Acidic Amino Acids Impart Enhanced Ca2+ Permeability and Flux in Two Members of the ATP-gated P2X Receptor Family

P2X receptors are ATP-gated cation channels expressed in nerve, muscle, bone, glands, and the immune system. The seven family members display variable Ca2+ permeabilities that are amongst the highest of all ligand-gated channels (Egan and Khakh, 2004). We previously reported that polar residues regulate the Ca2+ permeability of the P2X2 receptor (Migita et al., 2001). Here, we test the hypothesis that the formal charge of acidic amino acids underlies the higher fractional Ca2+ currents (Pf%) of the rat and human P2X1 and P2X4 subtypes. We used patch-clamp photometry to measure the Pf% of HEK-293 cells transiently expressing a range of wild-type and genetically altered receptors. Lowering the pH of the extracellular solution reduced the higher Pf% of the P2X1 receptor but had no effect on the lower Pf% of the P2X2 receptor, suggesting that ionized side chains regulate the Ca2+ flux of some family members. Removing the fixed negative charges found at the extracellular ends of the transmembrane domains also reduced the higher Pf% of P2X1 and P2X4 receptors, and introducing these charges at homologous positions increased the lower Pf% of the P2X2 receptor. Taken together, the data suggest that COO− side chains provide an electrostatic force that interacts with Ca2+ in the mouth of the pore. Surprisingly, the glutamate residue that is partly responsible for the higher Pf% of the P2X1 and P2X4 receptors is conserved in the P2X3 receptor that has the lowest Pf% of all family members. We found that neutralizing an upstream His45 increased Pf% of the P2X3 channel, suggesting that this positive charge masks the facilitation of Ca2+ flux by the neighboring Glu46. The data support the hypothesis that formal charges near the extracellular ends of transmembrane domains contribute to the high Ca2+ permeability and flux of some P2X receptors.

P2X receptors are ATP-gated cation channels expressed in nerve, muscle, bone, glands, and the immune system. The seven family members display variable Ca 2+ permeabilities that are amongst the highest of all ligand-gated channels (Egan and Khakh, 2004). We previously reported that polar residues regulate the Ca 2+ permeability of the P2X 2 receptor (Migita et al., 2001). Here, we test the hypothesis that the formal charge of acidic amino acids underlies the higher fractional Ca 2+ currents (Pf%) of the rat and human P2X 1 and P2X 4 subtypes. We used patchclamp photometry to measure the Pf% of HEK-293 cells transiently expressing a range of wild-type and genetically altered receptors. Lowering the pH of the extracellular solution reduced the higher Pf% of the P2X 1 receptor but had no effect on the lower Pf% of the P2X 2 receptor, suggesting that ionized side chains regulate the Ca 2+ fl ux of some family members. Removing the fi xed negative charges found at the extracellular ends of the transmembrane domains also reduced the higher Pf% of P2X 1 and P2X 4 receptors, and introducing these charges at homologous positions increased the lower Pf% of the P2X 2 receptor. Taken together, the data suggest that COO − side chains provide an electrostatic force that interacts with Ca 2+ in the mouth of the pore. Surprisingly, the glutamate residue that is partly responsible for the higher Pf% of the P2X 1 and P2X 4 receptors is conserved in the P2X 3 receptor that has the lowest Pf% of all family members. We found that neutralizing an upstream His 45 increased Pf% of the P2X 3 channel, suggesting that this positive charge masks the facilitation of Ca 2+ fl ux by the neighboring Glu 46 . The data support the hypothesis that formal charges near the extracellular ends of transmembrane domains contribute to the high Ca 2+ permeability and fl ux of some P2X receptors.

I N T R O D U C T I O N
A change in calcium concentration is an essential intracellular signal that initiates fundamental physiological processes such as secretion and contraction. To succeed in this role, the intracellular free Ca 2+ concentration ([Ca 2+ ] i ) is tightly controlled, and nature has devised an impressive array of proteins that regulate Ca 2+ transport across cell membranes (Berridge et al., 2003). Neurotransmitter receptors play an essential role in this process. Metabotropic receptors increase [Ca 2+ ] i by modulating voltage-gated Ca 2+ channels or emptying internal Ca 2+ stores (Ross et al., 2005). Ionotropic receptors, like the ATP-gated P2X receptor, take a more direct approach. P2X receptors are ligand-gated cation channels that increase [Ca 2+ ] i by forming a cationpermeable pore (Benham and Tsien, 1987). They are expressed in a diverse range of tissues and are particularly abundant in the nervous system (Khakh, 2001;Illes and Ribeiro, 2004;Khakh and North, 2006). Seven full-length subtypes (P2X 1-7 ) are expressed in mammals, and most are capable of forming both homo-and hetero meric receptors (Torres et al., 1999;North, 2002). The percentage of the ATP-gated current attributed to Ca 2+ (Pf%) varies from ‫3ف‬ to 15% depending on the subunit composition of the oligomeric receptor and the species of origin (Egan and Khakh, 2004). It is large enough to stimulate transmitter release from neurons (Pankratov et al., 2006), hormone release from endocrine glands (Troadec et al., 1998), and contraction of vascular smooth muscle (Ramme et al., 1987;Gitterman and Evans, 2001) and vas deferens (Brain et al., 2002).
