ain (2). The functional channel unit is thought to be a trimer (8 -11), a feature that is shared with acid-sensing ion channels (12). A low resolution structural study of the rat P2X 2 isoform has been published; electron microscopy of single particles gave rise to a structure with a volume of ϳ1200 nm 3 (13). However, the orientation of the molecule was not assigned, and there was no independent evidence to verify the dimensions. Moreover, atomic force microscopy (AFM) 3 studies suggest a smaller molecular volume of 490 nm 3 ; however, these studies modeled the channel as a flat spherical cap with a height of 2.9 nm (10,14). Because the thickness of the plasma membrane is ϳ7 nm, this implies that the AFM studies reported on molecules in a squashed conformation (10,14). Thus, the available data do not provide a consistent or satisfying view of the shape, architecture, or size of P2X receptors.
To further our structural knowledge of P2X receptors and to address the above discrepancies in estimates of molecular volume and size, we have explored two parallel experimental approaches. First, we measured the efficiency of fluorescence resonance energy transfer (FRET) between subunits within homomeric P2X receptors bearing yellow (YFP) or cyan (CFP) fluorescent protein tags at their C termini. Extensive previous studies by us and others have employed green fluorescent protein (GFP)-labeled P2X receptors (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25), demonstrating that tagging P2X 2, P2X 4, and P2X 5 receptors on the C termini with GFP does not affect receptor function. Second, we overexpressed P2X 4 in human embryonic kidney 293 (HEK293) cells and purified the trimeric form to homogeneity. We used single particle analysis and domain-specific labeling to generate the three-dimensional structure of human P2X 4 at a resolution of 21 Å, oriented with respect to the lipid bilayer.

EXPERIMENTAL PROCEDURES
Molecular and Cell Biology-HEK293 or HEK293T cells transiently expressing the appropriate wild-type and fluorescently labeled P2X receptor subunits were used. Some cDNAs were available from previous work (18 -20), and others were generated using standard cloning procedures. The resulting plasmids were verified by sequencing (P2X 1 -P2X 7, all tagged with either CFP or YFP). The plasmids were propagated in Escherichia coli DH5␣. The plasmid encoding human P2X 4 with a C-terminal EYMPME tag has been described previously (26). Human P2X 4 containing a C-terminal decahistidine tag was generated from this construct by PCR. HEK293 cells (obtained from American Type Culture Collection) were maintained in 75-cm 2 cell culture flasks (Corning) in Dulbecco's modified Eagle's medium/nutrient mixture F-12 with Glutamax (Invitrogen) supplemented with 10% fetal bovine serum and penicillin/streptomycin. Cells were grown in a humidified atmosphere of 95% air and 5% CO 2 at 37°C in a cell culture incubator. The cells were split 1 in 10 when confluence reached 60 -90%, which was generally every 4 days. Cells were prepared for transfection by plating onto 6-well plates at the time of splitting, 3-4 days before transfection. They were transfected at ϳ60% confluence. For transient expression in HEK293 cells, we used 0.5-1 g of plasmid cDNA and Effectene transfection reagent (Qiagen Inc.) for each well of a 6-well plate. The manufacturer's instructions were followed, with 4 l of enhancer and 10 l of Effectene were used for each transfection. The transfection efficiency was 40 -60%. The HEK293 stable cell line constitutively expressing human P2X 4 -His 10 was generated using electroporation and selection with 400 g/ml G418 (Invitrogen, Paisley, UK) for 14 days in 15-cm culture dishes. Single colonies were isolated using sterile greased stainless steel rings, trypsin-treated, and transferred to 24-well plates. Expression of P2X 4 -His 10 was verified by Western blotting according to standard protocols, and the highest expressing clone was selected for overexpression.
