molecular and cell biology  hek293 or hek293 t 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 ( p2x1-p2x7 , all 
  tagged with either cfp or yfp ) .
the plasmids were propagated in 
  escherichia coli dh5. the plasmid encoding human 
  p2x4 with a c - terminal eympme tag has been described previously 
  ( 26 ) .
human p2x4 
  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 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% co2 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 .
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 hek293 stable cell line constitutively 
  expressing human p2x4-his10 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 p2x4-his10 was verified by 
  western blotting according to standard protocols , and the highest expressing 
  clone was selected for overexpression .
we used an 
  olympus bx50 microscope equipped with a peltier cooled ( -15 c ) imago ccd 
  camera ( 640  480 pixels , each pixel 9.9  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 40 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
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 mgcl2 , 1 ( or 0.1 ) mm cacl2 , 
  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 4-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 ( 20 p.s.i .
= 6.89 kilopascals ) , we used 
  2 - 4-megaohm pipettes as described previously 
  ( 21 ) ; solution exchange 
  occurred in 10 ms .
membrane fractions were prepared from 
  2.5  10 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 .
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 .
purification of p2x4 trimers1-ml 
  protein - containing fractions from the nickel affinity column were concentrated 
  using 50-kda cutoff filters ( millipore , watford , uk ) and separated by 
  perfluorooctanoic acid ( pfo)-page according to published methods 
  ( 28 ) .
for preparative 
  pfo - page , samples were loaded onto 1.5-mm thick 8% ( w / v ) polyacrylamide gels 
  and run at 4 c at 10 ma .
p2x4 trimers ( 150 - 180 kda ) were 
  excised from nonstained gels and placed into 2-ml d - tube dialysis units 
  ( merck ) , and protein was eluted at 60 v for 2 h at 4 c .
protein samples 
  were diafiltered into phosphate - buffered saline containing 0.1% ( w / v ) ddm .
gold and lectin labeling  for gold labeling , purified 
  p2x4-his10 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 protein - associated gold particles . for lectin 
  labeling ,
purified p2x4-his10 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 p2x4-his10 particles ( in 
  48  48 pixel boxes ) and 2862 lectin - labeled particles ( in 64  64 
  pixel boxes ) were manually selected .
three - dimensional structures were 
  generated using the refined p2x4 structure as a starting model with 
  eight rounds of iterative refinement .
 protein samples ( 20 g / 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  / 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 .
the 
  black traces show agonist - evoked currents for wild - type homomeric 
  p2x1-p2x5 and p2x7 receptors . in each case
, 
  the agonist was applied for 20 - 30 s so that the time course of desensitization 
  could be monitored ( 100 m atp for 
  p2x1-p2x5 and 30 m benzoyl 
  ( bz)-atp for p2x7 ) .
note that the response 
  is similar for each receptor with or without the tag .
the bar graphs below 
  show peak current densities ; here again , the peaks for wild - type and 
  yfp - tagged receptors were similar .
p2x6 receptors were not examined 
  because they do not express in the plasma membrane .
the 
  black traces show agonist - evoked currents for wild - type homomeric 
  p2x1-p2x5 and p2x7 receptors . in each case
, 
  the agonist was applied for 20 - 30 s so that the time course of desensitization 
  could be monitored ( 100 m atp for 
  p2x1-p2x5 and 30 m benzoyl 
  ( bz)-atp for p2x7 ) .
note that the response 
  is similar for each receptor with or without the tag .
the bar graphs below 
  show peak current densities ; here again , the peaks for wild - type and 
  yfp - tagged receptors were similar .
p2x6 receptors were not examined 
  because they do not express in the plasma membrane .
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 .
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  ) . to approximate distances , we used the frster equation , e = 
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 \begin{equation*}r_{0}^{6}/(r_{0}^{6}+r^{6})\end{equation*}\end{document } , 
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 \begin{document } 
 \begin{equation*}r_{0}^{6}\end{equation*}\end{document } = ( 8.79  
  10)ndjda , 
  e is the experimentally determined fret efficiency , and r is 
  the distance between donor and acceptor fluorophores 
  ( 30 , 
  31 ) .
r0 
  includes terms for the donor quantum efficiency ( d ) , 
  the solvent refractive index ( n ) , overlap of the donor emission and 
  acceptor absorption spectra ( jda ) , and the orientation 
  factor (  ) .
r0 is the distance at which 
  e is 50% for any given fluorophore pair .
in the case of cfp and yfp , 
  this is 50  ( 5 nm ) .