Identifying the functional domains responsible for regulating Ca 2+ fl ux is complicated by the lack of a clear picture of channel architecture. Each receptor is an oligomer of three subunits, with individual subunits comprised of intracellular amino and carboxy termini linked by two transmembrane domains and an extracellular loop (North, 2002). Mutagenesis studies indicate a role for the pore-lining second transmembrane segment (TM2) in the cation permeability (Migita et al., 2001) and Ca 2+ current (Egan and Khakh, 2004) of at least one family member, the P2X 2 receptor. The fi rst transmembrane segment (TM1) also lines the pore, and mutagenesis of this domain is known to alter permeability and gating .
We previously reported that ‫%6ف‬ of the total current through the P2X 2 receptor is carried by Ca 2+ (Egan and Khakh, 2004), and that the ability of this receptor to select amongst cations involves a Ca 2+ -sensing Correspondence to Terrance Egan: egantm@slu.edu Abbreviations used in this paper: BU, bead unit; HEK, human embryonic kidney; Pf%, fractional Ca 2+ current; TM, transmembrane. domain made of three polar amino acids of TM2 (Migita et al., 2001). This domain is absent in the two P2X receptors (P2X 1 and P2X 4 ) that display higher Pf%s of 11-15%, suggesting that distinct loci underlie the divergent Ca 2+ currents of different family members. The aim of the present study was to identify the site(s) responsible for the elevated Ca 2+ current of the P2X 1 and P2X 4 receptors.

Molecular Biology and Cell Culture
We used wild-type rat P2X 3 , human P2X 4 (hP2X 4 ), human P2X 5 (hP2X 5 ), and zebrafi sh P2X 4.1 (zP2X 4.1 ) receptors, and C-terminal epitope-tagged rat P2X 1 and P2X 2 receptors (P2X 1 -EYMPME, P2X 2 -DYKDDDDK) that were made and expressed using conventional techniques. Addition of epitope did not change the EC 50 for ATP, the kinetics of the ATP-gated response (Torres et al., 1999), or, most importantly, the contribution of Ca 2+ fl ux to total current through the channel pore of either parent receptor (Table I). Point mutations were introduced with the Quikchange II site-directed mutagenesis kit (Stratagene) and verifi ed by automated DNA sequencing (Retrogen, Inc.). Chimerae were made as previously described (Haines et al., 2001a). All constructs were coexpressed with eGFP in human embryonic kidney-293 cells (HEK-293 or HEK-293T cells; American Type Culture Collection) using Effectene (QIAGEN) or Lipofectamine (Invitrogen) according to the manufacturers protocols. Transfected cells were maintained in Dulbecco's modifi ed Eagle's medium (Invitrogen) supplemented with 10% FBS (Invitrogen or HyClone), 2 mM glutamine, 50 U/ml penicillin G, and 50 μg/ml streptomycin (Invitrogen), and incubated for 24-48 h at 37°C in a humidifi ed, 5% CO 2 atmosphere. They were subsequently replated at low density onto poly-l-lysine-coated glass coverslips (Gold Seal; Becton Dickinson) the night before the experiment.

Patch-Clamp Photometry
Our use of the dye-overload method (Schneggenburger et al., 1993) is described in detail in Egan and Khakh (2004). In brief, we measured fractional calcium current (Pf%) by simultaneously measuring total membrane current and fl uorescence in cells loaded with a high concentration (2 mM) of the calcium-sensitive dye, K 5 fura-2 (for examples, see Fig. 2 A). Cells on coverslips were placed in a recording chamber mounted on the stage of a TE3000 epifl uorescence microscope (Nikon Instruments). ATP-gated current was recorded from single cells held at −60 mV using borosilicate glass recording electrodes (1B150F, World Precision Instruments), an Axopatch 200B amplifi er (Molecular Devices), ITC-16 data acquisition hardware (Instrutech), IGOR Pro software (Wavemetrics), and a G4 computer (Apple Computers). Electrodes had open-tip resistances of 1-3 MΩ and contained an intracellular solution of the following composition (in mM): 140 CsCl, 10 tetraethylammonium Cl, 10 HEPES, 2 K 5 fura-2 (Molecular Probes/Invitrogen), 4.8 CsOH, pH 7.35. Light emitted from the 100-W xenon lamp was passed through a 380-nm bandpass fi lter and directed through the microscope objective (HMC 40X ELWD Plan Fluor; Modulation Optics, Inc.) into the recording chamber by refl ection from the surface of a 400-nm dichroic long pass mirror. Light emitted by fura-2 was gathered by the objective, passed through the dichroic mirror and a 510-nm bandpass fi lter, and measured by a Model 714 Photomultiplier Detection System (Photo Technology International). We controlled the day-to-day variation in the sensitivity of the microscope/PMT by normalizing the fura-2 signal to a "bead unit" (BU). One BU equaled the average fl uorescence of seven Carboxy Bright Blue 4.6-μm microspheres (Polysciences) measured one at a time on the morning of that day's experiment (Schneggenburger et al., 1993;Frings et al., 2000). Subsequent measurements of ATP-evoked changes in the fura-2 fl uorescence of living cells were recorded as multiples of the daily BU. Cells were loaded with fura-2 by passive diffusion through the tip of the recording electrode. The time course of diffusion was measured in a subset of cells by monitoring the increase in intracellular fl uorescence that results from fura-2 entry (Pusch and Neher, 1988). The time constant of loading was 144 ± 16 s (n = 30), and a near steady-state concentration of intracellular fura-2 was reached within ‫01-9ف‬ min of patch disruption. These measurements suggest that 10 min is the minimum time needed to equilibrate the intracellular compartment with the contents of the pipette, and we waited at least this long before acquiring data. The extracellular bath solution was (in mM) 140 NaCl, 2 CaCl 2 , 1 MgCl 2 , 10 glucose, and 10 HEPES, titrated to pH 7.4 with 4 NaOH. HEPES was replaced by MEPS in experiments performed at pH 5. ATP was applied for 0.2-2.0 s once every 2-3 min using triple-barreled theta glass and a Perfusion Fast-Step System SF-77 (Warner Instruments). For each construct and experimental algorithm, we used a concentration of ATP that evoked an easily measured decrease in fl uorescence of fura-2 excited at 380 nm (∆F 380 ) but avoided the problem of dye saturation ( Table I).
