molecular and cell biology  hek 293 cells were maintained at 
  37 c and 5% co2 in growth medium ( hek - gm ) containing 
  dulbecco 's modified eagle 's medium / f-12 , 10% fetal calf serum , and 2 mm 
  l - glutamine ( invitrogen ) . human or mouse p2x1 subunits with a 
  c - terminal emypme epitope tag subcloned into pcdna3.1(+ ) were used as the 
  template for all mutagenesis reactions . the mutations were introduced using 
  quikchange site - directed mutagenesis ( stratagene ) , and the entire coding 
  region was confirmed by sequencing ( seqman ii ; dnastar ) . hek 293 cells were 
  plated out onto 35-mm petri dishes and allowed to attain cell density of 
  10 cells / cm before transfection . the cells were 
  transfected for 46 h using lipofectamine ( invitrogen ; see ref . 12 ) , with 0.5 g / ml 
  receptor dna and 0.05 g / ml of pegfp - n1 for subsequent cell visualization . the cells were then trypsinized , washed , reconstituted in hek - gm , and plated 
  out on glass coverslips at 1500 cells / cm . the 
  coverslips were maintained in 35-mm dishes containing 2 ml of hek - gm at 37 
  c and 5% co2 for 2448 h before recordings were 
  made . whole cell recording and application of agonists and 
  antagonists  all of the cells were pretreated with apyrase ( 2 
  units / ml , type vii ; sigma ) for at least 2 h before the recordings were 
  commenced . the coverslips with attached cells were placed in a recording 
  chamber mounted on the stage of an axiovert microscope ( carl zeiss ) . 
  extracellular recording solution containing 147 mm nacl , 3 
  mm kcl , 1 mm mgcl2 , 2 mm 
  cacl2 , 10 mm hepes , and 13 mm d - glucose , 13 
  ( ph adjusted to 7.4 with naoh ) was superfused at a rate of 5.5 ml / min . whole 
  cell recordings were made at room temperature ( 2023 c ) using an 
  epc9 amplifier , and data were collected using pulse software ( heka ) . patch electrodes and 
   puffer  electrodes ( for atp application ) were pulled from glass 
  pipettes ( harvard apparatus , edenbridge , uk ) on a vertical puller ( heka ) and 
  ranged from 6 to 9 m in resistance when filled with an 
  intracellular solution containing 147 mm nacl , 10 mm 
  hepes , 10 mm egta ( ph adjusted to 7.3 with naoh ) . atp solutions 
  were prepared on the day of recording by diluting 100 mm frozen 
  stock solution ( ph adjusted to 7.3 ) in the external recording solution . suramin and nf449 were prepared as 10 mm frozen stock and diluted 
  to the required concentration on the day of recording currents . atp was 
  applied via a glass puffer pipette ( 1-m tip diameter , 10 p.s.i . , 69 
  kpa ) using a pneumatic picopump ( pv830 ; world precision instruments ) . the tip 
  of the puffer pipette was positioned downstream from the cell with respect to 
  the direction of flow of the superfusing solution and temporarily repositioned 
  to a point 15 m from the cell only for the period of application . 
  with a concentration of atp > 1 m , second applications 2 min 
  after the first evoked a current that was less than 20% of the first response . therefore , all concentration - response relations shown in this study were 
  constructed from pooled data , in which atp ( 0.001100 m ) 
  was applied only once to each cell on each coverslip . accordingly , the same 
  data points for control concentration - response curves appear more than once in 
  the panels of figs . 2 , 
  3 , 
  4 , 
  5 . suramin and nf449 were 
  applied in the superfusing solution for 510 min prior to the 
  application from the puffer pipette of a solution containing both atp and the 
  appropriate antagonist . the currents evoked were then compared with those 
  observed in other cells with no antagonist pretreatment . numerical data are the means  s.e . for 
  the number of macrophages tested . the current traces were obtained using 
  axograph ( molecular devices ) , kaleidagraph ( synergy software , reading , pa ) , 
  and canvas ( acd systems ) software . concentration - response curves were fitted 
  using the nonlinear regression program from prism 4 ( graphpad , san diego , ca ) 
  to the mean values of the currents evoked at each concentration . the agonist 