notably , r0 contains a 
  term for the fluorophore dipole orientation factor (  ) , 
  which at 2/3 assumes random tumbling of the fluorophores 
  ( 30 ) .
this means that 
  r0 and the distance measurements reported here assume that 
  the fluorophores adopt all spherically random orientations .
given that we do 
  not know the relative orientation of the fluorophores on p2x2 
  channels , assuming a  value of 2/3 seems appropriate 
  ( 30 ) .
when interpreting fret 
  e measurements in terms of distance , it is important to consider that 
  in the case of cfp and yfp the fluorophore is buried in the center of a 
  -can with a width of 30  and a length of 45  
  ( 32 ) .
a further consideration 
  is that we do not know the absolute ratio of donors to acceptors during fret 
  at the single molecule level .
we have previously considered these issues in 
  depth ( 18 , 
  19 ) . for the fret experiments , 
  we chose plasma membrane regions of interest post hoc .
our criteria were that 
  regions of interest ( i ) were bright in both cfp and yfp images ; ( ii ) located 
  at the edge , presumably plasma membrane of the cell ; ( iii ) did not change or 
  drift in intensity during the control period ; and ( iv ) remained in focus 
  during the entire experiment .
tagged p2x receptors are functional  in past work , it has 
  been demonstrated that p2x2 , p2x4 , and p2x5 
  receptors tagged on their c termini with gfp function like their wild - type 
  counterparts in terms of atp - evoked currents and atp sensitivity 
  ( 15 - 25 ) . 
 
we started by recording atp - evoked currents from hek cells expressing cfp- and 
  yfp - tagged p2x receptors and compared the responses with wild - type p2x 
  receptors ( fig .
receptor 
  function was preserved for all receptors and the kinetics of their activation , 
  desensitization , and deactivation were not detectably affected for some 
  receptors . below and in fig .
2 , 
  we present a detailed characterization of these parameters for p2x4 
  receptors .
thus , as expected 
  ( 3 ) , yfp - tagged p2x1 
  and p2x3 receptors desensitized rapidly ( < 1 s ) ; p2x2 
  and p2x4 receptors desensitized slowly ( over tens of seconds ) ; and 
  p2x5 receptors expressed poorly ( 100 m atp used for 
  activation ) . in the case of p2x7 receptors , we recorded large 
  variability in 30 m benzoyl - atp - evoked currents . thus , in the 
  case of the wild - type or yfp - tagged receptors , the currents from single cells 
  were either monophasic or biphasic and displayed slow and variable kinetics 
  ( three exemplar traces are shown in fig . 
  1 ) .
these cell - to - cell differences for p2x7 receptors 
  have been well documented and may represent permeability changes 
  ( 33 ) , variable phosphorylation 
  ( 34 ) , interactions with 
  pannexin-1 channels ( 35 , 
  36 ) , or modulation by 
  calmodulin ( 37 ) .
the focus of 
  this study was not p2x7 receptors , so we can not yet discriminate 
  between these possibilities .
the key point here is that the wild - type and 
  yfp - tagged p2x7 receptors show similar overall responses , but in 
  future work it will be important to determine the most critical factors that 
  contribute to p2x7 response variability .
these overall features 
  show that yfp tagging does not largely affect the functional properties of 
  homomeric p2x receptors or peak currents 
  ( fig .
1 ) . we did not carry out 
  an extensive pharmacological analysis of each tagged receptor because this was 
  not the focus of this study and also because this has been reported already 
  with gfp - tagged p2x2 , p2x4 , and p2x5 
  receptors 
  ( 15 - 25 ) . 
  because the structure of cfp and yfp is similar to that of gfp and because 
  they differ by only a few residues 
  ( 32 ) , it is reasonable to 
  surmise that receptors that can be tagged with gfp can also tolerate cfp and 
  yfp without altering function .
published data show this is the case for 
  p2x2 and p2x4 receptors 
  ( 15 , 
  17 , 
  18 , 
  21 , 
  23 - 25 ) .
a , average 100 m atp - evoked current 
  traces for p2x4 , p2x4-cfp , and p2x4-yfp 
  receptors ( n = 9 ) , with bar graphs below showing the properties of 
  the currents .
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 .
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 ) . functional properties and fret for cfp- and yfp - tagged p2x4 
  receptors .
a , average 100 m atp - evoked current 
  traces for p2x4 , p2x4-cfp , and p2x4-yfp 
  receptors ( n = 9 ) , with bar graphs below showing the properties of 
  the currents .
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 .