As previously reported, we saw no effect of short applications of submaximal concentrations of ATP on the fura-2 fl uorescence of mock-transfected cells using the methods described above (Egan and Khakh, 2004).
The Pf% was calculated as follows: Q T is total charge and equal to the integral of the leak-subtracted ATP-gated transmembrane current. Q Ca is the part of Q T carried by Ca 2+ , and is equal to ∆F 380 divided by the calibration factor F max . F max was determined in a separate set of experiments as previously described (Egan and Khakh, 2004) and equaled 0.0185 ± 0.002 BU/nC (n = 12).

Ca 2+ and Cl − Permeability Measurements
We measured the relative permeabilities of Ca 2+ and Cl − to Cs + (P Ca /P Cs and P Cl /P Cs , respectively) using a reversal potentialbased method and the Goldman equations (Hille, 2004). Membrane current was recorded using an AxoPatch 200B amplifi er, indifferent electrodes suspended in 3 M KCl agar bridges in contact with the bath solution, and the broken-patch confi guration of the whole-cell voltage-clamp technique. The solution in the recording pipette was (in mM) 150 CsCl, 10 EGTA, and 10 HEPES, brought to pH 7.4 with CsOH. Open tip electrode resistances measured 0.7-2.0 MΩ. Giga-ohm seals were established in a standard bath solution of (in mM) 150 NaCl, 1 CaCl 2 , 1 MgCl 2 , 10 glucose, and 10 HEPES, pH 7.4 with NaOH, and subsequently switched to test solutions of different ionic compositions. To measure P Ca /P Cs , the bath solution was fi rst changed to one that contained predominately CsCl (in mM: 150 CsCl, 0.1 CaCl 2 , 1 MgCl 2 , 10 glucose, 10 HEPES), followed by one that contained predominately Ca 2+ (in mM: 112 CaCl 2 , 1 MgCl 2 , 10 glucose, 10 HEPES). We included 0.1 mM Ca 2+ in the CsCl solution to retard pore dilation because this process causes a time-dependent change in cation permeability that could alter P Ca /P Cs (Khakh et al., 1999;Chaumont and Khakh, 2006); in theory, addition of 0.1 mM Ca 2+ to a solution of 150 mM CsCl solution is expected to have a negligible effect on the reversal potential of ATP-gated current (Lewis, 1979). We changed the membrane voltage of cells bathed in each solution from −80 to 60 mV at a constant rate (1.4 V/s) before and during applications of ATP, and measured the membrane voltage corresponding to the zero-current level (i.e., the E rev ) from the leak-subtracted currents. We calculated P Ca /P Cs as where a Cs and a Ca are the activity coeffi cients of Cs + (0.75) and Ca 2+ (0.25), respectively, ∆E rev is E rev,Ca − E rev,Cs , T is temperature (295.15°K, equal to 22°C), and F and R are universal constants. We used bath solutions containing different concentrations of CsCl to measure P Cl /P Cs . The fi rst solution had the same concentrations (154 mM) of Cs + and Cl − as the pipette solution. We applied a voltage ramp to obtain E rev of the ATP-gated current and calculated the junctional offset as the deviation of the measured E rev from the expected zero current level of 0 mV. This was then P2X 1 -EE and P2X 2 -FLAG are the epitope-tagged receptors (P2X 1 -EYMPME and P2X 2 -DYKDDDDK, respectively) that served as templates for mutagenesis. The concentrations of agonist ([ATP]) used in the study were chosen to give measurable responses while avoiding dye saturation, and the pA/pFs are the average current densities measured at these concentrations. Wild-type (WT) P2X 1 and epitope-tagged P2X 1 -EE receptors did not differ in Pf%s, and the pooled data were used as control in subsequent statistical evaluations. Although the Pf% of the tagged P2X 2 -FLAG receptor measured here is the same as that of the WT P2X 2 receptor measured in our previous work reported elsewhere (Egan and Khakh, 2004), we did not pool these two datasets because they were obtained in separate studies performed at distinct locations (Cambridge, UK versus St. Louis, MO). Thus, all comparisons in the present study refl ect differences from the P2X 2 -FLAG receptor. In the three rightmost columns, we compare the Pf%s of mutants and chimerae to the parent receptors. Data that are signifi cantly different from P2X 1 , P2X 2 , and hP2X 4 receptors are marked "Yes" and data that show no difference are marked "No." All data were obtained at extracellular pH 7.4 except where noted. a Not different from the WT P2X 1 receptor. b Unpublished data (n = 10); Li, personal communication. c Data (n = 18) from Egan and Khakh (2004 subtracted from the E rev obtained in a "low" CsCl bath solution that contained (in mM) 30 CsCl, 0.1 CaCl 2 , 1 MgCl 2 , 210 sucrose, 10 glucose, and 10 HEPES. P Cl /P Cs was calculated as o is the ratio of the intracellular and extracellular ion activities (Mitchell et al., 1997). Activity coeffi cients were determined by interpolation of the tabulated data of Robinson and Stokes (1970) and equaled 0.82 and 0.72 for 30 and 150 mM CsCl, respectively.

