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• W4225137411 abstract "P2X receptors are a class of nonselective cation channels widely distributed in the immune and nervous systems, and their dysfunction is a significant cause of tumors, inflammation, leukemia, and immune diseases. P2X7 is a unique member of the P2X receptor family with many properties that differ from other subtypes in terms of primary sequence, the architecture of N- and C-terminals, and channel function. Here, we suggest that the observed lengthened β2- and β3-sheets and their linker (loop β2,3), encoded by redundant sequences, play an indispensable role in the activation of the P2X7 receptor. We show that deletion of this longer structural element leads to the loss of P2X7 function. Furthermore, by combining mutagenesis, chimera construction, surface expression, and protein stability analysis, we found that the deletion of the longer β2,3-loop affects P2X7 surface expression but, more importantly, that this loop affects channel gating of P2X7. We propose that the longer β2,3-sheets may have a negative regulatory effect on a loop on the head domain and on the structural element formed by E171 and its surrounding regions. Understanding the role of the unique structure of the P2X7 receptor in the gating process will aid in the development of selective drugs targeting this subtype. P2X receptors are a class of nonselective cation channels widely distributed in the immune and nervous systems, and their dysfunction is a significant cause of tumors, inflammation, leukemia, and immune diseases. P2X7 is a unique member of the P2X receptor family with many properties that differ from other subtypes in terms of primary sequence, the architecture of N- and C-terminals, and channel function. Here, we suggest that the observed lengthened β2- and β3-sheets and their linker (loop β2,3), encoded by redundant sequences, play an indispensable role in the activation of the P2X7 receptor. We show that deletion of this longer structural element leads to the loss of P2X7 function. Furthermore, by combining mutagenesis, chimera construction, surface expression, and protein stability analysis, we found that the deletion of the longer β2,3-loop affects P2X7 surface expression but, more importantly, that this loop affects channel gating of P2X7. We propose that the longer β2,3-sheets may have a negative regulatory effect on a loop on the head domain and on the structural element formed by E171 and its surrounding regions. Understanding the role of the unique structure of the P2X7 receptor in the gating process will aid in the development of selective drugs targeting this subtype. P2X receptors are ligand-gated nonselective cation channels activated by extracellular ATP (1Brake A.J. Wagenbach M.J. Julius D. New structural motif for ligand-gated ion channels defined by an ionotropic ATP receptor.Nature. 1994; 371: 519-523Crossref PubMed Scopus (837) Google Scholar, 2Valera S. Hussy N. Evans R.J. Adami N. North R.A. Surprenant A. et al.A new class of ligand-gated ion channel defined by P2x receptor for extracellular ATP.Nature. 1994; 371: 516-519Crossref PubMed Scopus (894) Google Scholar). To date, seven subtypes of the P2X receptor family have been identified as P2X1-P2X7 (1Brake A.J. Wagenbach M.J. Julius D. New structural motif for ligand-gated ion channels defined by an ionotropic ATP receptor.Nature. 1994; 371: 519-523Crossref PubMed Scopus (837) Google Scholar, 3Lewis C. Neidhart S. Holy C. North R.A. Buell G. Surprenant A. Coexpression of P2X2 and P2X3 receptor subunits can account for ATP-gated currents in sensory neurons.Nature. 