Cysteine Substitution Mutagenesis and the Effects of Methanethiosulfonate Reagents at P2X2 and P2X4 Receptors Support a Core Common Mode of ATP Action at P2X Receptors

Abstract

The agonist binding site of ATP-gated P2X receptors is distinct from other ATP-binding proteins. Mutagenesis on P2X1 receptors of conserved residues in mammalian P2X receptors has established the paradigm that three lysine residues, as well as FT and NFR motifs, play an important role in mediating ATP action. In this study we have determined whether cysteine substitution mutations of equivalent residues in P2X2 and P2X4 receptors have similar effects and if these mutant receptors can be regulated by charged methanethiosulfonate (MTS) compounds. All the mutants (except the P2X2 K69C and K71C that were expressed, but non-functional) showed a significant decrease in ATP potency, with >300-fold decreases for mutants of the conserved asparagine, arginine, and lysine residues close to the end of the extracellular loop. MTS reagents had no effect at the phenylalanine of the FT motif, in contrast, cysteine mutation of the threonine was sensitive to MTS reagents and suggested a role of this residue in ATP action. The lysine-substituted receptors were sensitive to the charge of the MTS reagent consistent with the importance of positive charge at this position for coordination of the negatively charged phosphate of ATP. At the NFR motif the asparagine and arginine residues were sensitive to MTS reagents, whereas the phenylalanine was either unaffected or showed only a small decrease. These results support a common site of ATP action at P2X receptors and suggest that non-conserved residues also play a regulatory role in agonist action. The agonist binding site of ATP-gated P2X receptors is distinct from other ATP-binding proteins. Mutagenesis on P2X1 receptors of conserved residues in mammalian P2X receptors has established the paradigm that three lysine residues, as well as FT and NFR motifs, play an important role in mediating ATP action. In this study we have determined whether cysteine substitution mutations of equivalent residues in P2X2 and P2X4 receptors have similar effects and if these mutant receptors can be regulated by charged methanethiosulfonate (MTS) compounds. All the mutants (except the P2X2 K69C and K71C that were expressed, but non-functional) showed a significant decrease in ATP potency, with >300-fold decreases for mutants of the conserved asparagine, arginine, and lysine residues close to the end of the extracellular loop. MTS reagents had no effect at the phenylalanine of the FT motif, in contrast, cysteine mutation of the threonine was sensitive to MTS reagents and suggested a role of this residue in ATP action. The lysine-substituted receptors were sensitive to the charge of the MTS reagent consistent with the importance of positive charge at this position for coordination of the negatively charged phosphate of ATP. At the NFR motif the asparagine and arginine residues were sensitive to MTS reagents, whereas the phenylalanine was either unaffected or showed only a small decrease. These results support a common site of ATP action at P2X receptors and suggest that non-conserved residues also play a regulatory role in agonist action. P2X receptors for ATP comprise a distinct family of ligandgated ion channels, with two transmembrane domains, intracellular amino and carboxyl termini and a large extracellular ligand binding loop. Seven P2X receptor genes have been identified (P2X1–7) and the subunits form functional homo- and heterotrimeric ion channels with a variety of phenotypes (1North R.A. Physiol. Rev. 2002; 82: 1013-1067Crossref PubMed Scopus (2473) Google Scholar). ATP is released from neurons, in response to shear stress, as well as from damaged cells. P2X receptors have been shown to be involved in a wide range of physiological roles including blood clotting (2Hechler B. Lenain N. Marchese P. Vial C. Heim V. Freund M. Cazenave J.-P. Cattaneo M. Ruggeri Z.M. Evans R.J. Gachet C. J. Exp. Med. 2003; 198: 661-667Crossref PubMed Scopus (194) Google Scholar), sensory perception (including pain (3Souslova V. Cesare P. Ding Y. Akopian A.N. Stanfa L. Suzuki R. Carpenter K. Dickenson A. Boyce S. Hill R. Nebenius-Oosthuizen D. Smith A.J. Kidd E.J. Wood J.N. Nature. 