Data Analysis
All data are presented as the mean ± SEM for the number of experiments stated in the text. Signifi cant differences amongst groups were determined using InStat (GraphPad Software) by one-way ANOVA with Tukey's post hoc, or Student's t test where appropriate. The P values of individual datasets are quoted in the text; values ≤0.05 were considered signifi cant. We used the pooled data of wild-type P2X 1 and P2X 1 -EE receptors as controls for comparisons to mutated receptors because we measured no difference in the Pf%s of these two groups (Table I).

R E S U LT S
The percentage of ATP current carried by Ca 2+ varies signifi cantly amongst members of the P2X receptor family.
In the present study, we fi rst focused on the P2X 1 and P2X 2 receptors because they transduce appreciably different Ca 2+ fl uxes. We measured Pf%s of 11.8 ± 0.5 and 6.7 ± 0.2% for the rat P2X 1 and P2X 2 receptors, respectively (Table I). These values are signifi cantly different (P < 0.001) from each other and in good agreement with previously published results (Rogers and Dani, 1995;Egan and Khakh, 2004). With this baseline established, we examined the effect of genetic manipulation of channel structure on Pf% to gain insight into the molecular basis of the variability in Ca 2+ fl ux throughout the P2X family.
To What Extent Do Individual Transmembrane Segments Infl uence Ca 2+ Flux?
Empirical data suggest that both TM1 and TM2 line the ion-conducting pore of the P2X 2 receptor . Although no such data exist for the P2X 1 receptor, ATP activates cationic currents through chimeric P2X 1 /P2X 2 proteins with swapped transmembrane domains (Werner et al., 1996;Haines et al., 2001a), and therefore it is reasonable to assume that the pores of both channels share a roughly similar design. If so, then the disparity in Ca 2+ fl ux amongst receptors might refl ect subtype-specifi c differences in the primary structures of their pore-lining domains. To determine the infl uence of individual segments, we investigated three chimeric channels in which one (P2X 2 -TM1 x1 and P2X 2 -TM2 x1 ) or both (P2X 2 -TM1/2 x1 ) of the transmembrane segments of the P2X 2 channel were replaced by equivalent stretches of the P2X 1 channel (Fig. 1, A and B). All three chimerae formed functional channels when expressed in HEK-293 cells (Fig. 1 C).
We found that swapping transmembrane segments had quantifi able effects on Pf% (Fig. 2). The chimeric channel containing the fi rst transmembrane segment of the P2X 1 channel in the P2X 2 background (P2X 2 -TM1 X1 ) had a Pf% equal to 9.3 ± 0.6%, which was signifi cantly greater (P < 0.001) than that of the wild-type P2X 2 channel. Substitution of both transmembrane segments (P2X 2 -TM1/2 X1 ) had no greater effect (Pf% of 8.9 ± 0.5%) than replacing TM1 alone. In stark contrast, the chimeric P2X 2 channel containing the TM2 of P2X 1 , (P2X 2 -TM2 x1 ) exhibited a Pf% of 2.6 ± 0.3% that was signifi cantly lower (P < 0.001) than both wild-type channels ( Table I). These results suggest either that: (1) both transmembrane domains contribute to the regulation of Ca 2+ current, each in their own way; or (2) construction of the chimerae produced nonspecifi c changes in the poreforming domains that affect Pf%. To explore these possibilities, we used the less invasive methods described below.
The Ca 2+ Flux of the P2X 1 Receptor Is pH Sensitive The considerable Ca 2+ fl uxes of many ligand-gated channels result from an interaction with negatively charged acidic amino acids in the mouth of the pore (Keramidas et al., 2004;Jensen et al., 2005). We looked for an effect of ionized side chains by measuring the Pf% of P2X 1 and P2X 2 receptors at a lower pH where a greater proportion of the COO − side chains would be protonated and hence neutral (Hille et al., 1975;Green and Andersen, 1991) (Fig. 3 A). The Pf% of the wild-type P2X 1 receptor at pH 5.0 was 5.8 ± 0.8%, a signifi cant (P < 0.001) reduction when compared with that measured at pH 7.4. In contrast, the Pf% of the wild-type P2X 2 receptor was unaffected by changing pH (Fig. 3 D). These data suggest that the electrostatic attraction of Ca 2+ by one or more acidic amino acids is responsible for the high Pf%s of the P2X 1 receptor, and that titratable carboxylates are unlikely to play a role in Ca 2+ fl ux through the P2X 2 receptor.
Acidic Residues Regulate the Ca 2+ Flux of the P2X 1 Receptor Next, we used site-directed mutagenesis to locate the acidic amino acid(s) responsible for the high Ca 2+ fl ux of the P2X 1 channel. The data obtained using chimeric receptors suggested that the search should fi rst center on the transmembrane domains. We discounted the conserved aspartate of TM2 because it is present in all family members (bold black residues of Fig. 1 B), and instead looked for differences in the primary sequences of the transmembrane domains of channels that show higher (P2X 1 , P2X 4 ) and lower (P2X 2 ) Ca 2+ fl uxes. P2X 1 and hP2X 4 channels have the highest Pf%s of all members of the extended family (Egan and Khakh, 2004). Both channels have acidic residues at homologous positions in TM1 (Glu 52 -P2X 1 and Glu 51 -hP2X 4 ) and TM2 (Asp 327 -P2X 1 and Asp 331 -hP2X 4 ) that are missing in the P2X 2 channel (bold red residues of Fig. 1 B).