1995; 377: 432-435Crossref PubMed Scopus (889) Google Scholar, 4Collo G. North R.A. Kawashima E. Merlo-Pich E. Neidhart S. Surprenant A. et al.Cloning OF P2X5 and P2X6 receptors and the distribution and properties of an extended family of ATP-gated ion channels.J. Neurosci. 1996; 16: 2495-2507Crossref PubMed Google Scholar, 5Soto F. Garcia-Guzman M. Gomez-Hernandez J.M. Hollmann M. Karschin C. Stühmer W. P2X4: an ATP-activated ionotropic receptor cloned from rat brain.Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3684-3688Crossref PubMed Scopus (304) Google Scholar, 6Chen C.C. Akopian A.N. Sivilotti L. Colquhoun D. Burnstock G. Wood J.N. A P2X purinoceptor expressed by a subset of sensory neurons.Nature. 1995; 377: 428-431Crossref PubMed Scopus (911) Google Scholar, 7Surprenant A. Rassendren F. Kawashima E. North R.A. Buell G. The cytolytic P2Z receptor for extracellular ATP identified as a P2X receptor (P2X7).Science (New York, N.Y.). 1996; 272: 735-738Crossref PubMed Scopus (1476) Google Scholar). Among them, P2X7 has many features that differ from other P2X subtypes, such as the fact that P2X7 requires very high concentrations of ATP for activation and has an EC50 value (the concentration that produces half of the maximal response) of approximately 0.3 to 1 mM under normal physiological conditions (2 mM extracellular Ca2+ and 1 mM extracellular Mg2+), whereas the EC50 values of other subtypes are generally around 0.5 to 10 μM (8North R.A. Surprenant A. Pharmacology of cloned P2X receptors.Annu. Rev. Pharmacol. Toxicol. 2000; 40: 563-580Crossref PubMed Scopus (603) Google Scholar). In terms of the channel desensitization of P2X receptors, P2X1 and P2X3 desensitize very quickly (milliseconds) after saturating ATP applications, whereas P2X2 and P2X4 are relatively slow (seconds); P2X7 does not desensitize at all (9Jarvis M.F. Khakh B.S. ATP-gated P2X cation-channels.Neuropharmacology. 2009; 56: 208-215Crossref PubMed Scopus (264) Google Scholar, 10Koshimizu T. Koshimizu M. Stojilkovic S.S. Contributions of the C-terminal domain to the control of P2X receptor desensitization.J. Biol. Chem. 1999; 274: 37651-37657Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), even though prolonged activation leads to pore dilation of P2X7, allowing permeation of small molecules with molecular weights up to 900 Da (11Nuttle L.C. Dubyak G.R. Differential activation of cation channels and non-selective pores by macrophage P2z purinergic receptors expressed in Xenopus oocytes.J. Biol. Chem. 1994; 269: 13988-13996Abstract Full Text PDF PubMed Google Scholar, 12Steinberg T.H. Newman A.S. Swanson J.A. Silverstein S.C. ATP4- permeabilizes the plasma membrane of mouse macrophages to fluorescent dyes.J. Biol. Chem. 1987; 262: 8884-8888Abstract Full Text PDF PubMed Google Scholar). The aforementioned unique properties make P2X7 considered as the most special member of the P2X family compared to other subtypes. P2X7 receptors are involved in many physiopathological functions, particularly its expression in lymphocytes, macrophages, and microglia, and its role in the immune response (13Di Virgilio F. Dal Ben D. Sarti A.C. Giuliani A.L. Falzoni S. The P2X7 receptor in infection and inflammation.Immunity. 2017; 47: 15-31Abstract Full Text Full Text PDF PubMed Scopus (525) Google Scholar). It has been shown that P2X7 receptors are required for the establishment of long-lived memory CD8+ cells (14Borges da Silva H. Beura L.K. Wang H. Hanse E.A. Gore R. Scott M.C. et al.The purinergic receptor P2RX7 directs metabolic fitness of long-lived memory CD8(+) T cells.Nature. 2018; 559: 264-268Crossref PubMed Scopus (127) Google Scholar). P2X7 receptors are expressed in neutrophils, and their activation leads to activation of nod-like receptor family pyrin domain-containing 3 inflammasome and secretion of interleukin-1β (IL-1β) (15Karmakar M. Katsnelson M.A. Dubyak G.R. Pearlman E. Neutrophil P2X7 receptors mediate NLRP3 inflammasome-dependent IL-1β secretion in response to ATP.Nat. Commun. 