  concentration required for the half - maximal responses ( ec50 ) is 
  expressed as its negative logarithm ( pec50 )  s.e . statistical significance between data were determined using unpaired 
  t tests and analysis of variance ( instat , graphpad software inc . , san 
  diego , ca ) , and differences were considered significant at the level of 
  p < 0.05 . chemicals all other chemicals used in the 
  present study were purchased from sigma or vwr international ltd.(poole , 
  uk ) . mouse receptors : effect of suramin and nf449atp applied for 
  1 s evoked concentration - dependent inward currents at wild type mouse 
  p2x1 ( mp2x1 ) receptors 
  ( fig . 2 ) . the pec50 
  was 6.5  0.12 ( n = 66 ) , which is similar to values reported 
  previously in hek 293 cells for human 
  ( 12 ) and rat receptors 
  ( 21 , 
  22 ) 
  ( fig . 2 and 
  table 1 ) . similar currents were 
  also evoked by -methylene - atp , with pec50 of 5.4 
   0.4 ( n = 16 ) . suramin ( 10 m ) had little or no 
  effect on the response evoked by 10 m atp 
  ( fig . 2 ) ; neither suramin nor 
  nf449 changed the holding current in the absence of applied atp . the 
  pec50 for atp in the presence of suramin was 6.2  0.53 
  ( n = 40 ) , which is not different ( p > 0.05 ) from that in 
  its absence ( table 1 ) . the 
  effectiveness of atp was not much altered by introduction of single lysine 
  residues at gln , gln , glu , or 
  asn or at all four positions , except in the case of 
  mp2x1[q111k ] , which was less sensitive to atp ( pec50 5.7 
   0.22 , n = 27 ) ( table 
  1 and fig . likewise , these mutations did not cause any marked 
  change in the peak amplitude of the current evoked by atp ( 100 
  m ) , except in the case of mp2x1[q111k ] 
  ( fig . 2b ) , where the 
  maximal peak response was 2-fold greater . in the presence of suramin ( 10 
  m ) , the atp concentration - response curve was significantly 
  ( 40-fold ) shifted to the right for mp2x1[e138k ] and 
  mp2x1[q111k , q127k , e138k , n148k ] ( mp2x1[four k ] ) . it was 
  much less shifted ( 7-fold ) for mp2x1[n148k ] and unaffected in 
  the other two cases ( mp2x1-[q111k ] and mp2x1[q127k ] ) . we 
  tested two concentrations of nf449 ( 3 and 300 nm ) most extensively . 
  when applied against currents evoked by 10 m atp , nf449 had a 
  very small effect at 3 nm but gave almost complete inhibition at 
  300 nm ( fig . concentration - response curves of atp evoked in the presence of 3 nm 
  nf449 showed a pec50 of 6.1  0.22 ( n = 20 ) 
  ( control was 6.5  0.12 , n = 66 ; 
  table 1 ) . the corresponding 
  value for 300 nm nf449 was considerably less than 4 , indicating 
  very complete antagonism ( fig . mp2x1[n138k ] and mp2x1[four k ] 
  were much more sensitive to antagonism by nf449 ( 3 nm ) than the 
  wild type receptor ( fig . in contrast , mp2x1[q127k ] and 
  mp2x1[n148k ] showed only minor changes in blockade by 3 or 300 
  nm nf449 ( fig . the effect on mp2x1[q111k ] was complicated by the 
  change in peak amplitude of the atp current that resulted from the point 
  mutation ( fig . 3b ) ; 
  however , there was no marked increase in the effectiveness of nf449 . replacement of the glutamic acid at position 138 with aspartic acid 
  ( mp2x1-[e138d ] ) resulted in a receptor that was similar to the wild 
  type receptor with respect to its sensitivity to suramin 
  ( table 1 ) . table 1summary pec50 data for wild type and mutants of mouse and 
  human p2x1 receptorsthe values are shown as the means  s.e . , and ( 10 m)nf 449 ( 3 nm)nf449 ( 300 nm)mouse p2x1  wild type 
   6.5  0.12 ( 66 ) 
   6.2  0.53 ( 40 ) 
   0.3 
   6.1  0.22 ( 20 ) 
   0.4 
   < 4 ( 11 ) 
   > 2.5 
   q111k 
   5.7  0.22 ( 22 ) 
   5.6  0.78 ( 14 ) 
   0.1 
   6.2  0.29 ( 17 ) 
   -0.5 
   < < 4 ( 14 ) 
   > > 0.5 
   q127k 
   7.1  0.26 ( 27 ) 
   6.3  0.48 ( 21 ) 
   0.8 
   6.1  0.35 ( 16 ) 
   1.0 
   < 4 ( 14 ) 
   > 3.1 
   e138k 
   6.5  0.33 ( 28 ) 
   4.9  0.14 ( 18 ) 
   1.6a < < 4 ( 14 ) 
   > 2a < < 4 ( 14 ) 
   > > 2.5 
   n148k 
   6.8  0.15 ( 25 ) 
   6.0  0.26 ( 20 ) 
   0.8 
   6.0  0.09 ( 13 ) 
   0.8 
   < 4 ( 14 ) 
   > 2.5 
   four - k 
   6.4  0.26 ( 33 ) 
   4.8  0.09 ( 19 ) 
   1.6a < 4 ( 14 ) 
   > 2a < < 4 ( 14 ) 
   > > 2.5 
 