We engineered P2X 1 mutants in which one or both of these were replaced by the amino acids that occupy the equivalent positions in the P2X 2 receptor (Gln 52 and Ser 326 ). Neutralizing fi xed charge in TM1 (P2X 1 -E52Q) signifi cantly (P < 0.001) reduced Pf% to 8.4 ± 0.4% (Fig. 3 B and Table I). Removing the fi xed charge in TM2 (P2X 1 -D327S) had no signifi cant effect (Pf% equals 10.5 ± 0.5%). However, neutralizing both charges (P2X 1 -DM) caused a greater reduction (6.1 ± 0.6%) than neutralizing the charge of either TM1 (P < 0.01) or TM2 (P < 0.001) alone, and the sum of the effects of the single mutations (42% reduction in Pf%) was roughly equal to the effect of the double mutation alone (48% reduction in Pf%). Further, lowering the pH to 5.0 signifi cantly (P < 0.01) reduced the Pf% of P2X 1 -E52Q to 6.0 ± 0.7% but had no effect on P2X 1 -DM (5.8 ± 1.6%), suggesting that the resident charge at , hP2X 4 (TM1, Gly 29 -Lys 52 ; TM2, Asp 331 -Leu 358 ), and zP2X 4 (TM1, Gly 32 -Lys 55 ; TM2, Asn 340 -Leu 367 ) receptors. The acidic amino acids that are the focus of this study (Glu 52 -P2X 1 , Glu 51 -P2X 4 , Asp 327 -P2X 1 , and Asp 331 -P2X 4 ) are marked with bold red letters. The conserved aspartate (corresponding to P2X 1 -Asp 350 ) found in all family members is marked with bold black letters. The polar residues of TM2 that regulate Ca 2+ current through the P2X 2 receptor (Migita et al., 2001) are marked with bold blue letters. Note that a polar residue occupies only one of the three corresponding positions of the P2X 1 (Ser 337 , underlined) and hP2X 4 (Ser 350 , underlined) receptors. The neighboring amino acids (His 45 and Glu 46 ) of the P2X 3 receptor are shown in bold green letters. (C) Currents recorded in the presence of ATP for the P2X 1 , P2X 2 , and the three chimeric receptors described in the text. ATP was applied for 2 s (open horizontal bars) at concentrations of 10 μM for the P2X 2 -TM1/2 X1 chimera and 3 μM for the others. 3 and 4, the sampling rate of the fl uorescence signals are reduced by decimation to 10 Hz, converted to Q Ca (where Q Ca =F 380 / (F max *BU)), normalized to peak Q T , and then multiplied by 100%. The amplitude of the steady-state value of each trace is equal to Pf%. (C) The bar graph shows the mean and standard error of the Pf% values. Values signifi cantly different from the P2X 1 and P2X 2 receptors are marked with "a" and "b," respectively. position 327 that is present in P2X 1 -E52Q but missing in P2X 1 -DM does affect Pf%. Taken together, these data argue that both Glu 52 and Asp 327 interact with Ca 2+ in the mouth of the pore, although the relative contribution of each amino acid is different.
We also performed the converse experiment of placing carboxylates at appropriate sites in the P2X 2 receptor. We generated three mutant P2X 2 receptors in which one or both of Gln 52 and Ser 326 were replaced by glutamate or aspartate, respectively (Fig. 3, C and D). The Pf% for the single mutants P2X 2 -Q52E and P2X 2 -S326D were 11.2 ± 0.8 and 11.0 ± 0.9%, respectively. Both values are significantly greater (P < 0.001) than the wild-type P2X 2 receptor and approximately equal to the wild-type P2X 1 channel. Placing both mutations in a single construct (P2X 2 -DM) did not increase Pf% more than either mutation alone. Lowering the pH to 5.0 signifi cantly (P < 0.001) reduced the Pf% of P2X 2 -DM to 6.6 ± 1.3%, (Fig.  3 A), suggesting that the increase in Pf% results from an electrostatic effect of the added charge and not an unintended change in protein structure.
The Pf% of the P2X 4 Receptor Decreases when Fixed Charge Is Removed Next, we looked at the effects of mutagenesis on the hP2X 4 receptor because it shows the highest Pf% of all P2X receptors (Egan and Khakh, 2004) and, like the rat P2X 1 receptor, has fi xed negative charge at the outer edges of its transmembrane segments. The Pf% of the wild-type hP2X4 receptor was 16.0 ± 1.0% (Fig. 4). We found that neutralizing both of the fi xed charges of the transmembrane segments by mutagenesis gave a receptor (hP2X 4 -DM) with a signifi cantly (P < 0.001) reduced Pf% of 8.6 ± 0.6%. In contrast, removing a single charge had no effect. Specifi cally, the hP2X 4 -E51Q and hP2X 4 -D332S mutants had Pf%s of 14.6 ± 1.1 and 13.0 ± 0.6%, respectively, that were no different than the wild-type receptor. In this respect, the single charge deletion mutants of the hP2X 4 receptors resemble the single charge addition mutants of the P2X 2 receptor; the presence of one charge per subunit is enough to maintain a high Ca 2+ fl ux through the pore.
As a fi nal test, we measured the Pf% of the wild-type zebrafi sh P2X 4.1 receptor (zP2X 4.1 ) (Kucenas et al., 2003). It lacks the two relevant formal charges found in hP2X 4 receptors (Fig. 1 B) and therefore naturally replicates the charge reduction of P2X 4 -DM. In keeping with our hypothesis, the Pf% of the zP2X 4.1 receptor at 7.3 ± 1.3% was signifi cantly smaller than the hP2X 4 receptor and not different from the rat P2X 2 and hP2X 4 -DM receptors (Fig. 4 B).