2016; 7: 10555Crossref PubMed Scopus (231) Google Scholar), indicating that P2X7 receptors are closely associated with inflammation. P2X7 plays a role in allograft transplant rejection, and inhibition of the P2X7 receptor or the nod-like receptor family pyrin domain-containing 3 inflammasome contributes to the induction of graft tolerance (16Amores-Iniesta J. Barberà-Cremades M. Martínez C.M. Pons J.A. Revilla-Nuin B. Martínez-Alarcón L. et al.Extracellular ATP activates the NLRP3 inflammasome and is an early danger signal of skin allograft rejection.Cell Rep. 2017; 21: 3414-3426Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). P2X7 in the central nervous system is involved in the neurodegenerative diseases, such as Alzheimer’s disease (17Chen X. Hu J. Jiang L. Xu S. Zheng B. Wang C. et al.Brilliant Blue G improves cognition in an animal model of Alzheimer's disease and inhibits amyloid-β-induced loss of filopodia and dendrite spines in hippocampal neurons.Neuroscience. 2014; 279: 94-101Crossref PubMed Scopus (56) Google Scholar), Huntington’s disease (18Díaz-Hernández M. Díez-Zaera M. Sánchez-Nogueiro J. Gómez-Villafuertes R. Canals J.M. Alberch J. et al.Altered P2X7-receptor level and function in mouse models of Huntington's disease and therapeutic efficacy of antagonist administration.FASEB J. 2009; 23: 1893-1906Crossref PubMed Scopus (185) Google Scholar), and multiple sclerosis (19Amadio S. Parisi C. Piras E. Fabbrizio P. Apolloni S. Montilli C. et al.Modulation of P2X7 receptor during inflammation in multiple sclerosis.Front. Immunol. 2017; 8: 1529Crossref PubMed Scopus (36) Google Scholar). In addition, P2X7 receptors are inextricably linked to cancer (20Adinolfi E. Raffaghello L. Giuliani A.L. Cavazzini L. Capece M. Chiozzi P. et al.Expression of P2X7 receptor increases in vivo tumor growth.Cancer Res. 2012; 72: 2957-2969Crossref PubMed Scopus (239) Google Scholar) and pain (21Sorge R.E. Trang T. Dorfman R. Smith S.B. Beggs S. Ritchie J. et al.Genetically determined P2X7 receptor pore formation regulates variability in chronic pain sensitivity.Nat. Med. 2012; 18: 595-599Crossref PubMed Scopus (274) Google Scholar). Therefore, P2X7 receptors are one of the important new drug targets in recent years (22Letavic M.A. Savall B.M. Allison B.D. Aluisio L. Andres J.I. De Angelis M. et al.4-Methyl-6,7-dihydro-4H-triazolo[4,5-c]pyridine-Based P2X7 receptor antagonists: optimization of pharmacokinetic properties leading to the identification of a clinical candidate.J. Med. Chem. 2017; 60: 4559-4572Crossref PubMed Scopus (34) Google Scholar, 23Calzaferri F. Ruiz-Ruiz C. de Diego A.M.G. de Pascual R. Méndez-López I. Cano-Abad M.F. et al.The purinergic P2X7 receptor as a potential drug target to combat neuroinflammation in neurodegenerative diseases.Med. Res. Rev. 2020; 40: 2427-2465Crossref PubMed Scopus (22) Google Scholar, 24Sperlágh B. Illes P. P2X7 receptor: an emerging target in central nervous system diseases.Trends Pharmacol. Sci. 2014; 35: 537-547Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar). The gating mechanism based on the structure of each P2X receptor subtype has been well studied. The first crystal structure of the zebrafish P2X4 (zfP2X4) receptor was determined in 2009 (25Kawate T. Michel J.C. Birdsong W.T. Gouaux E. Crystal structure of the ATP-gated P2X4 ion channel in the closed state.Nature. 2009; 460: 592-598Crossref PubMed Scopus (573) Google Scholar), and the structures of several other P2X subtypes have been determined in the last decade (26Hattori M. Gouaux E. Molecular mechanism of ATP binding and ion channel activation in P2X receptors.Nature. 