  e138d 
 
  5.7  0.28 ( 23 ) 
 
  5.6  0.07 ( 20 ) 
 
  0.1 
 
  6.6  0.28 
 
  -0.9 
 
  nt 
  human p2x1  wild type 
   6.5  0.06 ( 57 ) 
   5.1  0.15 ( 28 ) 
   1.4 
   6.2  0.24 ( 22 ) 
   0.3 
   < 4 ( 13 ) 
   > 2.5 
   k111q 
   6.5  0.21 ( 23 ) 
   5.4  0.51 ( 15 ) 
   1.1 
   6.4  0.17 ( 21 ) 
   0.1 
   < 4 ( 14 ) 
   > 2.5 
   k127q 
   6.8  0.09 ( 21 ) 
   5.3  0.37 ( 15 ) 
   1.5 
   6.4  0.3 ( 16 ) 
   0.4 
   < 4 ( 12 ) 
   > 2.8 
   k138e 
   6.9  0.18 ( 26 ) 
   5.9  0.18 ( 18 ) 
   1.0a 6.6  0.25 ( 24 ) 
   0.3 
   5.2  0.19 ( 18 ) 
   1.5a  k148n 
   6.6  0.22 ( 20 ) 
   5.0  0.17 ( 15 ) 
   1.6 
   6.2  0.20 ( 17 ) 
   0.4 
   < 4 ( 14 ) 
   > 2.6 
   no - k 
   6.4  0.09 ( 20 ) 
   5.4  0.08 ( 14 ) 
   1.0a 6.4  0.20 ( 19 ) 
   0 
   5 ( 15 ) 
   1.4a 
  k138r 
 
  6.2  0.23 ( 26 ) 
 
  4.8  0.14 ( 18 ) 
 
  1.4 
 
  6.4  0.18 ( 28 ) 
 