2000; 407: 1015-1017Crossref PubMed Scopus (392) Google Scholar, 4Tsuda M. Shigemoto-Mogami Y. Koizumi S. Mizokoshi A. Kohsaka S. Salter M.W. Inoue K. Nature. 2003; 424: 778-783Crossref PubMed Scopus (1271) Google Scholar, 5Chessell I.P. Hatcher J.P. Bountra C. Michel A.D. Hughes J.P. Green P. Egerton J. Murfin M. Richardson J. Peck W.L. Grahames C.B. Casula M.A. Yiangou Y. Birch R. Anand P. Buell G.N. Pain. 2005; 114: 386-396Abstract Full Text Full Text PDF PubMed Scopus (655) Google Scholar), bladder voiding (6Cockayne D.A. Hamilton S.G. Zhu Q.-M. Dunn P.M. Zhong Y. Novakovic S. Malmberg A.B. Cain G. Berson A. Kassotakis L. Hedley L. Lachnit W.G. Burnstock G. McMahon S.B. Ford A.P. Nature. 2000; 407: 1011-1015Crossref PubMed Scopus (882) Google Scholar), and taste (7Finger T.E. Danilova V. Barrows J. Bartel D.L. Vigers A.J. Stone L. Hellekant G. Kinnamon S.C. Science. 2005; 310: 1495-1499Crossref PubMed Scopus (594) Google Scholar)), as well as bone formation (8Ke H.Z. Qi H. Weidema A.F. Zhang Q. Panupinthu N. Crawford D.T. Grasser W.A. Paralkar V.M. Li M. Audoly L.P. Gabel C.A. Jee W.S. Dixon S.J. Sims S.M. Thompson D.D. Mol. Endocrinol. 2003; 17: 1356-1367Crossref PubMed Scopus (254) Google Scholar). As the receptors provide novel drug targets for a range of diseases (9Burnstock G. Pharmacol. Rev. 2006; 58: 58-86Crossref PubMed Scopus (525) Google Scholar) an understanding of the agonist binding site would be useful for rational drug design. However, it is clear that common ATP binding sequences, e.g. the Walker motif (10Walker J.E. Saraste M. Runswick M.J. Gay N.J. EMBO J. 1982; 1: 945-951Crossref PubMed Scopus (4252) Google Scholar), are not present in P2X receptors. In the absence of a crystal structure a site-directed mutagenesis approach has been used to gain insight into amino acids important in mediating the actions of ATP at P2X receptors. We have used the human P2X1 receptor and alanine mutagenesis of conserved residues in the extracellular loop (Fig. 1) to propose a model of the ATP binding site (reviewed in Refs. 11Vial C. Roberts J.A. Evans R.J. Trends Pharmacol. Sci. 2004; 25: 487-493Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar and 12Roberts J.A. Vial C. Digby H.R. Agboh K.C. Wen H. Atterbury-Thomas A.E. Evans R.J. Pflugers Arch. 2006; 452: 486-500Crossref PubMed Scopus (138) Google Scholar). Cysteine substitution mutagenesis and charged methanethiosulfonate (MTS) 3The abbreviations used are: MTS, methanethiosulfonate; MTSEA, (2-aminoethyl)methanethiosulfonate hydrobromide; MTSES, sodium (2-sulfonatoethyl)methanethiosulfonate; WT, wild type. compounds have been useful in investigating a variety of ion channels including the pore region (13Rassendren F. Buell G. Newbolt A. R.A. A. EMBO J. PubMed Scopus Google Scholar, J. PubMed Google Scholar) as well as residues involved in ATP at P2X receptors F. A. R.A. J. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, J.A. Evans R.J. J. PubMed Scopus Google Scholar). We have used this approach to study the of the amino acids the transmembrane to ATP action at the P2X1 receptor J.A. Evans R.J. J. PubMed Scopus Google Scholar). we have shown that for the mutant ATP and binding of is The mutant was also to MTS charged a cysteine residue it a charged of similar to lysine S.J. R.A. J. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). ATP at that the charged lysine at this position a role in the binding of the negatively charged phosphate of ATP J.A. Evans R.J. J. PubMed Scopus Google Scholar). In with alanine and cysteine mutants has also identified residues that are to play a role in ATP action (Fig. has to a model of the ATP binding site with the phosphate of ATP to residues and and the residues and is by a study on the P2X1 receptor that cysteine mutants of residues and can form an and this is in the of ATP B. J. Y. A. J. PubMed Scopus Google Scholar). The model is also by mutagenesis from P2X2 and P2X4 receptors F. A. R.A. J. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, M. H. N. Y. Sci. 2005; PubMed Scopus Google Scholar). However, an model of ATP action at the P2X4 receptor on a in structure to the has also been suggested M. Mol. Pharmacol. 