A Highly Conserved Salt Bridge Stabilizes the Kinked Conformation of β2,3-Sheet Essential for Channel Function of P2X4 Receptors

Wen-Shan Zhao,Meng-Yang Sun,Liang-Fei Sun,Yan Liu,Yang Yang,Li-Dong Huang,Ying-Zhe Fan,Xiao-Yang Cheng,Peng Cao,You-Min Hu,Lingyong Li,Yun Tian,Rui Wang,Ye Yu

doi:10.1074/jbc.m115.711127

Abstract

Significant progress has been made in understanding the roles of crucial residues/motifs in the channel function of P2X receptors during the pre-structure era. The recent structural determination of P2X receptors allows us to reevaluate the role of those residues/motifs. Residues Arg-309 and Asp-85 (rat P2X4 numbering) are highly conserved throughout the P2X family and were involved in loss-of-function polymorphism in human P2X receptors. Previous studies proposed that they participated in direct ATP binding. However, the crystal structure of P2X demonstrated that those two residues form an intersubunit salt bridge located far away from the ATP-binding site. Therefore, it is necessary to reevaluate the role of this salt bridge in P2X receptors. Here, we suggest the crucial role of this structural element both in protein stability and in channel gating rather than direct ATP interaction and channel assembly. Combining mutagenesis, charge swap, and disulfide cross-linking, we revealed the stringent requirement of this salt bridge in normal P2X4 channel function. This salt bridge may contribute to stabilizing the bending conformation of the β2,3-sheet that is structurally coupled with this salt bridge and the α2-helix. Strongly kinked β2,3 is essential for domain-domain interactions between head domain, dorsal fin domain, right flipper domain, and loop β7,8 in P2X4 receptors. Disulfide cross-linking with directions opposing or along the bending angle of the β2,3-sheet toward the α2-helix led to loss-of-function and gain-of-function of P2X4 receptors, respectively. Further insertion of amino acids with bulky side chains into the linker between the β2,3-sheet or the conformational change of the α2-helix, interfering with the kinked conformation of β2,3, led to loss-of-function of P2X4 receptors. All these findings provided new insights in understanding the contribution of the salt bridge between Asp-85 and Arg-309 and its structurally coupled β2,3-sheet to the function of P2X receptors.

Highlights

Significant progress has been made in understanding the roles of crucial residues/motifs in the channel function of P2X receptors during the pre-structure era
Disulfide cross-linking, electrophysiology, Western blotting, and molecular modeling, we suggest here the critical role of this highly conserved salt bridge in protein stability and channel gating via a mechanism of stabilizing the kinked conformation of the ␤2,3-sheet, a structural element covalently coupled with one residue of this salt bridge
Conserved Salt Bridge between Arg-312 and Asp-88 Is Essential for the Channel Function of the zebrafish P2X4 receptors (zfP2X4) Receptor—The crystal structure of the zfP2X4 receptor revealed that the guanidinium (RNHC(NH2)2ϩ) of Arg-312 in one subunit interacts with the anionic carboxylate (R-COOϪ) of Asp-88 in another subunit, with the NOO bond distances of 1.79 and 1.74 Å, respectively, forming a salt bridge, both at resting and open states (Fig. 1B)

Summary

Introduction

Significant progress has been made in understanding the roles of crucial residues/motifs in the channel function of P2X receptors during the pre-structure era. Salt Bridge between Arg-309 and Asp-85 Is Essential for rP2X4 Stability and Channel Gating Rather than Direct ATP Interaction and Channel Assembly—Given that the decreased channel response is generally attributed to lower protein expression or impaired channel gating, we measured the protein expression level of the rP2X4 receptor and its mutants, at both total and membrane expression levels.

Results

Conclusion