P2X receptors are a family of ATP-gated ion channels thought to have intracellular N and C termini and two transmembrane segments separating a large extracellular domain. We examined the involvement of the second putative transmembrane domain (TM2) of the P2X2 subunit in ion conduction, using the substituted cysteine accessibility method (SCAM). This method tests the ability of hydrophilic reagents such as Ag+ or the methanethiosulfonates to modify covalently the sulfhydryl side chains exposed to aqueous environments. ATP-gated current was measured in HEK293 cells transiently expressing either wild-type or functional mutant P2X2 receptors containing a cysteine substitution in or around TM2. Application of Ag+ to gating channels had no sustained effect on wild-type P2X2 (WT) but irreversibly altered whole-cell currents in 15 mutants. By contrast, bath application of (2-aminoethyl)methanethiosulfonate (MTSEA) to closed channels inhibited 8 of the 15 residues affected by Ag+ when the channel was gating. Inhibition of the closed channel was prevented in seven of eight mutants when membrane-permeant MTSEA was scavenged by 20 mM intracellular cysteine, indicating that these seven mutants lie on the intracellular side of the channel gate. Further, MTSEA inhibited current through G342C in the absence of intracellular cysteine but augmented the current when cysteine was present, suggesting that this residue may be part of the gate. Taken together, the data help to the identify a functional domain of the channel pore by mapping residues on either side of the channel gate.

P2X receptors are a family of ion channels gated by extracellular ATP. Each member of the family can form functional homomeric channels, but only P2X2 and P2X3 have been shown to combine to form a unique heteromeric channel. Data from in situ hybridization studies suggest that P2X1 and P2X5 may also co-assemble. In this study, we tested this hypothesis by expressing recombinant P2X1 and P2X5 receptor subunits either individually or together in human embryonic kidney 293 cells. In cells expressing the homomeric P2X1 receptor, 30 microM alpha,beta-methylene ATP (alpha,beta-me-ATP) evoked robust currents that completely desensitized in less than 1 sec, whereas alpha,beta-me-ATP failed to evoke current in cells expressing the homomeric P2X5 receptor. By contrast, alpha, beta-me-ATP evoked biphasic currents with a pronounced nondesensitizing plateau phase in cells that co-expressed both subunits. Further, the EC50 for alpha,beta-me-ATP was greater in cells expressing both P2X1 and P2X5 than in cells expressing P2X1 alone (5 and 1.6 microM, respectively). Heteromeric assembly was confirmed using a co-immunoprecipitation assay of epitope-tagged P2X1 and P2X5 subunits. In summary, this study provides biochemical and functional evidence of a novel channel formed by P2X subunit heteropolymerization. This finding suggests that heteromeric subunit assembly constitutes an important mechanism for generating functional diversity of ATP-mediated responses.

P2X receptors are simple polypeptide channels that mediate fast purinergic depolarizations in both nerve and muscle. Although the depolarization results mainly from the influx of Na ؉ , these channels also conduct a significant Ca 2؉ current that is large enough to evoke transmitter release from presynaptic neurons. We sought to determine the molecular basis P2X receptors are a ubiquitous family (P2X 1 -P2X 7 ) of ligandgated ion channels activated by physiological concentrations of extracellular ATP (1). Six of the seven family members form functional homomeric receptors that are distinguished by their phenotypic response to ATP and their relative sensitivity to a range of purinergic agonists and antagonists (2). Individual subunits coassemble into oligomers (3), although the number of subunits needed to form a complete complex is still debated (4, 5). Both homomeric and heteromeric receptors are implicated in the physiological responses of ATP (1, 6), and recent transgenic experiments demonstrate that mice lacking individual subtypes display distinct phenotypes suggesting that P2X receptors are linked to such diverse phenomena as ejaculation (7), pain (8, 9), and inflammation (10).We know more about the biophysics of homomeric P2X 2 receptors than other family members primarily because this subunit shows a relatively slow rate of desensitization; the slow desensitization results in sustained responses to applications of ATP that are easy to study under voltage-clamp. Like all family members, P2X 2 receptors are freely permeable to small cations, impermeable to anions, and display a strong preference for Ca 2ϩ over Na ϩ (11,12). Furthermore, they show a conductance sequence to alkali metal cations that differs from the relative mobility of these ions in water, suggesting that the receptor distinguishes among similarly sized ions (13). The molecular basis of this selection is unknown. Each P2X 2 subunit has two transmembrane-spanning domains (14), and scanning cysteine mutagenesis studies demonstrate that the second of these, TMD2, 1 forms a hydrated surface of the channel pore (15, 16). In or near TMD2 are two conserved aspartates (Fig. 1) that may facilitate cation transport through the pore by altering the local concentrations of cations and anions. In addition, TMD2 contains a number of pore-lining, polar amino acids that may regulate cation current by solvating ions within the pore (16).To identify the specific amino acids of TMD2 that play a role in monovalent cation and Ca 2ϩ permeability, we measured relative permeability sequences from the reversal potentials of ATP-gated currents. Acidic and polar amino acids within TMD2 of the P2X 2 receptor were mutated one at a time to alter the charge, polarity, and/or volume of the side chain. P Ca /P Cs was measured first because the high Ca 2ϩ permeability suggests that sites within the pore confer a favorable electrostatic profile for Ca 2ϩ transport. Then, relative monovalent cation permeabilities were measured for all mutants displaying altered Ca 2ϩ permeabi...