2012; 485: 207-212Crossref PubMed Scopus (380) Google Scholar, 27Kasuya G. Fujiwara Y. Takemoto M. Dohmae N. Nakada-Nakura Y. Ishitani R. et al.Structural insights into divalent cation modulations of ATP-gated P2X receptor channels.Cell Rep. 2016; 14: 932-944Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 28Karasawa A. Kawate T. Structural basis for subtype-specific inhibition of the P2X7 receptor.Elife. 2016; 5e22153Crossref PubMed Scopus (142) Google Scholar, 29Mansoor S.E. Lu W. Oosterheert W. Shekhar M. Tajkhorshid E. Gouaux E. X-ray structures define human P2X(3) receptor gating cycle and antagonist action.Nature. 2016; 538: 66-71Crossref PubMed Scopus (142) Google Scholar, 30McCarthy A.E. Yoshioka C. Mansoor S.E. Full-length P2X7 structures reveal how palmitoylation prevents channel desensitization.Cell. 2019; 179: 659-670.e613Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 31Kasuya G. Yamaura T. Ma X.-B. Nakamura R. Takemoto M. Nagumo H. et al.Structural insights into the competitive inhibition of the ATP-gated P2X receptor channel.Nat. Commun. 2017; 8: 876Crossref PubMed Scopus (52) Google Scholar, 32Wang J. Wang Y. Cui W.W. Huang Y. Yang Y. Liu Y. et al.Druggable negative allosteric site of P2X3 receptors.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: 4939-4944Crossref PubMed Scopus (47) Google Scholar). The overall three-dimensional (3D) structure of the P2X receptor and the way that each subunit is folded are essentially the same for all subtypes: each P2X subtype exhibits a chalice-like trimeric structure containing a large hydrophilic extracellular structural domain, two transmembrane helices and intracellular termini, and the individual subunits of each P2X subtype resemble “dolphins”. Nevertheless, the gating differences between P2X subtypes are not fully understood, especially the unique P2X7 receptor. In addition to its function and properties being specific among P2X subtypes, the structure of P2X7 is unique, starting with the long intracellular N- and C-terminals of P2X7. The C-terminus of P2X7 is involved in regulating many functions of the P2X7 receptor, including protein–protein interactions, phosphorylation, and post-translational modifications (33Costa-Junior H.M. Sarmento Vieira F. Coutinho-Silva R. C terminus of the P2X7 receptor: treasure hunting.Purinergic Signal. 2011; 7: 7-19Crossref PubMed Scopus (83) Google Scholar). In contrast to other P2X receptors, the C-terminus of P2X7 has a unique cysteine-rich region called the C-cys anchor that prevents P2X7 receptor desensitization (30McCarthy A.E. Yoshioka C. Mansoor S.E. Full-length P2X7 structures reveal how palmitoylation prevents channel desensitization.Cell. 2019; 179: 659-670.e613Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 34Allsopp R.C. Evans R.J. Contribution of the juxtatransmembrane intracellular regions to the time course and permeation of ATP-gated P2X7 receptor ion channels.J. Biol. Chem. 2015; 290: 14556-14566Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar), and approximately 20 amino acid residues at the C-terminus form a unique structure called the “cytoplasmic ballast”. The N-terminal of P2X7 is important for Ca2+-influx (35Liang X. Samways D.S. Wolf K. Bowles E.A. Richards J.P. Bruno J. et al.Quantifying Ca2+ current and permeability in ATP-gated P2X7 receptors.J. Biol. Chem. 2015; 290: 7930-7942Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar), channel activation facilitation (34Allsopp R.C. Evans R.J. Contribution of the juxtatransmembrane intracellular regions to the time course and permeation of ATP-gated P2X7 receptor ion channels.J. Biol. Chem. 2015; 290: 14556-14566Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar), and the activation of extracellular signal–regulated kinases (36Amstrup J. Novak