  0.2 
 
  nt 
  alargest differences from corresponding control . summary pec50 data for wild type and mutants of mouse and 
  human p2x1 receptors the values are shown as the means  s.e . , and the numbers of cells 
  tested are shown in parentheses . figure 2.introduction of lysine residues into the mouse receptor can increase 
  sensitivity to suramin . a , representative traces of membrane 
  currents evoked by atp ( 10 m ) , applied for 1 s as indicated by 
  the horizontal bars . b , atp concentration - response curves 
  for wild type mouse p2x1 receptors and for receptors with one or 
  four lysines introduced at the positions indicated . note the increased effectiveness of suramin as an antagonist in the case of 
  mouse p2x1[e138k ] and of mouse p2x1[4k ] receptors . a , representative traces of membrane 
  currents evoked by atp ( 10 m ) , applied for 1 s as indicated by 
  the horizontal bars . b , atp concentration - response curves 
  for wild type mouse p2x1 receptors and for receptors with one or 
  four lysines introduced at the positions indicated . note the increased effectiveness of suramin as an antagonist in the case of 
  mouse p2x1[e138k ] and of mouse p2x1[4k ] receptors . human receptors : effect of suramin and nf449application of 
  atp for 1 s evoked concentration - dependent inward currents with a 
  pec50 of 6.5  0.06 ( n = 57 ) at human 
  p2x1 receptors ( hp2x1 ) , which is similar to values 
  reported previously in hek 293 cells 
  ( 12 ) 
  ( fig . 4 and 
  table 1 ) . inward currents were 
  also evoked by -methylene - atp , with a pec50 of 5.0 
   0.33 ( n = 17 ) . the action of atp at hp2x1 
  receptors was effectively antagonized by suramin ( 10 m ) 
  ( fig . the 
  concentration - response curve to atp was shifted 30-fold to the right , 
  giving a pec50 of 5.1  0.15 ( n = 28 ) 
  ( fig . 4b and 
  table 1 ) . mutation of each of 
  the four lysine residues in hp2x1 receptors to the corresponding 
  amino acid in the mouse receptor did not produce any significant change in the 
  effectiveness of atp , either in the single substitutions or in the quadruple 
  mutation ( hp2x1[no k ] ) ( table 
  1 ) . suramin was a less effective antagonist in the case of 
  hp2x1[k138e ] and hp2x1[no k ] ; in this case the rightward 
  shift was 10-fold . on the other hand , the effectiveness of suramin ( 10 
  m ) was little different in the case of hp2x1[k127q ] 
  and hp2x1[k148n ] ( where the rightward shifts were 38- and 
  42-fold ) . the case of hp2x1[k111q ] was less easy to interpret , 
  because suramin also reduced the maximum effect of atp by 30% 
  ( fig . 4b ) . at wild type hp2x1 receptors , nf449 produced a 
  concentration - dependent antagonism . at 3 nm there was a small 
  rightward shift ( pec50 6.2  0.24 ( n = 22 ) compared 
  with the control of 6.5  0.06 ( n = 57 ) . at 300 nm 
  there was almost complete inhibition ( nf449 at 30 nm had an 
  intermediate effect ; data not shown ) ( fig . the major effect of removing the lysine residues from the 
  hp2x1 receptor was to reduce the effectiveness of nf449 ( 300 
  nm ) as an atp antagonist , in the case of hp2x1[k138e ] 
  and hp2x1[no k ] ( fig . 5 ) , whereas for the substitutions k111q , k127q , and k148n , there 
  was little change in the effectiveness of nf449 at 300 nm . the 
  results with nf449 at 3 nm were consistent with the reduced 
  antagonism in the case of hp2x1[k138e ] and hp2x1[no k ] 
  ( pec50 values of 0.3 and 0.0 , respectively ) 
  ( table 1 ) , but these results 
  are complicated by the finding that 3 nm nf449 also reduced the 
   maximum  atp response at hp2x1 receptors with the 
  single lysine substitutions ( fig . 
  5 ) . figure 3.introduction of lysine residues into the mouse receptor can increase 
  sensitivity to nf449 . a , representative traces of membrane 
  currents evoked by atp ( 10 m ) , applied for 1 s as indicated by 
  the horizontal bars . b , atp 
  concentration - response curves for wild type mouse p2x1 receptors 
