for P2X4, P2X4-CFP, and P2X4-YFP receptors (n = 9), with bar graphs below showing the properties of the currents. (The kinetic values are time constants.) pF, picofarads. B, concentration-response curves for wild-type and CFP/YFP-tagged receptors. C, images of two HEK cells expressing P2X4-CFP and P2X4-YFP before and after photodestruction of the acceptor YFP. Note that the fluorescence intensity of the YFP channel decreases following photodestruction, whereas that of the CFP channel increases. The images were from the 30-min photodestruction time point. D, time course of YFP photodestruction and CFP photorecovery for whole-cell and near membrane regions. Graphs such as these are used to determine FRET e. YFP photodestruction is plotted versus CFP photorecovery, and the data are fit with a straight line and extrapolated to 100% YFP photodestruction. The intersection with the y axis represents the FRET e (see Fig. 3).
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fig2: Functional properties and FRET for CFP- and YFP-tagged P2X4 receptors. A, average 100 μm ATP-evoked current traces for P2X4, P2X4-CFP, and P2X4-YFP receptors (n = 9), with bar graphs below showing the properties of the currents. (The kinetic values are time constants.) pF, picofarads. B, concentration-response curves for wild-type and CFP/YFP-tagged receptors. C, images of two HEK cells expressing P2X4-CFP and P2X4-YFP before and after photodestruction of the acceptor YFP. Note that the fluorescence intensity of the YFP channel decreases following photodestruction, whereas that of the CFP channel increases. The images were from the 30-min photodestruction time point. D, time course of YFP photodestruction and CFP photorecovery for whole-cell and near membrane regions. Graphs such as these are used to determine FRET e. YFP photodestruction is plotted versus CFP photorecovery, and the data are fit with a straight line and extrapolated to 100% YFP photodestruction. The intersection with the y axis represents the FRET e (see Fig. 3).

Mentions: Imaging Data Analysis—For donor dequenching experiments, the FRET efficiency (e) was calculated as e = (1 - (IC-before/IC-after)) × 100, where IC-before is the donor fluorescence intensity before photodestruction and IC-after is the intensity after photodestruction. The photodestruction of the YFP proceeds with kinetics similar to the dequenching of the donor (see Fig. 1C), and plotting the photorecovery versus photodestruction yields a linear plot (see Fig. 2). We used such linear plots and extrapolated to 100% acceptor photodestruction to calculate the maximum donor dequenching for epifluorescence microscopy: e is given by the y axis intercept. In a specific set of experiments, we also calculated FRET e from just one point of photodestruction and extrapolated to 100% destruction. We did this to ensure that the estimates of e were not affected by the method used to measure FRET (see Fig. 4), particularly for P2X1 and P2X7 receptors (see “Results”).


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)

Functional properties and FRET for CFP- and YFP-tagged P2X4 receptors. A, average 100 μm ATP-evoked current traces for P2X4, P2X4-CFP, and P2X4-YFP receptors (n = 9), with bar graphs below showing the properties of the currents. (The kinetic values are time constants.) pF, picofarads. B, concentration-response curves for wild-type and CFP/YFP-tagged receptors. C, images of two HEK cells expressing P2X4-CFP and P2X4-YFP before and after photodestruction of the acceptor YFP. Note that the fluorescence intensity of the YFP channel decreases following photodestruction, whereas that of the CFP channel increases. The images were from the 30-min photodestruction time point. D, time course of YFP photodestruction and CFP photorecovery for whole-cell and near membrane regions. Graphs such as these are used to determine FRET e. YFP photodestruction is plotted versus CFP photorecovery, and the data are fit with a straight line and extrapolated to 100% YFP photodestruction. The intersection with the y axis represents the FRET e (see Fig. 3).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig2: Functional properties and FRET for CFP- and YFP-tagged P2X4 receptors. A, average 100 μm ATP-evoked current traces for P2X4, P2X4-CFP, and P2X4-YFP receptors (n = 9), with bar graphs below showing the properties of the currents. (The kinetic values are time constants.) pF, picofarads. B, concentration-response curves for wild-type and CFP/YFP-tagged receptors. C, images of two HEK cells expressing P2X4-CFP and P2X4-YFP before and after photodestruction of the acceptor YFP. Note that the fluorescence intensity of the YFP channel decreases following photodestruction, whereas that of the CFP channel increases. The images were from the 30-min photodestruction time point. D, time course of YFP photodestruction and CFP photorecovery for whole-cell and near membrane regions. Graphs such as these are used to determine FRET e. YFP photodestruction is plotted versus CFP photorecovery, and the data are fit with a straight line and extrapolated to 100% YFP photodestruction. The intersection with the y axis represents the FRET e (see Fig. 3).
Mentions: Imaging Data Analysis—For donor dequenching experiments, the FRET efficiency (e) was calculated as e = (1 - (IC-before/IC-after)) × 100, where IC-before is the donor fluorescence intensity before photodestruction and IC-after is the intensity after photodestruction. The photodestruction of the YFP proceeds with kinetics similar to the dequenching of the donor (see Fig. 1C), and plotting the photorecovery versus photodestruction yields a linear plot (see Fig. 2). We used such linear plots and extrapolated to 100% acceptor photodestruction to calculate the maximum donor dequenching for epifluorescence microscopy: e is given by the y axis intercept. In a specific set of experiments, we also calculated FRET e from just one point of photodestruction and extrapolated to 100% destruction. We did this to ensure that the estimates of e were not affected by the method used to measure FRET (see Fig. 4), particularly for P2X1 and P2X7 receptors (see “Results”).

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