I. P2X7 receptor activates extracellular signal-regulated kinases ERK1 and ERK2 independently of Ca2+ influx.Biochem. J. 2003; 374: 51-61Crossref PubMed Scopus (79) Google Scholar). In addition, despite binding the U-shape of ATP and key residues recognized by ATP (K64, K66, T189, N292, R294 and K311, rP2X7 numbering) are conserved in the P2X receptor family, but recent structures have shown that the ATP-binding pocket of the P2X7 receptor is narrower than that of the human P2X3 (hP2X3) receptor (29Mansoor S.E. Lu W. Oosterheert W. Shekhar M. Tajkhorshid E. Gouaux E. X-ray structures define human P2X(3) receptor gating cycle and antagonist action.Nature. 2016; 538: 66-71Crossref PubMed Scopus (142) Google Scholar, 30McCarthy A.E. Yoshioka C. Mansoor S.E. Full-length P2X7 structures reveal how palmitoylation prevents channel desensitization.Cell. 2019; 179: 659-670.e613Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Thus, P2X7 is structurally unique in many ways compared to other subtypes, and all of these unique structural elements are associated with functions that distinguish it from other subtypes. Here, we find another unique structural element of P2X7. By comparison of structure and sequence, we found that the β2 and β3 sheets of P2X7 and their linker (loop β2,3) are longer compared to other subtypes. The question of whether the longer β2,3-sheet and loop β2,3 of the P2X7 receptor have a unique function compared to other P2X receptor subtypes remains unclear. In this study, we show that the redundant sequence between the P2X7 receptor β2,3 and its corresponding structural elements plays an important role in both expression and channel gating of the P2X7 receptor, which is unique among the P2X family. Sequence alignment analysis of the P2X receptor family revealed that residues 73 to 80 of the P2X7 receptor are redundant compared to other subtypes (Fig. 1C). In the recently determined resting-state structure of rP2X7 (PDB ID: 6U9V), no stable conformation of this region was observed in this disordered region; however, in the open state, this region folded into a longer β2,3 in the cryo-electron microscopic (Cryo-EM) structure of the P2X7 receptor than that of the P2X3 receptor (Fig. 1, A and B and PDB IDs: 6U9W and 5SVK). The superposition of the resting and open states showed that the longer β2,3 region shifted to the right in the open state (Fig. 1, A and B). To verify whether the longer β2,3 structure in the P2X7 receptor is necessary for channel function, we deleted residues 73 to 80 and obtained Δ(73–80). Δ(73–80) mutant significantly reduced the 1 mM ATP-induced currents (70.9 ± 57.8 and 250 ± 61 pA/pF, for Δ(73–80) and wildtype, WT, n = 4 and 13, respectively, p < 0.01, Δ(73–80) versus WT; Fig. 2, A and B). We also performed a smaller deletion of this fragment and obtained three truncations, Δ(73–74), Δ(75–76), and Δ(77–80), where Δ(73–74) dramatically decreased the function of the channel. However, mutations in N73A and N74A at individual positions did not have as strong an effect as that caused by deleting both of them (Fig. 2, A and B). Moreover, the EC50 values of Δ(73–80) (208.7 ± 22.6 μM), Δ(73–74) (1762.3 ± 5.8 μM), Δ(75–76) (335.4 ± 88.2 μM), E73A (33.9 ± 1.7 μM), and N74A (26.6 ± 0.2 μM) were significantly changed compared to rP2X7 WT (98.5 ± 37.3 μM) (Fig. 2C). These results suggest that this redundant structure may not facilitate interactions with surrounding residues through individual amino acids but rather participate in channel gating together as an integrated whole. In addition to gating modulation, there are many factors that can affect the maximum current of a channel. First, we analyzed the membrane expression of these mutants. We found that Δ(73–80), Δ(73–74), Δ(75–76), and Δ(77–80) had