  and for receptors with one or four lysines introduced at the positions 
  indicated . note the increased effectiveness of nf449 
  ( 3 nm ) as an antagonist in the case of mouse p2x1[e138k ] 
  and of mouse p2x1[4k ] receptors . a , representative traces of membrane 
  currents evoked by atp ( 10 m ) , applied for 1 s as indicated by 
  the horizontal bars . left panels , control . center and 
  right panels , in nf449 ( 3 and 300 nm ) . b , atp 
  concentration - response curves for wild type mouse p2x1 receptors 
  and for receptors with one or four lysines introduced at the positions 
  indicated . note the increased effectiveness of nf449 
  ( 3 nm ) as an antagonist in the case of mouse p2x1[e138k ] 
  and of mouse p2x1[4k ] receptors . figure 4.removal of lysine residues from the human mouse receptor can reduce 
  sensitivity to suramin . a , representative traces of membrane 
  currents evoked by atp ( 10 m ) , applied for 1 s as indicated by 
  the horizontal bar . b , atp concentration - response curves 
  for wild type mouse p2x1 receptors and for receptors with one or 
  four lysines removed at the positions indicated . note the decreased effectiveness of suramin as an antagonist in the case of 
  human p2x1[k138e ] and also for human p2x1[no k ] 
  receptors . removal of lysine residues from the human mouse receptor can reduce 
  sensitivity to suramin . a , representative traces of membrane 
  currents evoked by atp ( 10 m ) , applied for 1 s as indicated by 
  the horizontal bar . b , atp concentration - response curves 
  for wild type mouse p2x1 receptors and for receptors with one or 
  four lysines removed at the positions indicated . note the decreased effectiveness of suramin as an antagonist in the case of 
  human p2x1[k138e ] and also for human p2x1[no k ] 
  receptors . substitution of lysine by arginine at position 138 
  ( hp2x1[k138r ] ) resulted in a receptor that was not different from 
  wild type hp2x1 receptors in its sensitivity to suramin and nf449 
  ( table 1 ) . this study presents substantial differences between the sensitivity to 
  suramin of mouse and human p2x1 receptors . we tested the hypothesis 
  that this difference resulted from four lysine residues that are present in 
  the human p2x1 receptor ectodomain but that are replaced by neutral 
  or negatively charged residues in the mouse receptor 
  ( fig . 1 ) . lysine residues have 
  been shown to play a critical role in p2x receptor function . this is 
  particularly so for positions 68 and 309 of the human p2x1 receptor 
  ( 23 ) ( and many other p2x 
  receptors ( 24 ) , where any 
  replacement of the lysine results in a channel that is barely or not at all 
  activated by atp . it seems rather unlikely that these residues are in or near 
  an atp - binding site because they are not at all conserved among p2x 
  subunits . in the present work , the effectiveness of the agonist atp was not different 
  between the two species and was not consistently affected by any of the 
  mutations used ( table 1 ) . for 
  example the substitution q111k in the mp2x1 receptor reduced the 
  atp pec50 by 0.8 log units , but the converse mutation k111q in the human 
  receptor had no effect ( table 
  1 ) . in our discussion of the relative effects of the antagonists 
  suramin and nf449 , we have measured effects independently of these changes in 
  the effectiveness of atp ( table 
  1 ) . suramin ( 10 m ) was a much more effective blocker at the 
  human receptor than at the mouse receptor . it shifted the atp 
  concentration - response curve 3-fold in the mouse and 40-fold in the human 
  ( table 1 ) . introduction of a 
  single lysine into the mouse sequence ( e138k ) resulted in a receptor that was 
  at least as sensitive to suramin as the wild type human receptor ( which 
  contains lysine at this position ) ( fig . 
  2 ) . this was true whether the lysine was introduced alone or in 
  combination with three others ( q111k , q127k , and n148k ) . conversely , in the 
  wild type human receptor suramin caused a 25-fold shift in the atp 