Scanning cysteine mutagenesis was used to identify potential pore-forming residues in and around the first transmembrane domains of ionotropic P2X 2 receptor subunits. Twenty-eight unique cysteine-substituted mutants (R28C-Y55C) were individually expressed in HEK293 cells by lipofection. Twenty-three of these were functional as assayed by application of ATP to transfected voltage-clamped cells. Individual mutants varied in their sensitivity to ATP; otherwise, currents through functional mutant receptors resembled those of the homomeric wild-type (WT) receptor. In five (H33C, R34C, I50C, K53C, and S54C) of 23 functional mutants, coapplication of 30 M ATP and 500 nM Ag ϩ irreversibly inhibited inward current evoked by subsequent applications of ATP alone. These inhibitions did not result in a lateral shift in the agonist concentration-response curve and are unlikely to involve a modification of the agonist binding site. Two (K53C and S54C) of the five residues modified by Ag ϩ applied in the presence of ATP when the channels were gating were also modified by 1 mM (2-aminoethyl)methanethiosulfonate applied in the absence of ATP when the channels were closed. These data suggest that domains near either end of the first transmembrane domain influence ion conduction through the pore of the P2X 2 receptor. Key words: ATP; scanning cysteine mutagenesis; purinergic; ion channel; ligand-gated; methanethiosulfonateATP is unusual in its ability to influence cell activity from both the intracellular and extracellular compartments. Intracellular hydrolysis of ATP to adenosine 5Ј-diphosphate and inorganic phosphate provides the energy needed to drive a wide range of energetically unfavorable chemical reactions and is an important source of phosphate in many biosynthetic reactions (Alberts et al., 1998). Extracellular ATP modulates cell excitability by activating membrane-bound P2 purinoceptors (Ralevic and Burnstock, 1998). One branch of this family, the P2X receptors, is a class of ligand-gated ion channels that conduct the flow of cations across the cell surface membranes of a wide variety of tissues (Khakh et al., 2001). Conduction occurs when the ion channel opens as a result of agonist occupation of an extracellular binding site. The molecular mechanism by which occupation evokes channel gating remains a mystery, attributable in part to an incomplete mapping of the functional domains of the receptor complex, including the agonist binding site and the channel pore. The general location of the ion-conducting pore can be inferred from recent experiments that examined the secondary structure of individual isoforms. P2X receptors incorporate at least three equivalent subunits (Kim et al., 1997;Nicke et al., 1998) and are homomeric or heteromeric in composition (Torres et al., 1999). Each subunit within a complex crosses the membrane twice in such a way that the intracellular N and C termini are linked by a large ectodomain (Torres et al., 1998), and one or both of the short intramembraneous domains probably line the ion-conduc...

Based on pharmacological properties, the P2X receptor family can be subdivided into those homo-oligomers that are sensitive to the ATP analog ␣␤-methylene ATP(␣␤meATP) (P2X 1 and P2X 3 ) and those that are not (P2X 2 , P2X 4 , P2X 5 , P2X 6 , and P2X 7 ). We exploited this dichotomy through the construction of chimeric receptors and site-directed mutagenesis in order to identify domains responsible for these differences in the abilities of extracellular agonists to gate P2X receptors. Replacement of the extracellular domain of the ␣␤meATP-sensitive rat P2X 1 subunit with that of the ␣␤meATP-insensitive rat P2X 2 subunit resulted in a receptor that was still ␣␤meATP-sensitive, suggesting a non-extracellular domain was responsible for the differential gating of P2X receptors by various agonists. Replacement of the first transmembrane domain of the rat P2X 2 subunit with one from an ␣␤meATP-sensitive subunit (either rat P2X 1 or P2X 3 subunit) converted the resulting chimera to ␣␤meATP sensitivity. This conversion did not occur when the first transmembrane domain came from a non␣␤meATP-sensitive subunit. Site-directed mutagenesis indicated that the C-terminal portion of the first transmembrane domain was important in determining the agonist selectivity of channel gating for these chimeras. These results suggest that the first transmembrane domain plays an important role in the agonist operation of the P2X receptor.P2X receptors are ligand-gated ion channels activated by extracellular ATP. Although these receptors were first described almost 20 years ago (1), the lack of useful pharmacological tools has greatly hampered the elucidation of the roles that these receptors play in ongoing physiological functions (2, 3). Recent advances in the molecular biology of these receptors have led to a resurgence of interest, and have served to illustrate how little is actually known about these proteins. Indeed, results from in situ hybridization and immunochemical studies demonstrate that these subunits have a widespread distribution throughout the body, being present in almost all tissues (for a review, see Ref. 4), suggesting that they may have more extensive functions than appreciated previously.To date, a total of seven individual subunit genes have been cloned and their products characterized (4). These subunits have been demonstrated to form homo-and/or hetero-oligomeric receptors (5) that are non-selective cation channels with a high permeability to Ca 2ϩ (6), a property that confers the potential for important functions in excitable and secretory cells. Recent investigation into the structural features of the subunits has helped elucidate the role of the transmembrane domains in ion permeation through the channel (7-9), and have identified a number of extracellular residues as affecting the binding of agonists and antagonists (10 -13). Nevertheless, the domains involved in ligand-induced opening of the channel (gating) have not yet been delineated, nor have the domains responsible for the pharmacological fingerprints of...