The Second Transmembrane Domain of P2X7 Contributes to Dilated Pore Formation

Abstract

Activation of the purinergic receptor P2X7 leads to the cellular permeability of low molecular weight cations. To determine which domains of P2X7 are necessary for this permeability, we exchanged either the C-terminus or portions of the second transmembrane domain (TM2) with those in P2X1 or P2X4. Replacement of the C-terminus of P2X7 with either P2X1 or P2X4 prevented surface expression of the chimeric receptor. Similarly, chimeric P2X7 containing TM2 from P2X1 or P2X4 had reduced surface expression and no permeability to cationic dyes. Exchanging the N-terminal 10 residues or C-terminal 14 residues of the P2X7 TM2 with the corresponding region of P2X1 TM2 partially restored surface expression and limited pore permeability. To further probe TM2 structure, we replaced single residues in P2X7 TM2 with those in P2X1 or P2X4. We identified multiple substitutions that drastically changed pore permeability without altering surface expression. Three substitutions (Q332P, Y336T, and Y343L) individually reduced pore formation as indicated by decreased dye uptake and also reduced membrane blebbing in response to ATP exposure. Three others substitutions, V335T, S342G, and S342A each enhanced dye uptake, membrane blebbing and cell death. Our results demonstrate a critical role for the TM2 domain of P2X7 in receptor function, and provide a structural basis for differences between purinergic receptors.

Functional characterization of mutants in the transmembrane domains of the rat P2X7 receptor that regulate pore conductivity and agonist sensitivity

“…Recently, it has been demonstrated that the Q332P, Y336T, and Y343L mutants of P2X7R exhibited decreased dye uptake, whereas the V335T, S342G, and S342A mutants exhibited enhanced dye uptake during sustained agonist application (Sun et al . ). Another study revealed that the T348K, D352N, and D352K mutant receptors displayed increased relative permeability to chloride ions and promoted the entry of the large negative dye fluorescein‐5‐isothiocyanate (Browne et al .…”

“…; Sun et al . ). The role of TM1 in the dilation of the P2X7R pore has also been suggested (Nicke et al .…”

“…The gating hypothesis of P2X2R dilation suggests that the permeability changes of this channel occur at the interface between TM1 and TM2 of neighboring subunits Khakh and Egan 2005). Recently, several residues that modulate dye uptake have also been identified in the porelining TM2 of P2X7R (Browne et al 2013;Sun et al 2013). The role of TM1 in the dilation of the P2X7R pore has also been suggested (Nicke et al 2009) but crucial residues in TM1 have yet to be determined.…”

Comparative study of the P2X gene family in animals and plants

“…However, subtype-specific motifs are mainly located in the C-terminus, such as motifs 27 and 35 in mammalian P2X6, which is thought to be involved in desensitization [56]. A series of P2X7-specific motifs (motifs 19,24,23,20,16, and 21) was found, which can constitute a longer TM2 domain enabling P2X7 receptors to provide a pathway that allows the passage of larger organic cations [57,58]. In addition, some primitive species (Aqu-p2xa, Sko-p2xa, Nve-p2xa) share a series of conserved motifs with advanced species, while some homologous short motifs were also detected in Fig.…”

Localization of the gate and selectivity filter of the full-length P2X7 receptor

“…The cytolytic activity of human P2X7R (hP2X7R) has been attributed to a time-dependent dilation of the integral ion channel based on macroscopic current recordings of various cell types (7)(8)(9)(10)(11). Particularly revealing was the observation that substituting T348 and D352 with basic residues in the channellining second transmembrane domain (TM2) of the rat P2X7R (rP2X7R) simultaneously increased the permeability of the normally cationic channel for Cl − and an acidic fluorescent dye with an effective diameter of >10 Å (12).…”