  concentration - response curve ; this was only 10-fold for the human receptors in 
  which lys was replaced by glutamate , either alone or in 
  combination with three others ( k111q , k127q , and k148n ) 
  ( table 1 ) 
  ( fig . 4 ) . figure 5.removal of lysine residues from the human can reduce sensitivity to 
  nf449 . a , representative traces of membrane currents evoked by 
  atp ( 10 m ) , applied for 1 s as indicated by the horizontal 
  bar . b , atp concentration - response curves 
  for wild type human p2x1 receptors and for receptors with one or 
  four lysines removed at the positions indicated . note the decreased effectiveness of nf449 ( 300 nm ) 
  as an antagonist in the case of human p2x1[k138e ] and also for 
  human p2x1[no k ] receptors . a , representative traces of membrane currents evoked by 
  atp ( 10 m ) , applied for 1 s as indicated by the horizontal 
  bar . b , atp concentration - response curves 
  for wild type human p2x1 receptors and for receptors with one or 
  four lysines removed at the positions indicated . note the decreased effectiveness of nf449 ( 300 nm ) 
  as an antagonist in the case of human p2x1[k138e ] and also for 
  human p2x1[no k ] receptors . the 
  inhibition observed with 3 nm nf449 was rather small for the wild 
  type mouse p2x1 receptor but became quite striking when a lysine 
  was introduced at position 138 ( mp2x1[n138k ] and 
  mp2x1[four k ] ) ( fig . conversely , 300 nm nf449 strongly blocked the wild 
  type human p2x1 receptor but had much less effect on 
  hp2x1[k138e ] or hp2x1[no - k ] . taken together these 
  reciprocal changes in the effectiveness of suramin and nf449 indicate that the 
  most important contributor to their blocking action is the residue at position 
  138 . these findings were further supported by the restoration of antagonism 
  profiles of wild type p2x1 receptors , by substituting like for like 
  amino acids at glu ( mouse ) and lys ( human ) , 
  mp2x1[e138d ] and hp2x1[k138r ] mutants , respectively . nf449 has eight distributed sulfonate moieties , whereas suramin has six , but 
  the results indicate that it is the same lysine residue that is mostly 
  involved in each case . it is tempting to interpret these results in the 
  context of an electrostatic interaction between antagonist and receptor , 
  although the mutation could also induce a conformational change that 
  influences antagonist binding indirectly . at this position , in contrast to the 
  other three , the mutations cause charge reversal as compared with simple 
  charge addition or charge removal . indeed , the identity of the amino acid at 
  any of the four positions studied in the present work is remarkably degenerate 
  among seven human p2x subunits ; position 111 is occupied by five different 
  residues , position 128 is occupied by five different residues , position 138 is 
  occupied by six different residues , and position 148 is occupied by three 
  different residues . in contrast , the lysine residues of the p2x1 
  receptor that likely contribute to atp - binding site ( at positions 68 , 70 and 
  309 ) are much more conserved 
  ( 2528 ) . suramin blocks rat p2x2 receptors at concentrations very similar 
  to those that were effective in the present study 
  ( 12 , 
  29 ) . however , the rat 
  p2x2 receptor sequence does not possess any lysine residues that 
  correspond in position to those of the human p2x1 receptor . on the 
  other hand , the rat p2x4 receptor is notable for its very weak 
  sensitivity to blockade by suramin 
  ( 13 ) ; it also lacks lysines at 
  the corresponding positions . however , in the case of the p2x4 
  receptor , the human form is more sensitive than the rat . soto et al . 
  ( 14 ) showed that replacing a 
  glutamine residue at position 78 with lysine , which is the residue at the 
  equivalent position in the human sequence , could increase the effectiveness of 
  suramin . this also does not align with any of the four lysines studied in the 