Neutralizing Fixed Charge Alters P Ca /P Cs but Not P Cl /P Cs A caveat to using patch-clamp photometry to draw conclusions about the Ca 2+ fl ux of cation channels is that it does not account for the possibility that some mutations may render the channels anion permeable. With the exception of the human and chick P2X 5 receptors, most wild-type P2X receptors are cation selective and therefore impermeable to anion . However, it is possible that some mutations increase Cl − permeability, and under the conditions of our experiments (equimolar [Cl − ] on either side of the membrane and a holding potential of −60 mV), such an effect would lead to an apparent inward current caused by effl ux of Cl − , a larger Q T , and a reduction in Pf% that does not refl ect a change in Ca 2+ fl ux per se. To differentiate a change in Ca 2+ permeability from a change in Cl − permeability, we acquired reversal potential data from ATP-gated currents obtained from cells bathed in a range of extracellular solutions (see Materials and methods).
Switching the bath solution from one that contained 150 mM CsCl to one that contained 112 mM CaCl 2 caused a rightward shift in the reversal potential of the ATPgated current of the wild-type P2X 1 and hP2X 4 receptors ( Fig. 5 A) that translated to P Ca /P Cs values of 3.6 ± 0.2 and 4.6 ± 0.4, respectively (Table II). The shifts were smaller for P2X 1 -DM and hP2X 4 -DM receptors that lack the relevant formal charges, and the resulting P Ca /P Cs s were signifi cantly (P < 0.001) lower than the wild-type templates at 2.6 ± 0.1 and 2.8 ± 01, respectively. They were not signifi cantly different from that of the P2X 2 receptor that measured 2.9 ± 0.1 (Table II). Again, these data support our contention that formal charge infl uences the Ca 2+ dynamics of P2X 1 and P2X 4 receptors.
In contrast, Cl − permeability was not affected by the mutagenesis (Fig. 5 B). We fi rst measured the P Cl /P Cs of the hP2X 5 receptor because it shows a signifi cant Cl − permeability (Bo et al., 2003). As expected, we found that switching to the 30 mM CsCl bath solution caused a ‫-6ف‬mV shift in E rev ; the calculated P Cl /P Cs was 0.66 ± 0.12 (Fig. 5 B). We then measured the Cl − permeability of wild-type and double mutant P2X 1 and P2X 4 receptors. Shifting the extracellular solution from one that contained 150 mM CsCl to one that contained 30 mM CsCl caused an approximately −30 mV shift in the ATP-gated current in all four constructs, demonstrating that both the wild-type and mutant receptors are largely impermeable to anions (Table II). These data support the hypothesis that the changes in Pf% that we measure after mutagenesis refl ect specifi c effects on Ca 2+ fl ux.

P2X 3 Has Fixed Charge but a Relatively Low Pf%
The glutamate of TM1 is conserved in only one other P2X subtype as Glu 46 of the P2X 3 receptor. However, the P2X 3 receptor exhibits the lowest Ca 2+ fl ux of all family members (Egan and Khakh, 2004). Unlike P2X 1 and P2X 4 receptors, the P2X 3 receptor contains a positively charged amino acid, His 45 , immediately upstream to the conserved Glu 46 residue (green residues of Fig. 1 B). His 45 may provide a countercharge in the pore that serves to partially shield the neighboring Glu 46 or simply repel Ca 2+ , thus attenuating the ability of the carboxylate to attract Ca 2+ . To test this hypothesis, we constructed a mutant P2X 3 receptor in which His 45 was replaced by the tyrosine found at the homologous position of the P2X 1 receptor. This produced a small but signifi cant (P < 0.05) increase in Pf% to 6.7 ± 0.5% compared with the wild-type P2X 3 control of 4.8 ± 0.3% (Table I).
We also generated mutant P2X 1 receptors in which Tyr 51 was replaced by either a basic histidine (P2X 1 -Y51H) or a neutral valine (P2X 1 -Y51V). We found that the Pf% of P2X 1 -Y51H was signifi cantly reduced (6.3 ± 0.8%) compared with the wild-type P2X 1 receptor. This decrease was unlikely to be caused by a gross disruption of channel structure because substituting neutral valine (the corresponding residue of P2X 2 ) for Tyr 51 had no effect on Pf% (Table I). Taken together, the data suggest that the identity of the amino acid just upstream of the conserved charges of P2X 1 and P2X 3 receptors helps to determine the magnitude of the Ca 2+ fl ux through the channel pore.