significantly reduced numbers of membrane proteins (Fig. 3, A and B), which suggests that the reduction in the maximum currents of Δ(73–80), Δ(73–74), and Δ(75–76) channels may be caused by the reduction in surface expression of channels. However, there was no significant difference in the surface expression of these mutants, such as Δ(73–74), whose maximum current density was significantly reduced, but whose membrane expression was the same as that of E73A and N74A (maximum current density of E73A and N74A was only slightly lower or not different from that of WT, Fig. 2, A and B). In addition, the surface/total ratio was also calculated (Fig. 3C) and correlation analysis showed no correlation between surface/total ratio and current density (R2 = 0.48, p = 0.08, Pearson's correlation, Fig. 3D). These results indicate that some reduction in membrane expression did not contribute mainly to the reduction in the maximum current density of the mutants. In addition, we treated cells transfected with rP2X7 WT and Δ(73–80) with cyclohexanone (20 μg/ml) for different times to see if there was an effect on protein stability. Even after 10 h of cyclohexanone treatment, the amount of protein was not affected (Fig. 3, E and F). Once factors of protein surface expression and stability were excluded, we inferred that the reduction in the maximum current may be caused by gating changes in the channel due to the lack of the longer β2,3 structure of P2X7. The β2,3-sheet in the structure of each P2X receptor is sandwiched between the upper body domain of the same subunit and the head domain of the adjacent subunit (Fig. 1, A and B). In addition, the conformation of the β2,3-sheet of rP2X7 changed considerably from the resting state to the open state. In the resting state, the exact conformation cannot be seen due to insufficient resolution, but it should not be a stable β-sheet (30McCarthy A.E. Yoshioka C. Mansoor S.E. Full-length P2X7 structures reveal how palmitoylation prevents channel desensitization.Cell. 2019; 179: 659-670.e613Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Therefore, residues 73 to 80 of rP2X7 may have a loose loop-like secondary structure in the resting state, while in the open state, they form a rigid β-sheet (Fig. 1A). Notably, the longer region (residues 73–80) of the β2,3-sheet in P2X7 is closer to the upper body domain of the same subunit than that of other subtypes (Fig. 1, A and B). The superposition of the resting and open state structures of rP2X7 revealed that the conformation of the loop 168 to 175 (loop 1) in the upper body domain of the same subunit changed significantly, while the longer β2,3 shifted to the right, as evidenced by the apparent flip-flopping of the side chains of amino acids A170, E171, and E172 (Fig. 4A). We further mutated each of these three amino acids and found that mutation of E171A significantly reduced the function of the channel (3.5 ± 1.4 and 314 ± 79 pA/pF for E171A and WT, respectively, p < 0.01, E171A versus WT; Fig. 4, B and C) even when the concentration of ATP was increased to 30 mM and it did not further increase the current density (2.1 ± 1.5 pA/pF for E171A, saturated ATP). E171D, a mutation with more similar side chain properties, still significantly impaired channel activation of rP2X7, suggesting a rigid requirement of this position for rP2X7 activation (112 ± 42, 151 ± 60, and 173 ± 62 pA/pF after the treatment of 1, 10, and 30 mM ATP, respectively, in E171D; Fig. 4D). E171D significantly changed the apparent ATP affinity of rP2X7 (EC50 = 127 ± 41 and 752 ± 18 μM for rP2X7 WT and E171D, respectively; Fig. 4E). In addition, Western blot experiments showed that E171A did not cause abnormal surface expression of rP2X7 (Fig. 4, F and G). These results suggest