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 ) .
functional properties of cfp- and yfp - tagged p2x4 
  receptors  in the remainder of this study , we focused on homomeric 
  p2x4 receptors , and so we began by determining if cfp or yfp 
  tagging affects the overall properties of p2x4 receptors 
  ( fig .
2a shows average 100 m atp - evoked currents 
  from cells expressing wild - type p2x4 , p2x4-yfp , or 
  p2x4-cfp receptors .
the traces are shown as picoamps / picofarads and 
  clearly show that cfp or yfp tagging did not significantly affect the peak 
  responses or the overall shape of the atp - evoked current waveforms ( n 
  = 9 ) .
2a plot the average rise time ( milliseconds ) , percent 
  desensitization in 2 s of atp , and decay time ( milliseconds ) after atp removal 
  for wild - type and cfp / yfp - tagged p2x4 receptors .
we next examined 
  the atp concentration dependence of the atp - evoked responses for wild - type and 
  cfp / yfp - tagged receptors .
2b shows that the concentration - response curves 
  superimpose with ec50 values of 3 m and hill 
  slopes of 1.5 ( n = 8) .
these values are similar to previous work 
  on wild - type p2x4 receptors 
  ( 38 ) .
overall , our data with 
  cfp- and yfp - tagged receptors reproduce past work with gfp - tagged 
  p2x4 receptors , where it was demonstrated that gfp did not affect 
  p2x4 responses 
  ( 15 ) .
receptor - specific fret for homomeric p2x receptors  to 
  measure fret efficiency ( e ) , we studied homomeric p2x receptors 
  tagged with cfp and yfp and expressed in single living hek cells .
we focused 
  on plasma membrane regions of interest and used donor dequenching 
  ( 30 ) to estimate fret 
  e for p2x receptors in these regions 
  ( 19 , 
  39 , 
  40 ) . in 
  fig . 2 ( b and 
  c ) , we present representative images and graphs for 
  p2x4-cfp / p2x4-yfp receptors .
we measured a decrease in 
  p2x4-yfp fluorescence intensity with a time course that followed 
  that of the p2x4-cfp increase when the acceptor yfp was optically 
  destroyed with 525 nm light ( fig . 
  2c ) .
maximum p2x4-cfp photorecovery when 
  p2x4-yfp was 100% photodestroyed corresponds to fret e , 
  which in the case of p2x4-cfp / p2x4-yfp channels near the 
  cell perimeter was 30  5% ( n = 8) .
we repeated these 
  experiments to establish the level of fret noise that arises as cfp and yfp 
  randomly approach a distance of 20 - 70  within the cell .
the 
  measured fret efficiency for cytosolic cfp and yfp and p2x6-cfp and 
  p2x6-yfp receptors within hek cells was 3% 
  ( fig .
3a ) . in these 
  experiments , we used p2x6 receptors as a negative control because 
  they do not express in the plasma membrane and are unassembled 
  ( 41 ) .
we next extended the 
  work by measuring fret e for all homomeric p2x receptors in plasma 
  membrane regions .
3 . these experiments 
  clearly establish that fret e for receptors in plasma membrane 
  regions is subunit - specific . in the case of p2x6 receptors ,
we 
  could not measure fret e for membrane regions because these channels 
  are retained within the cell in intracellular compartments 
  ( 41 ) .
figure 3.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 frster equation relating fret e ( y axis ) and 
  interfluorophore distance ( x axis ) , with fret e data for all 
  homomeric p2x receptors superimposed .
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  is taken 
  as 2/3 and assumes random tumbling as discussed by us and others 
  ( 19 , 
  30 ) .
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 frster equation relating fret e ( y axis ) and 
  interfluorophore distance ( x axis ) , with fret e data for all 
  homomeric p2x receptors superimposed .
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  is taken 
  as 2/3 and assumes random tumbling as discussed by us and others 
  ( 19 , 
  30 ) .
we explored the relationship of the experimentally determined fret 
  e for p2x receptors in the membrane to the dependence of fret between 
  cfp and yfp and interfluorophore distance ( in angstroms ) . for this comparison
, 
  we used the frster equation and an r0 value of 50 
   .
3i plots fret e for all homomeric p2x receptors 
  along with how fret e falls ( as 1/r ) with 
  distance between the chromophores of cfp and yfp .
the numbers next to the data 
  points indicate the length of the cytosolic c - terminal tail for each of the 
  receptors . from these data ,
the fret e 
  for p2x receptors ( and thus one assumes the interfluorophore distance ) is 
  related to the length of the c - terminal tail .
for instance , the channels with 
  the shortest c - terminal tails display the lowest fret e 
  ( e.g. p2x1 and p2x3 , c - terminal tail lengths of 
  44 and 42 residues ) , whereas the channels with the longest c - terminal tails 
  display the highest fret e ( e.g. p2x2 and 
  p2x7 , c - terminal tail lengths of 118 and 238 residues ) . 