D I S C U S S I O N
We identifi ed acidic residues at the outer ends of the transmembrane domains that contribute to the high fractional Ca 2+ current of ATP-gated P2X 1 and P2X 4 receptors. We found that acidifi cation decreased the relatively large Ca 2+ current of the P2X 1 receptor but had no effect on the smaller Ca 2+ current of the P2X 2 receptor, suggesting that only the former uses acidic amino acids to discriminate amongst permeant cations. (We also looked at the effect of a lower pH on the hP2X 4 receptor. The concentration-response curve of the P2X 4 receptor shifts to the right at lower pHs, necessitating the use of a very high (>1 mM ATP) concentration of ATP to evoke a measurable ligand-gated current. At these high concentrations, applications of ATP evoked biphasic changes in intracellular calcium that did not follow the time course of the integrated current, making measurements of Pf% untenable.) The magnitude of the reduction is surprising when one considers the predicted degree of protonation of the COO − side chains. We expected to measure a modest reduction in Pf% upon lowering the pH to 5.0, a value close to the pK a of glutamate and aspartate in aqueous solution, and where half of the carboxyl groups are protonated and thus unable to attract Ca 2+ (Falke et al., 1994). Instead, we found that acidifi cation produced a Pf% equal to that of mutant receptors lacking both formal charges (i.e., P2X 1 -DM). These data suggest that the pK a s of Glu 52 and Asp 327 of the P2X 1 receptor may be signifi cantly higher than predicted from measurements of pure aqueous solutions of amino acids, an idea in keeping with published reports that the local microenvironment of a protein greatly infl uences the degree of ionization of its constituent amino acids (Fersht, 1985;Klockner et al., 1996;Seifert et al., 1999;Petsko and Ringe, 2004). Of particular interest is the positive charge provided by a conserved lysine (e.g., Lys 53 of the P2X 1 receptor) that sits immediately downstream of Glu 52 . Placing positive charge close to an acidic amino acid is enough to shift the latter's pK a to 7.0 or higher (Falke et al., 1994). We have not pursued a study of this lysine because it is fully conserved and therefore unlikely to play a role in determining the dissimilar Ca 2+ fl uxes measured across the family. However, its role in the absolute magnitude of the Ca 2+ fl ux should be considered in future experiments.
The idea that individual P2X receptors use distinct domains for cation selection is supported by the marked changes in Pf% observed in P2X 1 /P2X 2 chimerae. Replacing the TM1 of the less Ca 2+ -permeable P2X 2 receptor with that of the more permeable P2X 1 receptor The raw data traces show ATP-gated currents evoked in solutions that contained either Cs + (blue traces) or Ca 2+ (red traces) as the dominant extracellular cation. The change in E rev measured after switching from the Cs + to Ca 2+ solution was smaller for the double mutant receptors (right traces) than it was for their wild-type templates (left traces), suggesting that eliminating formal charge decreased P Ca /P Cs . (B) The raw data traces show ATP-gated currents evoked in extracellular solutions that contained either 150 mM (blue traces) or 30 mM CsCl (red traces). For the wild-type P2X 1 and hP2X 4 recep-tors (top and middle left graphs), the change in E rev measured upon switching to the low CsCl solution was ‫33-03ف‬ mV as expected for a channel that showed a low Cl − permeability. The same was true for the double mutant receptors that lacked the relevant formal charges of acidic amino acids (right traces), showing that mutagenesis did not change P Cl /P Cs . The change in E rev was smaller in hP2X 5 receptors that show a signifi cant Cl − permeability (bottom left graph). signifi cantly enhanced Ca 2+ fl ux, as did addition of a single formal negative charge to the extracellular end of TM1 (P2X 2 -Q52E). These data are important for three reasons: fi rst, they add weight to the proposition that TM1 lines the outer segment of the ion channel (Haines et al., 2001b); second, they show for the fi rst time that TM1 makes a measurable contribution to cation selection; and third, they suggest that the divergent Ca 2+ currents of the P2X 1 and P2X 2 subtypes result in part from differences in the primary sequences of their TM1s.
The case of TM2 is more complex. We found that adding fi xed negative charge to TM2 of the P2X 2 receptor increased Pf% only when Thr 336 , Thr 339 , and Ser 340 are unperturbed (compare P2X 2 -S326D to P2X 2 -TM2 X1 ), a fi nding that supports previous reports that the polar residues of TM2 play an important role in Ca 2+ fl ux in this receptor subtype (Egan and Khakh, 2004). Two of these are replaced by neutral amino acids in the P2X 2 -TM2 X1 chimera, thus depriving the parent P2X 2 receptor of polar side chains that may provide the countercharge needed to partially dehydrate Ca 2+ at a constriction within the pore (Migita et al., 2001). If so, then P2X 1 receptors either use other means to overcome the energy barrier provided by the constriction or lack it entirely. An alternative explanation is that the decrease in Pf% in the P2X 2 -TM2 X1 chimera refl ects nonspecifi c changes in the topology of the pore brought about by swapping one TM2 with another. This latter possibility is diffi cult to ignore, and additional experiments are needed to fi rmly establish a role for polar amino acids in ion permeation in some P2X receptors.
Many classes of ligand-gated cation channels use the formal charge of acidic amino acids positioned near the pore to facilitate Ca 2+ transport into cells (Keramidas et al., 2004;Jensen et al., 2005). P2X receptors have three sites that could serve a similar function. One of these is fully conserved in TM2 (see bold black letters in Fig. 1  B) and therefore unlikely to explain the differences in Ca 2+ fl ux measured here. Two nonconserved sites are present in P2X 1 and P2X 4 receptors but not in the P2X 2 receptor, and we found that removing both charges signifi cantly decreased the Pf% and P Ca /P Cs without affecting P Cl /P Cs . Thus, our data are consistent with a model that uses the oxygen atoms of COO − side chains to form an electrostatic ring that attracts cations. The attraction is stronger for Ca 2+ than for Na + , and therefore could explain the high Ca 2+ permeability and fl ux of P2X 1 and P2X 4 receptors. The location of the ring in the permeation pathway is unknown because the inner and outer limits of the pore are poorly defi ned . We favor the hypothesis that it lies in the extracellular vestibule for the following two reasons. First, such a model is consistent with those that explain the high Ca 2+ fl uxes of other ligand-gated cation channels (Barry and Lynch, 2005). Second, models that incorporate the formal charges of aspartate and glutamate side chains into Ca 2+ binding sites within the pore often result in channels that show Ca 2+ block of monovalent current (Voets et al., 2004), and no such effect is reported for either the P2X 1 or P2X 4 receptors.