that residue E171 in the region adjacent to the longer β2,3 region (residues 73–80) is also critical for the gating process of P2X7. Does E171, like the longer β2,3 redundant region, has a unique effect on the maximum current of rP2X7 compared to other members of the P2X family? To explore this idea, we performed a sequence alignment analysis and found that E171 is highly conserved in the P2X family (Fig. 5A). Therefore, we performed the same point mutation in the identical position for other P2X subtypes. There are no or only small ATP-induced currents in the P2X5 and P2X6 subtypes, and thus, no mutations were made to these two subtypes. In P2X1 to P2X4, mutations at this position resulted in significant changes (∼10-fold) in the EC50 values of P2X1, P2X3, and P2X4 (EC50 = 0.67 ± 0.03 and 4.39 ± 0.84 μM for P2X1 WT and E168A, respectively; 0.48 ± 0.31 and 0.64 ± 0.24 μM for P2X3 WT and E156A, respectively; 7.04 ± 4.41 and 52.0 ± 18.6 μM for P2X4 WT and E168A, respectively; Fig. 5, B, F and H). For the P2X2 subtype, mutation E167A did not affect the EC50 of ATP (EC50 = 24.0 ± 7.8 and 27.5 ± 3.9 μM for P2X2 WT and E167A, respectively; Fig. 5D). Unlike P2X7, none of the mutations in these subtypes altered the maximum current density of ATP (Fig. 5, C, E, G and I), suggesting that E171 of rP2X7 plays a different role in channel function than other subtypes, at least for P2X1-4. Although both β2,3 and E171 in rP2X7 affect channel gating and both are specific to the P2X7 subtype, whether the two are interrelated needs further verification. To address this issue, we explored the effects of these two regions of P2X7 on channels in different species, including human P2X7 (hP2X7), panda P2X7 (pdP2X7), dog (dP2X7), guinea pig P2X7 (gpP2X7), mouse P2X7 (mP2X7), bovine P2X7 (bP2X7), and chicken P2X7 (ckP2X7). In these genes, ckP2X7 has a different sequence, i.e., it does not have a longer β2,3-sheet like other P2X7, and the identical E171 position is Q159 (Fig. 6A). As expected, the mutant ckP2X7Q159A does not affect the maximum current of this channel (353 ± 121 and 387 ± 164 pA/pF for ckP2X7Q159A and ckP2X7WT, respectively; p > 0.05, ckP2X7Q159A versus ckP2X7WT; Fig. 6, B and C). Since other species have the longer β2,3 and a conserved glutamate at the same position as 171, we deleted the redundant region of β2,3 and mutated residues at the corresponding positions of E171 in these species to obtain pdP2X7Δ(73-80), pdP2X7E171A, hP2X7Δ(73-80), hP2X7E171A, dP2X7Δ(73-80), dP2X7E171A, gpP2X7Δ(73-80), gpP2X7E171A, mP2X7Δ(73-80), mP2X7E171A, bP2X7Δ(75-82), and bP2X7E173A. Removing the redundant sequence, pdP2X7Δ(73-80), does not affect the maximum current of this channel, and therefore, the mutation in pdP2X7E171A does not affect the maximum current either (325 ± 55 and 260 ± 59 pA/pF for pdP2X7E171A and pdP2X7WT, respectively; pdP2X7E171A versus pdP2X7WT, p > 0.05; Fig. 6, D and E). As well, the surface/total expression ratios of pdP2X7Δ(73-80) and pdP2X7E171A were comparable to that of pdP2X7WT(Fig. 7, A–C). In contrast, deletions corresponding to rP2X7 are similar in the other five species: mutations corresponding to the β2,3 redundant region and E171 position both significantly affect the maximum current of the P2X7 channel in these species (28.3 ± 10.7, 3.5 ± 2.6, and 74.9 ± 35.4 pA/pF for hP2X7Δ(73-80), hP2X7E171A, and hP2X7WT, respectively, p < 0.01, n = 11, 7, and 7, Fig. 6, F and G; 73.1 ± 52.7, 65.0 ± 36.7, and 222 ± 72 pA/pF for dP2X7Δ(73-80), dP2X7E171A, and dP2X7WT, respectively, p < 0.01, n = 6, 4, and 5, Fig. 6, H and I; 0.54 ± 0.54, 41.3 ± 28.5, and 356 ± 100 pA/pF for gpP2X7Δ(73-80), gpP2X7E171A, and gpP2X7WT, respectively, p < 0.01, n = 3, 4, and 3, Fig. 6, J and K; 91.3 ± 20.5, 5.8 ± 3.4, and 146 ± 12 pA/pF for