 
p2x5 sits intermediate in this trend , consistent with its 
  c - terminal tail being of intermediate length ( 96 residues ) .
p2x4 
  with a c - terminal tail length of 33 residues is the receptor that is slightly 
  shifted in the overall trend .
overall , our data show a surprising trend 
  between fret e for p2x receptors and the c - terminal tail length , 
  implying that the distance between the tips of the c - terminal tails for p2x 
  receptors near the membrane is larger than at the tips of those with longer 
  tails . put another way , the cytosolic domain of p2x2 and 
  p2x7 receptors narrows from its origins at the plasma membrane to 
  its tip ( fig .
3j ) . 
  using this method , the distance between the c - terminal tails of functional 
  p2x4 receptors in the plasma membrane of single living cells was 
  calculated to be 5.6 nm . all of the fret experiments shown in 
  fig .
3 were carried out using 
  identical imaging / analysis methods and in parallel to reduce any variability 
  between experiments . in the case of p2x2-p2x6 receptors , 
 
however , in the case 
  of p2x1 and p2x7 receptors , the linear plots were less 
  esthetically pleasing .
we could think of no precise scientific reason to 
  discard these data and present them faithfully in 
  fig .
the relevant issue here 
  is whether the fret e estimates for p2x1 and 
  p2x7 receptors were adversely affected . to address this ,
we 
  specifically determined if estimates of fret e were adversely 
  affected by prolonged photodestruction that is needed for the linear plots in 
  fig .
3 . we measured fret 
  e for all homomeric p2x receptors from only one point of 
  photodestruction and compared the estimated fret e values with those 
  determined with extensive photodestruction 
  ( fig .
2 , c and 
  d , and fig . 
  3 ) . fig .
thus , although there is some 
  variability between p2x receptors in the extent to which the cfp photorecovery 
  is fit by linear functions ( compare plots in 
  fig .
this is consistent with past work for 
  a variety of fret constructs , including p2x2 receptors , where we 
  showed using three - cube linear unmixing that fret e could be reliably 
  estimated by donor dequenching ( figs . 
  3 and 
  4 ) 
  ( 18 ) .
the graph shows the 
  relationship between fret e estimates from experiments such as those 
  in fig .
3 when extensive 
  photobleaching was employed ( 30 min ) and fret e estimates when 
  only 5 min of photodestruction was used .
the graph shows strong agreement in 
  fret e values for these two donor dequenching methods ( see 
   results  for further details ) .
the graph shows the 
  relationship between fret e estimates from experiments such as those 
  in fig .
3 when extensive 
  photobleaching was employed ( 30 min ) and fret e estimates when 
  only 5 min of photodestruction was used .
the graph shows strong agreement in 
  fret e values for these two donor dequenching methods ( see 
   results  for further details ) .
figure 5.purification and 21  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  .
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 )
h , structure 
  of human p2x4 at 21  resolution displayed at two volume 
  thresholds ( 5.6 , light blue inner core ; and 3 , dark 
  blue outer shell ) above the mean density .
purification and 21  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  .
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 .
h , structure 
  of human p2x4 at 21  resolution displayed at two volume 
  thresholds ( 5.6 , light blue inner core ; and 3 , dark 
  blue outer shell ) above the mean density .
we next sought to generate a three - dimensional structure of purified human 
  p2x4 protein .
first , because fret is an 
  ensemble measurement and makes a number of assumptions about the fluorophore 
  orientations , a three - dimensional structure could be used to verify the fret 
  e measurements .
second , the estimates of distance by fret may be used 
  as a  barometer  to ascertain that the p2x4 structure 
  was of a size appropriate for the channel in the membrane .
this is important 
  because the published single particle and afm studies differ largely on the 
  dimensions of p2x receptors ( see the introduction ) .
functional expression and purification of human 
  p2x4-his10 from hek cells  the expression of 
  human p2x4-his10 was verified by western blotting and 
  compared with that of p2x4-eympme 
  ( 26 ) .
both the c - terminal 
  p2x4 epitope and the his tag were readily detected , and total 
  protein expression levels appeared to be similar to those of 
  p2x4-eympme ( data not shown ) .
whole - cell currents were evoked by a 
  2-s application of 1 , 10 , and 100 m atp in perforated 
  patch - clamp configuration ; peak current densities to 100 m atp 
  were 40  4 pa / picofarads for p2x4-eympme and 45  6 
  pa / picofarads for p2x4-his10 ( n = 8) . 
