A Highly Conserved Salt Bridge Stabilizes the Kinked Conformation of 2,3-Sheet Essential for Channel Function of P2X4 Receptors

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 2,3-sheet that is structurally coupled with this salt bridge and the 2-helix. Strongly kinked 2,3 is essential for domain-domain interactions between head domain, dorsal fin domain, right flipper domain, and loop 7,8 in P2X4 receptors. Disulfide cross-linking with directions opposing or along the bending angle of the 2,3-sheet toward the 2-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 2,3-sheet or the conformational change of the 2-helix, interfering with the kinked conformation of 2,3, 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 2,3-sheet to the function of P2X receptors.