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

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 ␤2,3-sheet that is structurally coupled with this salt bridge and the ␣2-helix.

Structural insights into the competitive inhibition of the ATP-gated P2X receptor channel

“…Electrophysiological measurements were performed on HEK-293 cells, 24–48 h after transfection. The conventional whole-cell configuration under the voltage clamp at room temperature (23 ± 2 °C) was used to obtain the electrophysiological recordings 36, 45 . Patch pipettes were pulled from glass capillaries, using the two-stage puller PP-830 (Narishige Co., Ltd.), and the resistance between the recording electrode filled with pipette solution and the reference electrode in bath solution ranged from 3 to 5 MΩ.…”

“…During electrophysiological recordings, 80–90% of the series resistance was compensated and the membrane potential was held at −60 mV throughout the experiment. ATP solutions were prepared in the batch buffer within 2 h of use, and applied using a fast pressure-driven, computer-controlled microperfusion system, OctaFlow08P (ALA Scientific Instruments) 45 . ATP currents were normalized to the cell membrane capacitance.…”

Three Decades of Chloride Intracellular Channel Proteins: From Organelle to Organ Physiology

“…11.21.2; Legg-E'silva et al, 2012). The E-R salt bridge is known to modulate open probability of numerous ion channels (Leng, MacGregor, Dong, Giebisch, & Hebert, 2006;Moroni & Thiel, 2006;Zhao et al, 2016). Acidic pH, as in the case of the K + channel, breaks the critical salt bridge between different domains, thereby directly modulating CLIC channel activity.…”

Molecular mechanism underlying the subtype-selectivity of competitive inhibitor NF110 and its distinct potencies in human and rat P2X3 receptors

“…The homology models of rP2X3 and mutants were constructed based on the hP2X3 structure (PDB ID code 5SVJ) [35] with the program MODELLER according to previous descriptions [37,38,40,41]. ClustalW2 was used to align and manually adjust the target sequences to match published alignments [35].…”