 
ec50 values were 2.9  0.2 m for 
  p2x4-eympme and 2.7  0.3 m for 
  p2x4-his10 ( n = 8) .
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 . 
 
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 l 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 g of pure 
  p2x4 trimer was recovered from 2.5  10 cells 
  ( equivalent to a confluent monolayer of 5000 cm in area ) .
pure 
  protein was diluted to 25 g / ml , applied to electron microscopic ( em ) 
  grids , and negatively stained with 2% ( w / v ) uranyl acetate .
an image field for 
  wild - type human p2x4 is shown in 
  fig .
single 
  particles of the p2x4 receptor were relatively homogeneous , with 
  the majority ( 70% ) appearing either nearly circular , with a radius of 
  8 nm , or nearly rectangular , with a short side of 8 nm and a long side of 
  12 nm .
we interpreted these particles as either top / bottom views or side 
  views , respectively . in total , 6826 particles were manually selected in 48 
   48 pixel boxes for image processing using the eman software package 
  ( 29 ) .
reprojections of the 
  c3-symmetrized three - dimensional structure , paired with their corresponding 
  unsymmetrized class averages , are shown in 
  fig .
representations of the structure of human p2x4 were generated and 
  are shown in fig . 5h . 
 
the resolution of the wild - type structure was 21  as judged by 
  fourier shell correlation of structures calculated from sets of even- and 
  odd - numbered particles ( fig . 
 
gold and lectin labeling  to orient our structure , the 
  c - terminal his tags of human p2x4 were labeled using 1.8-nm 
  diameter ni - nta - nanogold particles and washing our em grids in a nanogold 
  suspension to prevent unbinding during sample preparation .
a high off - rate for 
  this probe has recently been described 
  ( 42 ) .
reprojections of the c3-symmetrized three - dimensional structure , paired with 
  their corresponding unsymmetrized class averages , are shown in 
  fig .
gold 
  particles were clearly visible , with some classes containing two attached gold 
  particles per trimer .
the gold - labeled particles were significantly smaller 
  than the wild - type particles , most noticeably in areas near where the gold was 
  bound , due to the strong negative density of the gold overshadowing the 
  protein density within its vicinity .
the extensive glycosylation present on human p2x4 receptors 
  enabled labeling of the ectodomain with l. culinaris lectin at a 1:1 
  molar ratio .
analyzed by transmission electron microscopy , the single 
  particles were heterogeneous ( fig . 
 
6b ) , which was expected considering the potential number 
  of lectin - binding sites per p2x4 molecule .
2862 particles were 
  manually selected in 64  64 pixel boxes , and reprojections of the 
  c3-symmetrized three - dimensional structure , paired with their corresponding 
  unsymmetrized class averages , are shown in 
  fig .
. three - dimensional structure of human p2x4 at 21  
  resolution  the overall shape of the molecule was globular and 
  somewhat reminiscent of a torpedo ( fig . 
 
5h ) ; in the gold - labeled structure , three strong negative 
  densities corresponding to the 1.8-nm diameter gold spheres were clearly 
  averaged into the density map toward the narrow end of the molecule 
  ( fig .
6e ) , implying 
  that the propeller - like structure at the top of the molecule corresponded to 
  the ectodomain .
the distance between the centers of the gold particles was 6.1 
  nm ( fig .
6e , black 
  arrow ) , which agrees well with the data from fret experiments ( 5.6 nm ) 
  ( fig .
that we had assigned the correct orientation , the 
  lectin - labeled particle clearly displayed two regions of additional protein 
  density per monomer at the propeller end of the molecule 
  ( fig .
6f ) . the 
  molecular mass of l. culinaris lectin tetramers is 49 kda , but the 
  additional volume observed in the labeled structure was smaller than expected 
  for three tetramers .
this is probably because lectin bound to p2x4 
 n - linked glycan chains present at different positions in the 
  ectodomain , and furthermore , flexibility of each glycan chain could also lead 
  to a smearing out of the lectin location .
such variability would lead to some 
  averaging out of the lectin location by single particle analysis so that only 
  the core of the lectin binding to p2x4 would be observed in the 
  final structure . at a threshold of 5.6 above the mean density 
  ( fig .