Epifluorescence FRET Microscopy-We have previously described the setup (19). We used an Olympus BX50 microscope equipped with a Peltier cooled (Ϫ15°C) Imago CCD camera (640 ϫ 480 pixels, each pixel 9.9 ϫ 9.9 m), epifluorescence condenser, control unit (containing ISC and PDC boards), and a Polychrome IV monochromator (all from Till Photonics). The light from the monochromator was led to the microscope through a quartz fiber light guide. The hardware was controlled by a personal computer, an appropriate frame grabber (TILL Photonics), and macros driven by TILLvisION Version 3.3 software. The cells were viewed with a 40ϫ water immersion objective lens with a numerical aperture of 0.8 (Olympus). We used the following filters for acquiring CFP or YFP images (all from Glen Spectra, Stanmore, UK; order is dichroic, emitter in nanometers): CFP (455DRLP, 480AF30) and YFP (525DRLP, 545AF35). To photodestroy the YFP, we used 525 nm light from the monochromator and a 525DRLP dichroic.
Electrophysiology-HEK293 cells were used for recordings 24 -48 h post-transfection. Cells were gently mechanically dispersed and plated onto glass coverslips 2-12 h before use. We included this step (27) to ensure adequate voltage clamp during reversal potential measurements from single spherical cells. The extracellular recording solution comprised 150 mM NaCl, 1 mM MgCl 2, 1 (or 0.1) mM CaCl 2, 10 mM HEPES, and 10 mM glucose (pH 7.4), and the pipette solution comprised 154 mM NaCl (or CsCl or CsF), 11 mM EGTA, and 10 mM HEPES. Whole-cell voltage-clamp recordings were made with ϳ4-megaohm borosilicate glass electrodes (World Precision Instruments) using an Axopatch-1D or Axopatch 200 amplifier controlled by a computer running pCLAMP 8.1 or 10 software via a Digidata 1322 interface (Molecular Devices). Data were filtered at 0.5-2 kHz and digitized at 1-5 kHz. Drugs were applied to single cells by pressure ejection using a Picospritzer (Intracell Ltd.) or in the bathing medium, which flowed at 2-3 ml/min. For pressure application (ϳ20 p.s.i., 1 p.s.i. ϭ 6.89 kilopascals), we used 2-4-megaohm pipettes as described previously (21); solution exchange occurred in ϳ10 ms.
Protein Purification-Membrane fractions were prepared from 2.5 ϫ 10 8 cells and solubilized in phosphate-buffered saline containing 1% (w/v) ␤-D-dodecyl maltoside (DDM; Merck, Nottingham, UK) and protease inhibitors (Complete-EDTA, Roche Applied Science). Samples were diluted into buffer A (150 mM NaCl, 20 mM sodium phosphate (pH 7.4), 150 mM imidazole, and 0.1% (w/v) DDM) and incubated overnight at 4°C with nickel-Sepharose beads (Amersham Biosciences, Chalfont St. Giles, UK) to capture His-tagged protein. Beads were packed into columns and washed with 20 column volumes of buffer A; 50% pure His-tagged protein was eluted in buffer A containing 500 mM imidazole.
Gold and Lectin Labeling-For gold labeling, purified P2X 4 -His 10 in 0.1% DDM was incubated for 10 min at room temperature with a 10:1 molar excess of 1.8-nm diameter nickel-nitrilotriacetic acid (Ni-NTA)-Nanogold particles (Nanoprobes, Yaphank, NY). Samples were adsorbed onto grids and washed with Nanogold suspension to prevent unbinding of the proteinassociated gold particles. For lectin labeling, purified P2X 4 -His 10 in 0.1% DDM was mixed with Lens culinaris lectin (Sigma, Poole, UK) at a molar ratio of 1:1 and immediately applied to grids. After transmission electron microscopy and image processing, 1966 gold-labeled P2X 4 -His 10 particles (in 48 ϫ 48 pixel boxes) and 2862 lectin-labeled particles (in 64 ϫ 64 pixel boxes) were manually selected. Three-dimensional structures were generated using the refined P2X 4 structure as a starting model with eight rounds of iterative refinement.
Structure Determination by Electron Microscopy and Single Particle Analysis-Protein samples (20 g/ml) were adsorbed onto glow-discharged carbon-coated copper grids and nega-tively stained with 2% (w/v) uranyl acetate. Transmission electron microscopy images were recorded on Kodak SO-163 film at a calibrated magnification of ϫ43,500 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 ϫ 48 pixel boxes. Using the