2005; PubMed Scopus Google Scholar). In this study to whether P2X receptor subunits a common of ATP action we at P2X2 and P2X4 receptors whether cysteine mutagenesis of the residues to in the P2X1 receptor model have effects on ATP or the action of MTS mutations for residues were the mutagenesis a human P2X2 receptor or as the P2X2 receptor of the of the with the as A. M. P. A. M. and F. to with the of the mutant was and used as the for S.J. J. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). of the mutations and absence of in the P2X2 and P2X4 mutant was by of in and mutant were to as S. S. Evans R.J. J. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). were with of an and at in sodium was to a of were on a with a to and as S. S. Evans R.J. J. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). in response to P2X receptor were by with in the ATP was a ATP to was at this were were with the Hill is is agonist is the Hill is and is the of agonist of the is the of the that had ATP were with ATP to the of were for and on the In the are to the of the of study the effect of MTS compounds on ATP at wild and cysteine ATP was and either or sodium were at the to to with ATP the as F. A. R.A. J. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, J.A. Evans R.J. J. PubMed Scopus Google Scholar). MTS reagents were in to MTS reagents were for with the or response The effects of the MTS reagents on the P2X2 receptor mutants were the of the compounds that the receptors. The effects of the MTS compounds on the mutant P2X4 receptors were a The P2X4 receptor has been shown to be to and agonist S.J. J. 2002; PubMed Google Scholar) for the mutant used in this study S.J. J. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, S.J. Evans J. Sci. 2005; PubMed Scopus Google Scholar). whether be used to P2X4 mutant receptors we the for in the of and of to or was to of the of P2X4 receptors. was by and of to an of ATP the absence of had no effect on the of P2X4 receptor of the mutants were equivalent to was present the that in for the mutant receptors from of cysteine residues and that cysteine residues were in the of the MTS reagents have a and this that the used is and was reagent present the to the receptors. These results suggest that the of MTS effects of is to from of receptors to the and are to the of P2X4 mutant receptors. We have for effects on an of ATP the of the MTS reagents as this to from the and of the The effects of MTS reagents on the to ATP for P2X2 receptors were of at for in the absence of was no effect on P2X4 receptors the effects of on were by of ATP the for in and of ATP were the with MTS reagents of wild and mutant receptors were by of and proteins. were from with wild or mutant receptor in and at by with and was used to the of wild or mutant receptor to the is to the and can only at the with wild or mutant receptor were with in for and with were in and the was with as S.J. Evans R.J. Mol. Pharmacol. 2002; PubMed Scopus Google Scholar). All were with and to for to were on a to and for for the or P2X4 are shown as and mutant agonist potency, and MTS reagent were by of by for of mutants the in mutant response for on with MTSEA, were with to the of for of Cysteine on ATP at P2X2 human P2X2 receptors ATP with an of (Fig. and 1) similar to that for this receptor J. PubMed Google Scholar, F. A. R.A. J. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, R.J. C. Buell G. S. R.A. A. Mol. Pharmacol. Google Scholar). were also for and showed a decrease in ATP (Fig. The of to a of ATP were by for the mutant and for this receptor the of to and the is ATP. At mutants and ATP small that the for these receptors is were in response to ATP for mutants K69C and of the of and of P2X2 and mutant receptors with showed that was at an in a in was a in for and that in with were to an effect on and not to the these results are consistent with from similar mutants for the P2X1 receptor J.A. Vial C. Digby H.R. Agboh K.C. Wen H. Atterbury-Thomas A.E. Evans R.J. Pflugers Arch. 2006; 452: 486-500Crossref PubMed Scopus (138) Google of the effects of cysteine substitution on P2X2 and P2X2 effect effect not not in a of MTS on Cysteine P2X2 P2X receptors the conserved cysteine residues in the extracellular are to form S.J. Evans R.J. Mol. Pharmacol. 