5h , light 
  blue inner core ) , the human p2x4 receptor ectodomain displayed 
  cavities with lateral fins running alongside them .
this volume threshold 
  corresponds to a molecular mass of 220 kda and a volume of 270 nm , 
  allowing for one p2x4 trimer in addition to a detergent micelle 
  ( assuming a protein density of 1.35 g / ml and no significant internal cavities 
  within the structure ) . at 3 above the mean density 
  ( fig . 5h , dark 
  blue outer core ) , the cavities disappeared , and the lateral fins were 
  more pronounced .
this density corresponds to a molecular mass of 410 kda and a 
  volume of 506 nm .
our value for the molecular volume of human 
  p2x4 of 270 nm is consistent with the value of 409 
  nm obtained for rat p2x2 using afm imaging ( trimer 
  masses of 165 kda for human p2x4 and 210 kda for rat 
  p2x2 ) ( 10 ) .
the 
  transmembrane domains are most likely shrouded in a torus - shaped detergent 
  micelle , which could explain the bulbous nature of the p2x4 
  three - dimensional structure in the region below the fins that we associate 
  with the ectodomain .
however , we can not rule out the possibility that regions 
  of the ectodomain re - enter the transmembrane region and contribute to the 
  protein content of the transmembrane space .
the main results of this study are ( i ) we developed methods to 
  overexpress p2x4 receptors for structural work ; ( ii ) we 
  determined the shape , architecture , and size of a single human p2x receptor ; 
  and ( iii ) we verified the dimensions with independent measurements 
  using fret .
we initially chose to work on all homomeric p2x receptors using 
  fret microscopy and measured the proximity between their c - terminal tail tips 
  at 5.3 - 6.4 nm .
we then focused on p2x4 receptors for electron 
  microscopy because of its favorable expression profile 
  ( 43 ) .
the single particle 
  studies provide data on the overall shape and size of a single p2x receptor 
  and on the approximate distance between the c - terminal tail tips at 6.1 nm . 
 
the agreement for proximity between fret and electron microscopy attests to 
  the reliability of the methods .
frster theory of energy transfer makes 
  several assumptions about the mutual orientation of the fluorophores ( notably 
  the  value ) . in the usual case ,  is 
  taken to equal 2/3 , which implies adoption of all spherically random 
  orientations between the fluorophores 
  ( 30 ) .
the agreement for 
  distance estimates by fret and single particle imaging suggests that fret is 
  useful as a molecular caliper for membrane - embedded p2x channels .
this is 
  consistent with previous pioneering fret studies by nashmi et al . 
  (
39 ) of neuronal nicotinic 
  receptors and comparisons of distance to the structural model of the muscle 
  nicotinic receptor ( 19 , 
  39 , 
  40 ) .
a , particle field from an electron micrograph of negatively stained 
  ( 2% ( w / v ) uranyl acetate ) single particles of human 
  p2x4-his10 following a 10-min incubation with 1.8-nm 
  diameter ni - nta - nanogold particles .
single particles are indicated by 48 
   48 pixel square boxes ( top / bottom views ) and 48 pixel radius 
  circles ( side views ) .
b , particle field of human 
  p2x4 following incubation with a 1:1 molar ratio of l. 
  culinaris lectin .
single particles are indicated by 64  64 pixel 
  square boxes ( top / bottom views ) and 64 pixel radius circles 
  ( side views ) .
c and d , class averages generated by single particle 
  analysis of gold - labeled ( c ) and lectin - labeled ( d ) particle 
  data sets .
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 left column ) are 
  displayed .
the location of gold particles in the unsymmetrized class averages 
  is indicated by the white arrowheads in the first box ( c ) . 
 
box sizes are 234  234  for the gold - labeled data set and 312 
   312  for the lectin - labeled data set .
e , top , bottom , 
  and side views of gold - labeled p2x4 showing the position of the 
  c - terminally attached gold particles .
the distance between the centers of the 
  gold particles ( indicated by the black arrow ) was 6.1 nm .
f , top , bottom , and side views of lectin - labeled 
  p2x4 showing the small regions of additional density imparted by 
  lectin binding to the ectodomain .
a , particle field from an electron micrograph of negatively stained 
  ( 2% ( w / v ) uranyl acetate ) single particles of human 
  p2x4-his10 following a 10-min incubation with 1.8-nm 
  diameter ni - nta - nanogold particles .
single particles are indicated by 48 
   48 pixel square boxes ( top / bottom views ) and 48 pixel radius 
  circles ( side views ) .
b , particle field of human 
  p2x4 following incubation with a 1:1 molar ratio of l. 
  culinaris lectin .
single particles are indicated by 64  64 pixel 
  square boxes ( top / bottom views ) and 64 pixel radius circles 
  ( side views ) .
c and d , class averages generated by single particle 
  analysis of gold - labeled ( c ) and lectin - labeled ( d ) particle 
  data sets .
