Conformational changes during human P2X7 receptor activation examined by structural modelling and cysteine-based cross-linking studies

Abstract

The P2X7 receptor (P2X7R) is important in mediating a range of physiological functions and pathologies associated with tissue damage and inflammation and represents an attractive therapeutic target. However, in terms of their structure-function relationships, the mammalian P2X7Rs remain poorly characterised compared to some of their other P2XR counterparts. In this study, combining cysteine-based cross-linking and whole-cell patch-clamp recording, we examined six pairs of residues (A44/I331, D48/I331, I58/F311, S60/L320, I75/P177 and K81/V304) located in different parts of the extracellular and transmembrane domains of the human P2X7R. These residues are predicted to undergo substantial movement during the transition of the receptor ion channel from the closed to the open state, predictions which are made based on structural homology models generated from the crystal structures of the zebrafish P2X4R. Our results provide evidence that among the six pairs of cysteine mutants, D48C/I133C and K81C/V304C formed disulphide bonds that impaired the channel gating to support the notion that such conformational changes, particularly those in the outer ends of the transmembrane domains, are critical for human P2X7R activation.Electronic supplementary materialThe online version of this article (doi:10.1007/s11302-016-9553-0) contains supplementary material, which is available to authorized users.

Identification of residues in the first transmembrane domain of the P2X7 that regulates receptor trafficking, sensitization, and dye uptake function

“…In the case of P2X7, alanine A44 could form hydrophobic interaction with isoleucine I331, which cysteine substitution, I331C, has been shown to exhibit a delayed deactivation time course (Pippel et al, 2017); the distances are 4.0 Å and 10.6 Å in the closed and open states, respectively (Figure 8d). However, application of DTT to the doubly mutated receptor A44C/I331C did not increase the agonist‐induced current (Caseley et al, 2017). It has been suggested that A44 is nestled more deeply in the TM1 domain and is not well positioned toward I331 to form a disulfide bond (Caseley et al, 2017).…”

“…However, application of DTT to the doubly mutated receptor A44C/I331C did not increase the agonist‐induced current (Caseley et al, 2017). It has been suggested that A44 is nestled more deeply in the TM1 domain and is not well positioned toward I331 to form a disulfide bond (Caseley et al, 2017). Thus, the role of A44 in P2X7 sensitization and dye uptake remains to be explained.…”

Protein disulfide isomerase-mediated S-nitrosylation facilitates surface expression of P2X7 receptor following status epilepticus

“…Since PDI inhibits N -methyl- d -aspartate receptor (NMDAR) activity via sulfhydration (reduction) of disulfide bonds [ 7 , 22 ], it is plausible that the L-NAME would also attenuate microglial activation and DG astroglial apoptosis by facilitating PDI-mediated reduction of surface P2X7R. However, P2X7R-mediated currents are unaffected by the reduction of disulfide bonds on surface P2X7R [ 47 ]. Therefore, our findings suggest that PDI-mediated S -nitrosylation of P2X7R may reinforce SE-induced microglial activation and DG astroglial loss by increasing its surface expression, independent of activities of PDI and surface P2X7R.…”

Structural basis for the functional properties of the P2X7 receptor for extracellular ATP

“…While the structures reveal that the upper body remains overall relatively immobile, there is increasing functional evidence to support that conformational changes occur in the upper body during P2X7 receptor activation. Simultaneous cysteine substitutions of Lys 81 and Val 304 in the human P2X7 receptor hindered receptor activation that was reversed by treatment with the reducing agent dithiothreitol (DTT) [48], indicating that these two residues or positions are in close vicinity in the closed state and move apart distantly in the open state. Replacement of Val 87 in the human P2X7 receptor with the corresponding isoleucine in the rat P2X7 receptor enhanced current responses to ATP and BzATP [49], suggesting that residue at this position influences the local conformational change differently in the rat and human receptors.…”

“…3d ). In the human P2X7 receptor, simultaneous introduction of cysteine substitution at Asp 48 and Ile 331 , located in the extracellular end of the TM1 and TM2 α-helices, respectively, prevented receptor activation, which was rescued by treatment with DTT [ 48 ]. Thus, these two residues or positions are in close proximity in the closed state and distance significantly from each other in the open state, supporting occurrence of considerable conformational changes in the external vestibule.…”