2002; PubMed Scopus Google Scholar, J. 2002; PubMed Google Scholar). The cysteine has been used to at the of cysteine mutants to receptor of of MTS reagents that the cysteine residue is in the and play an important role in receptor D.A. M. A. Science. PubMed Scopus Google Scholar). charged MTS reagents charged and negatively charged sodium have been used to residues involved in ATP action at P2X receptors F. A. R.A. J. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, J.A. Evans R.J. J. PubMed Scopus Google Scholar) as with the charge of the phosphate of ATP or with ATP in physiological to positive At P2X2 receptors a small of in the response to an of ATP as for this receptor (13Rassendren F. Buell G. Newbolt A. R.A. A. EMBO J. PubMed Scopus Google Scholar, J. PubMed Google Scholar). had no effect on the of ATP and to on had a similar effect at and mutant P2X2 receptors At was an in to of on this was to of that it results from a of the effect for and an and a in the response to an of ATP that was on that it results from of the At the of was in response to from to a large decrease in ATP at this for in the absence of agonist in a large in of and a in the response to ATP and a response was from a as not for to for MTSEA, (Fig. to a of ATP were small for the mutant and were the of for a was a large in the of and the for ATP in the of was As was to the to mutants with small we whether had effect on mutants receptor be at the but or no was in response to of ATP. At the to ATP were to of this effect was on mutants K69C and were not The negatively charged MTS reagent had no effect on at P2X2 receptors as F. A. R.A. J. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). had no effect on ATP at mutant P2X2 receptors MTSES, the of response to an of ATP by for mutants and a decrease was at These effects were of that the reagent was to the cysteine mutants K69C and K71C in the of MTSES, ATP not a of Cysteine on ATP at P2X4 P2X4 receptor with the mutation was used as the for these The mutation was to of the receptor S.J. J. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar) and the of ATP by in the present ATP at the receptor with an of similar to that for P2X4 receptors Zhang Y. M. Burnstock G. R. PubMed Scopus Google Scholar, G. C. G. R.A. A. EMBO J. PubMed Scopus Google Scholar, J. Physiol. 2005; PubMed Scopus Google Scholar) and this be to as P2X4 At cysteine mutants was a decrease in ATP and mutants were and the for ATP was by and the mutants and not at the and for these mutants the ATP is as and had with WT, this was not by a in either or of the receptors (Fig. the mutant the in from a in of the receptor to the (Fig. of MTS on Cysteine P2X4 charged had no effect on at P2X4 receptors consistent with that S.J. R.A. J. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). were also unaffected by for the mutants of the conserved phenylalanine residues and At and by an of ATP were to of and at mutant were by In contrast, at mutants a charged lysine residue was to cysteine and of the of the ATP response The of negatively charged had no effect on the of ATP at or and mutant P2X4 receptors At the of the mutants and the of ATP by These results suggest that the charge at this position with the action of ATP by the negatively charged phosphate These effects of and are consistent for the P2X2 and P2X4 receptors. However, in to the P2X2 receptor the effects of MTS reagents were on that either the reagent was not the receptor or that receptors were to the the is that P2X4 receptors to the S.J. J. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, S.J. J. 2002; PubMed Google Scholar, S.J. Evans J. Sci. 2005; PubMed Scopus Google Scholar) we to whether with the MTS reagents had an In as the MTS reagents were and ATP the as to whether the are in the or ligand these we mutant P2X4 receptors in in the of to ATP in the for by had no effect on the of P2X4 receptors with with the was a significant in the of for the mutants and and of ATP only and with These results that with MTS reagents are to the equivalent results to MTSES, and suggest that of MTS reagents results from of mutant P2X4 receptors to the These results also that these cysteine residues are in the absence of is consistent with on the P2X2 receptor that MTS effects at the region and are by ATP F. A. R.A. J. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar) and on the P2X1 receptor that mutants and are in the absence of ATP and this is of ATP J.A. Evans R.J. J. PubMed Scopus Google Scholar). these results that the MTS reagents are at cysteine residues that are in the absence of mutants and in an in the of the response to ATP we of We that with for the absence of by and of ATP in the absence of in an in ATP at the receptors with of and for and and (Fig. These results suggest that positive charge at these an important role in ATP The of this study was to whether a model of the ATP binding site on mutagenesis on the P2X1 receptor be to other P2X receptor family and to this to if the actions of ATP be in a similar by MTS The for the effects of cysteine mutants on ATP at P2X2 and P2X4 receptors support residues involved in ATP action. are three conserved lysine residues in the extracellular that have been suggested to be involved in ATP binding at P2X receptors. Mutagenesis of these residues has shown a decrease in agonist at a variety of receptors with the of the mutations in a in to and in e.g. P2X2 are the of at agonist F. A. R.A. J. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). At P2X4 receptors ATP at and was on with MTSEA, consistent with the that the of at cysteine a of similar and charge to lysine S.J. R.A. J. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). In contrast, were by with negatively charged that the charge at these three an important role in ATP action at the P2X2 receptors a response to ATP was not MTS at mutants K69C and that is common to the receptors is that the decrease in ATP at the cysteine mutant of the residue equivalent to in the P2X1 receptor can be by with MTSEA, consistent with an important role of positive charge at this study on the P2X2 receptor has suggested the lysine residue can play a role in of of the was on the that mutation of this residue of the mutant P2X2 and that mutation ATP by and the of the response with L. S.J. R.A. J. PubMed Scopus Google Scholar). In the study the to P2X2 and P2X4 not it is not to whether was a in the of the At the P2X1 receptor the in ATP at and mutants not in a decrease in the of the response J.A. Evans R.J. J. PubMed Scopus Google Scholar, S. S. Evans R.J. J. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar) as would have been if an effect on of the receptor has been for the in D. J. Pharmacol. PubMed Scopus Google Scholar). These results suggest that the effects of mutation of this conserved residue can be as a effect on ATP binding at the P2X1 receptor J.A. Evans R.J. J. PubMed Scopus Google Scholar) and an effect on at the P2X2 receptor L. S.J. R.A. J. PubMed Scopus Google Scholar). that in the P2X2 receptor the lysine residue with amino that is not conserved at the P2X1 receptor to these results support that the three charged lysine residues play an important role in ATP action to the ATP binding it would be to have an ATP-binding positive charge important in agonist The mutations of conserved in P2X2 and in the P2X4 receptor to a and decrease in ATP at the receptors. is consistent with on P2X1 and P2X4 receptors J.A. Evans R.J. J. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, H. M. J. PubMed Scopus Google Scholar) and that this conserved residue to ATP action. The MTS reagents used are of similar ATP and can gain to the ATP binding The effects of the MTS compounds at cysteine mutants of the threonine residue and for P2X2 and suggest that this region of the receptor is to these reagents and the of effect at the phenylalanine residues is to that this residue is either not close to the ATP binding or is involved in with other residues that The that the phenylalanine not play a role in binding of the of ATP as we have suggested for the P2X1 receptor J.A. Evans R.J. J. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). However, cysteine substitution to in agonist action at the The of a to the phenylalanine have an effect on or a to the of threonine in the conserved FT motif to cysteine in a and decrease in ATP at P2X2 and P2X4 receptors. on the P2X2 receptor have shown that alanine substitution at this residue ATP by F. A. R.A. J. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). is the a mutation has been at this position for the P2X4 receptors and it a similar of agonist action as P2X1 and P2X2 receptors. At P2X2 and P2X4 receptors the response was by These results that of the at this conserved threonine residue can to the FT motif is a conserved lysine residue that has been shown to be involved in ATP in P2X1 and in P2X2 that to alanine in S. S. Evans R.J. J. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar) and decreases in ATP F. V. A. R.A. J. Full Text Full Text PDF PubMed Scopus Google this region in the of the extracellular loop of the a role in of ATP potency, whether this results from an effect on agonist binding to be is clear is that residues involved in agonist action at P2X receptors are not close to the transmembrane and the pore of the At the conserved NFR motif cysteine substitution at and ATP by >300-fold and in at P2X2 and P2X4 receptors. The asparagine to cysteine mutation had the effect at P2X2 and P2X4 receptors with of by either MTS reagent as for P2X1 receptors J.A. Evans R.J. J. PubMed Scopus Google Scholar). The of ATP at the mutants of the conserved arginine by is the for P2X1 and P2X4 but the P2X2 receptor J.A. Evans R.J. J. PubMed Scopus Google Scholar and this that the P2X1 and P2X4 receptors are similar in that the MTS reagents with the action of ATP in a that is charge However, for P2X2 the results are consistent with the of charge at this position the phenylalanine of the NFR motif was with cysteine was only a in ATP potency, no effect on and MTS reagents were that at P2X2 and P2X4 receptors the of the asparagine and arginine residues is important in the actions of ATP the phenylalanine of are At P2X1 receptors and a effects of decrease in ATP with no effect on the response were with the equivalent mutations J.A. Evans R.J. J. PubMed Scopus Google Scholar, B. J. Y. A. J. PubMed Scopus Google Scholar). these results that whereas are the are also in of ATP the P2X receptor subunits the effects of on cysteine substitution of the arginine of the NFR is consistent with in the of P2X receptors and that non-conserved amino acids are with agonist of for the effects of MTS reagents at P2X receptor cysteine mutants in a The present study that the NFR region an important role in of ATP action at P2X2 and P2X4 receptors. We have for P2X1 that in ATP at cysteine mutants of the NFR motif are to from effects on agonist binding at and effects on binding and for and J.A. Evans R.J. J. PubMed Scopus Google Scholar). The of this region to the agonist binding site is by a B. J. Y. A. J. PubMed Scopus Google Scholar) that the phenylalanine is close to the conserved residue in the P2X1 receptor and cysteine mutants and can form an and this is by that residues are close to the ATP binding a study on P2X2 receptors that the of ATP for the mutant of the P2X2 receptor results from an effect on agonist binding L. S.J. R.A. J. PubMed Scopus Google Scholar). the NFR region be involved in coordination of ATP we had suggested that the phenylalanine residues in the NFR motif were involved in binding of the of ATP. However, the present results from P2X2 and P2X4 receptors with in ATP for cysteine mutants of these and the of effect of MTS reagents on ATP that the conserved residue not to agonist The of mutants of the arginine of the NFR motif for P2X1 and P2X4 receptors by positive and negatively charged MTS reagents that arginine is not involved in binding of the phosphate of ATP. that asparagine and arginine are involved in binding of the of ATP. In results support the that mammalian P2X receptors a common agonist binding site with the charge of ATP by charged lysine residues and the NFR motif involved in binding of the (Fig. The conserved FT motif also to agonist whether this results from an effect on agonist binding to be