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 left column ) are 
  displayed .
the location of gold particles in the unsymmetrized class averages 
  is indicated by the white arrowheads in the first box ( c ) . 
 
box sizes are 234  234  for the gold - labeled data set and 312 
   312  for the lectin - labeled data set .
e , top , bottom , 
  and side views of gold - labeled p2x4 showing the position of the 
  c - terminally attached gold particles .
the distance between the centers of the 
  gold particles ( indicated by the black arrow ) was 6.1 nm .
f , top , bottom , and side views of lectin - labeled 
  p2x4 showing the small regions of additional density imparted by 
  lectin binding to the ectodomain .
the side 
  view of the structure of wild - type human p2x4 is shown in light 
  green ( intracellular region at the bottom ) .
additional density imparted 
  by the lectin labeling , assumed to be regions of glycosylation on the 
  ectodomain , is shown as a dark pink mesh , and the gold particles 
  marking the position of the c termini ( c - term ) are also displayed 
  toward the bottom of the molecule .
the approximate position of a toroidal ddm 
  micelle surrounding the membrane - spanning domain ( msd ) is also 
  included in the model .
the side 
  view of the structure of wild - type human p2x4 is shown in light 
  green ( intracellular region at the bottom ) .
additional density imparted 
  by the lectin labeling , assumed to be regions of glycosylation on the 
  ectodomain , is shown as a dark pink mesh , and the gold particles 
  marking the position of the c termini ( c - term ) are also displayed 
  toward the bottom of the molecule .
the approximate position of a toroidal ddm 
  micelle surrounding the membrane - spanning domain ( msd ) is also 
  included in the model .
one of the key issues in the p2x field is the need to obtain direct 
  structural information for this novel class of membrane proteins .
this is a 
  nontrivial issue because no p2x receptors have been found in bacteria , and 
  there are no other receptors with similar structure based on sequence 
  analysis .
this calls for the development of approaches that can be used for 
  the expression and purification of p2x receptors . in this work ,
we have both 
  developed an efficient expression and purification system for human 
  p2x4 and significantly improved the quality and resolution of 
  existing structural data available for p2x receptors .
our methods should also 
  enable relatively straightforward purification of larger quantities of human 
  p2x4 for higher resolution structure studies .
preparative 
  electrophoresis has been used extensively for the purification of membrane 
  protein complexes ( 44 ) , and in 
  at least one case , this method has yielded sufficient quantities for 
  three - dimensional crystallization 
  ( 45 ) .
the use of our method , 
  by us and others , will allow high resolution structural information on p2x 
  receptors .
selective purification by preparative electrophoresis of a band 
  corresponding in apparent molecular mass to a trimeric form of human 
  p2x4 strongly suggested the application of c3 symmetry in image 
  processing and three - dimensional reconstruction . since initial pioneering work 
  with blue native page by nicke et al . 
 
( 8) , virtually all recent 
  evidence also indicates that trimers are the functional unit of p2x receptors 
  ( 9 - 11 ) . 
 
in addition , we were able to label the c - terminal his tags on each monomer 
  with 1.8-nm gold particles .
although three gold particles attached to a single 
  trimeric particle were only infrequently observed , probably because of a 
  relatively weak binding or a rapid off - rate 
  ( 42 ) , many particles were 
  doubly labeled , and after classification and averaging , a 120 angle was 
  subtended between the two gold labels in the classes corresponding to 
   face - on  views along the c3 axis of rotational symmetry 
  ( fig .
labeling of the large , multiply glycosylated ectodomain with plant lectin 
  confirmed that the  propeller-like domain , previously identified 
  in the low resolution structural analysis as being at the opposite end of the 
  molecule to the ni - nta - gold label , probably corresponded to this extracellular 
  region . a composite model based on all the em data is shown in 
  fig .
5 . note that the expected 
  dimensions of a torus - shaped ddm micelle are indicated , which would leave a 
  relatively small volume at its center corresponding to the transmembrane 
  regions of p2x4 . the final structure is therefore expected to be 
  torpedo - shaped , with a narrow transmembrane domain and compact ectodomain that 
  does not display a central cavity or pore .
approximate maximum dimensions were 
  8 nm in diameter and 12 nm along the longest axis , coincident with the c3 
  symmetry axis .
under our experimental conditions , in which p2x4 was 
  solubilized in detergent , the position of the membrane - spanning domain could 
  not be accurately determined ; however , we have indicated its approximate 
  position in fig .
it is 
  probable that the transmembrane domains were protected from the aqueous 
  environment by a detergent micelle , which could possess a molecular mass of in 
  excess of 50 kda .
however , our structure does not inform us about protein 
  organization within the transmembrane region , and it is possible that regions 
  of the ectodomain may form re - entrant loops , increasing the protein content 
  within the transmembrane space . in the model shown in 
  fig .