mP2X7Δ(73-80), mP2X7E171A, and mP2X7WT, respectively, p < 0.05, n = 4, 4, and 4, Fig. 6, L and M; 1.5 ± 1.7, 4.4 ± 3.2, and 185 ± 53 pA/pF for bP2X7Δ(75-82), bP2X7E173A, and bP2X7WT, respectively, p < 0.01, n = 4, 3, and 3, Fig. 6, N and O). In addition, we examined the membrane expression-to-total expression ratios of other three different species of P2X7 (dP2X7, mP2X7, and bP2X7) and their corresponding mutants (dP2X7Δ(73-80), mP2X7Δ(73-80), and bP2X7Δ(75-82)) by Western blot. dP2X7Δ(73-80) membrane expression was somewhat decreased compared to WT (Fig. 7, D–F). However, there was no significant difference between mP2X7Δ(73-80) and bP2X7Δ(75-82) and WT (Fig. 7, G–L). These results suggest that the deletion of the longer β2,3 in P2X7 causes a decrease in membrane expression (as in rP2X7) that varies among species. Based on the aforementioned results, there is a relationship between these two structural elements, i.e., when the redundant region influences the maximum current, the E171 position also has an effect; when there is no redundant region or the redundant region has no effect on the maximum current, then the E171 also has no effect. Moreover, this linkage does not appear to be strongly associated with redundant regions affecting protein membrane expression or not. Next, we investigated the mechanism of the interrelationship between the β2,3 redundant region and E171. Since the β2,3 redundant region/E171 of pdP2X7 and rP2X7 have a diametrically opposed role in the channel activation and the crystal or cryo-EM structures of both have been determined, we used both species of P2X7 for the following studies. rP2X7 structural superposition in the resting and open states showed that hydrogen bonding interactions were formed between R294 and E171 in both the resting and open states, except that in the resting state R294....E171 salt bridge is positioned further away from the ATP-binding site than in the open state (Fig. 8A). In this process, ATP attracts R294, prompting E171 to move and its side chain orientation to change, resulting in the R294...E171 salt bridge as a whole toward the ATP-binding site, further leading to the aforementioned conformational flip of E171 in the loop 1 (residues 168–175, rP2X7 numbering) (Fig. 8A). In addition, the head domain movement of the P2X7 receptor is upward upon ATP binding, and there is a clear positional shift in a segment of the loop structure in the head domain (which we call loop 2, residues 121–139, rP2X7 numbering), in contrast to that of other subtypes. Interestingly, in the apo structure of the pdP2X7 receptor, the position of E171 and the conformation of loop 2 are similar to the open structure of rP2X7, but different from that of the resting state (Fig. 8B). The difference led us to speculate that the redundant β2,3 structure could facilitate the conformational transition from the resting to the open state of E171 and surrounding structural elements in the rP2X7 receptor and that the differences between rat and panda species may be caused by sequence differences in the β2,3-sheet. Therefore, we replaced the β2,3 region of rP2X7 with the corresponding region of pdP2X7 to construct rP2X7CH1 (Fig. 8C). Such a replacement resulted in a certain reduction in the maximum channel current (82.9 ± 35.4 and 204 ± 57 pA/pF for rP2X7CH1 and rP2X7WT, respectively, p < 0.01, rP2X7CH1 versus rP2X7WT, n = 7 and 6; Fig. 8, D and E). On this" @default.
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• W4225137411 date "2022-06-01" @default.
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• W4225137411 title "The long β2,3-sheets encoded by redundant sequences play an integral role in the channel function of P2X7 receptors" @default.
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