  present work . taken together with the weak sequence conservation in this part 
  of the receptor , the results seem more consistent with the provision of a 
  nonspecific cloud of positivity where the attachment of a suramin ( or nf449 ) 
  can inhibit the conformational changes required for gating . in the present work , the agonist atp was applied to the cells by ejection 
  of a solution of known concentration for 1 s , from a pipette positioned 
  15 m from the cell . furthermore , in addition to reducing the 
  peak amplitude of the response , both antagonists ( but particularly nf449 ) 
  consistently prolonged the decay phase of the inward current ( figs . 
  2 , 
  3 , 
  4 , 
  5 ) . this was observed for the 
  wild type and all of the mutant forms examined ( data not shown ) . p2x1 receptors undergo pronounced desensitization when atp ( or 
  -methylene - atp ) is applied in m range 
  ( 11 , 
  30 ) , so that under the present 
  experimental conditions it is not possible to say whether the primary effects 
  of the antagonists result primarily from binding to open , closed , or 
  desensitized states . suramin was first reported to inhibit native p2x receptors in smooth muscle 
  of the mouse vas deferens ( 7 ) . 
  therefore , the comparative lack of effect on the antagonism by suramin ( 10 
  m ) in native mouse p2x1 receptors 
  ( 19 , 
  20 ) and confirmed in 
  heterologous expressed recombinant p2x1 receptors in the present 
  study was unexpected . there is thus a difference between the weak blockade by 
  suramin of homomeric mouse p2x1 receptors ( present study ) , mouse 
  macrophage receptors ( 19 ) , and 
  mouse megakaryocyte receptors 
  ( 20 ) and the stronger block 
  observed in the mouse vas deferens 
  ( 7 ) . however , vas deferentia 
  removed from mice bred without p2x1 receptor subunits do not 
  respond to added atp ( 31 ) . a 
  possible explanation for these apparently discrepant results might be that the 
  p2x receptor of the mouse vas deferens is actually a heteromeric protein that 
  includes a p2x1 subunit . the results certainly call into question 
  the block by suramin as any kind of defining characteristic of nonhuman 
  p2x1 receptors present in native cells and tissues .
p2x1 receptors belong to a family of cation channels gated by 
  extracellular atp ; they are found inter alia in smooth muscle , 
  platelets , and immune cells . 
 suramin has been widely used as an antagonist at 
  p2x receptors , and its analog 
  4,4,4,4-[carbonylbis(imino-5,1,3-benzenetriylbis(carbonylimino ) ) ] 
  tetrakis - benzene-1,3-disulfonic acid ( nf449 ) is selective for the 
  p2x1 subtype . 
 human and mouse p2x1 receptors were 
  expressed in human embryonic kidney cells , and membrane currents evoked by atp 
  were recorded . 
 atp ( 10 nm to 100 m ) was applied only 
  once to each cell , to avoid the profound desensitization exhibited by 
  p2x1 receptors . 
 suramin ( 10 m ) and nf449 
  ( 3300 nm ) effectively blocked the human receptor . 
 suramin 
  had little effect on the mouse receptor . 
 suramin and nf449 are polysulfonates , 
  with six and eight negative charges , respectively . 
 we hypothesized that 
  species differences might result from differences in positive residues 
  presented by the large receptor ectodomain . 
 four lysines in the human sequence 
  ( lys111 , lys127 , lys138 , and 
  lys148 ) were changed individually and together to their 
  counterparts in the mouse sequence . 
 the substitution k138e , either alone or 
  together with k111q , k127q , and k148n , reduced the sensitivity to block by 
  both suramin and nf449 . 
 conversely , when lysine was introduced into the mouse 
  receptor , the sensitivity to block by suramin and nf449 was much increased for 
  e138k , but not for q111k , q127k , or n148k . 
 the results explain the marked 
  species difference in antagonist sensitivity and identify an ectodomain lysine 
  residue that plays a key role in the binding of both suramin and nf449 to 
  p2x1 receptors .