We suggest the simple hypothesis that the formal charges of both TM1 and TM2 infl uence the fl ux of Ca 2+ through P2X 1 and hP2X 4 channels in a nonadditive fashion. We found that adding charge to either transmembrane domain signifi cantly enhanced Ca 2+ fl ux of the P2X 2 receptor, whereas removing the charge of either Glu 51 or Asp 332 had only a small effect on the Pf% of the hP2X 4 receptor. The results are therefore consistent, for together they suggest that (1) a single charge is suffi cient to cause the elevated Ca 2+ permeability noted in the P2X 2 -DM and wild-type hP2X 4 receptors; and (2) there is very little summation in terms of increasing Pf% when both charges are present in the outer parts of TM1 and TM2.
For the P2X 1 receptor, it appears that Glu 52 of TM1 might play a larger role than Asp 327 of TM2. We found that removing Glu 52 signifi cantly reduced the Pf% of the P2X 1 receptor, whereas removing Asp 327 had no effect, as might be expected if Asp 327 is innocuous. Although these fi ndings seem to invalidate the dual-charge hypothesis discussed above, two experiments suggest that Asp 327 does infl uence Ca 2+ fl ux through the channel. First, subtracting both charges (i.e., P2X 1 -DM) has a signifi cantly greater effect on the Pf% than removing either Glu 52 or Asp 327 alone. Second, acidifi cation decreases the Pf% of P2X 1 -E52Q but has no effect on P2X 1 -DM, and the only difference between these two mutants is the presence of Asp 327 . It is diffi cult to explain why eliminating the glutamate of TM1 significantly decreases the Pf% of P2X 1 but not P2X 4 receptors, except to say that structural differences in TM2 may make the Asp 327 of the P2X 1 receptor either less accessible to Ca 2+ or less charged than either the Asp 332 of the hP2X 4 receptor or the Asp 326 of the P2X 2 -S326D mutant.
P2X 1 , P2X 2 , and P2X 4 receptors have remarkably different phenotypes and display unique agonist and antagonist profi les, rates of desensitization, Ca 2+ permeabilities, and traffi cking (North, 2002). If function follows structure, then structural differences between these subtypes are to be expected. Fig. 6 shows cartoon representations of P2X 1 , P2X 2 and P2X 4 receptors that incorporate two different models of Ca 2+ transport. We assume that each channel is comprised of three subunits (Nicke et al., 1998;Aschrafi et al., 2004;Barrera et al., 2005;Mio et al., 2005), and that each subunit contributes two transmembrane segments to the pore (North, 2002). In P2X 1 and P2X 4 receptors, Ca 2+ current is enhanced by a ring of charge made of glutamate and aspartate residues positioned in the outer mouth of the pore. These residues could function to accumulate extracellular Ca 2+ in the permeation pathway by a simple electrostatic attraction, or form a binding site that selects Ca 2+ over monovalent cations (Hille, 2004;Jensen et al., 2005). As mentioned above, the latter hypothesis seems doubtful because, unlike P2X 2 receptors, Ca 2+ does not block monovalent cation current through P2X 1 receptors as expected for a binding site model (Evans et al., 1996). The extracellular ring of charge is missing in the P2X 2 receptor (Fig. 6, C and D). Instead, polar residues in the middle of the pore provide the countercharge needed to dehydrate Ca 2+ in a narrow part of the pore (Migita et al., 2001). In this model, the narrow constriction provides an energy barrier that impedes current fl ow through the channel, and the relative conductance of cations is determined by their ability to momentarily "bind" to the polar side chains as they shed water. The presence of an intrapore binding site in P2X 2 receptors is supported by two fi ndings. First, ATP-gated currents saturate with increasing concentrations of extracellular Na + (Ding and Sachs, 1999b). Second, monovalent current is blocked by low millimolar concentrations of extracellular Ca 2+ (Nakazawa and Hess, 1993;Ding and Sachs, 1999a).
One question then remains: how well do these two mechanisms describe the Ca 2+ current of other family members? Fixed charge is conserved in the highly Ca 2+permeable P2X 1 and P2X 4 receptors, but is also present in the less permeable P2X 3 and P2X 7 receptors. We have not investigated P2X 7 receptors so far, but experiments with P2X 3 receptors suggest again that each subunit uses a slightly different method to select amongst cations. We suggest that the Pf% of the P2X 3 receptor is relatively low because of the presence of a unique His 45 just upstream of Glu 46 . The positive charge it supplies might serve to partially neutralize the negative charge of Glu 46 and hinder the electrostatic interaction with Ca 2+ . However, it is worth noting that neutralizing His 45 did not elevate the Pf% of the P2X 3 receptor to a value equal to that of P2X 1 receptor, suggesting that additional factors contribute to the lower Pf% measured for P2X 3 receptor. Figure 6. Schematic representations of the ion permeable pores of trimeric P2X 1 , P2X 2 , and P2X 4 receptors. Residues in TM1 are marked with black letters, and residues in TM2 are marked with red letters. The leftmost panels show side (A and E) and top (B and F) views of P2X 1 and P2X 4 receptors. The mouth of the pore is surrounded by an outer ring of six acidic amino acids (two from each of three subunits). The outer ring is missing in the P2X 2 receptor (C and D). Also shown are the three polar residues within TM2 of the P2X 2 receptor that are thought to form a polar ring at a narrow section of the channel pore. Only one of these sites is occupied by a polar amino acid in the P2X 1 and P2X 4 receptors. TM1 also contains a conserved tyrosine (Tyr 43 ) that may be properly positioned to contribute to the polar ring.