7 , the c - terminal 
  decahistidine tag is positioned close to the start of the membrane - spanning 
  domain , a location that would place it close to the polar headgroup region of 
  the inner leaflet of the plasma membrane .
our work represents the first direct structural study of a human p2x 
  receptor , but should be discussed in the light of previous work on rat 
  p2x2 .
( 13 ) purified rat 
  p2x2 from an insect cell line and determined a structure by 
  transmission electron microscopy and single particle analysis .
their structure 
  resembled an inverted three - sided pyramid , 21.5 nm in height and 20 nm in side 
  length , corresponding to a molecular volume of 1200 nm ; this 
  is 4.5-fold greater than our measurement of 270 nm .
the authors 
  stated that their model contained a large , stain - permeable cavity ; however , as 
  the orientation of the particle was not assigned , it was not possible to state 
  which portion of the molecule contained the cavity in their experiments .
the 
  authors assumed that this portion of their structure corresponded to the 
  ectodomain by comparison with the topology map of the receptor .
however , our 
  data clearly show that the ectodomain of human p2x4 is a highly 
  compact structure , such as has been shown in the recently published structure 
  of the trimeric acid - sensing ion channel 1 
  ( 12 ) .
given the high degree of 
  sequence homology between rat p2x2 and human p2x4 , it 
  seems unlikely that the structures at the monomeric level would be so 
  radically different in size and shape . a possible explanation for the 
  discrepancy may be that the rat p2x2 particles were composed of a 
  higher order quaternary structure .
four trimeric p2x2 complexes of 
  the dimensions described here could potentially associate into a large complex 
  with tetragonal symmetry .
such a tetramer of trimers , composed of 12 
  p2x2 polypeptides , would account for the 4-fold greater volume 
  of the rat p2x2 particles versus expectations and the very 
  large ( 20-nm side length ) overall dimensions of the pyramidal particles .
they found that in 
  the presence of 1 mm atp , particles of 10 nm in diameter were 
  observed , with evidence suggesting the presence of a central pore .
the 
  diameter of these particles is more consistent with those observed for human 
  p2x4 in our study ( 8-nm diameter along the c3 axis ) ; however , at 
  the resolution of our study , we could not observe a central pore .
it is 
  possible that afm , which measures the surface topography of the specimen , is 
  detecting an indentation in p2x2 that is not present in 
  p2x4 or , alternatively , that the negative staining and em method do 
  not access this surface topography in p2x4 .
they provided evidence for the trimeric nature of 
  rat p2x2 by measuring the average angle ( 120 ) between 
  peripheral particles interpreted as antibodies attached to the ( three ) c 
  termini of their p2x2 specimen .
their estimate of the volume of the 
  receptor was 409 nm , which is consistent with our measurement for 
  human p2x4 of 270 nm , allowing for the difference in 
  molecular mass between the two receptor subtypes ( 165 kda for human 
  p2x4 trimers versus 210 kda for rat p2x2 
  trimers ) . in summary
, we have introduced an efficient method for purification of 
  human p2x4 that has been overexpressed in human cells .
this 
  methodology may be readily applicable to other members of this family and 
  could be of interest for the purification of other membrane proteins where 
  isolation of a single oligomeric form is desirable .
this method has allowed us 
  to study the overall structure and topology of p2x4 and has helped 
  to interpret existing structural data for this unique and intriguing family of 
  nonselective cation channels .
our work has enabled visualization of some 
  global structural features of these receptors and paves the way for future 
  studies .
this work should be viewed as an initial and necessary step toward 
  the longer term goal of obtaining high resolution structural information for 
  p2x receptors . from this perspective
, it is worth considering that spectacular 
  progress has been made using cryoelectron microscopy studies of nicotinic 
  receptors since initial views of the quaternary structure published in 1985 
  ( 46 ) to a recent 4  
  resolution model ( 47 ) .
 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 p2x4 receptors was 5.6 nm . 
 single particle 
  analysis of purified p2x4 receptors was used to determine the 
  three - dimensional structure at a resolution of 21 ; 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 p2x4 
  is a globular torpedo - like molecule with an approximate volume of 270 
  nm3 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 . 
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION