Intersubunit physical couplings fostered by the left flipper domain facilitate channel opening of P2X4 receptors

Abstract

P2X receptors are ATP-gated trimeric channels with important roles in diverse pathophysiological functions. A detailed understanding of the mechanism underlying the gating process of these receptors is thus fundamentally important and may open new therapeutic avenues. The left flipper (LF) domain of the P2X receptors is a flexible loop structure, and its coordinated motions together with the dorsal fin (DF) domain are crucial for the channel gating of the P2X receptors. However, the mechanism underlying the crucial role of the LF domain in the channel gating remains obscure. Here, we propose that the ATP-induced allosteric changes of the LF domain enable it to foster intersubunit physical couplings among the DF and two lower body domains, which are pivotal for the channel gating of P2X4 receptors. Metadynamics analysis indicated that these newly established intersubunit couplings correlate well with the ATP-bound open state of the receptors. Moreover, weakening or strengthening these physical interactions with engineered intersubunit metal bridges remarkably decreased or increased the open probability of the receptors, respectively. Further disulfide cross-linking and covalent modification confirmed that the intersubunit physical couplings among the DF and two lower body domains fostered by the LF domain at the open state act as an integrated structural element that is stringently required for the channel gating of P2X4 receptors. Our observations provide new mechanistic insights into P2X receptor activation and will stimulate development of new allosteric modulators of P2X receptors.

Druggable negative allosteric site of P2X3 receptors

“…The crystal structures for the open states of P2X4 and P2X3 receptors clearly show a downward shift of the LF domain toward the LB−DF domain of the neighboring subunit compared with the structures at the apo state, suggesting that the downward motion of the LF domain upon ATP binding may constitute an important step of P2X channel gating by its endogenous ligand, ATP (9)(10)(11). Previously, combining computational stimulation and mutational studies, we showed that manipulations that prevented the motion between the LF and DF domains all disrupted ATP gating of P2X4 (23,27). Not surprisingly, by allowing a disulfide bond formation between the LF and DF domains of P2X3, the activation of P2X3 K201C/V274C by ATP was also largely suppressed (34).…”

“…Furthermore, the salt bridge between R264 and D266 ( Fig. 2 A and B), a conserved structure at the beginning of LF domains of all P2X receptor subtypes (10,23), is crucial for holding the LF domain in place as the R264A (IC 50 = 0.72 ± 0.01 μM; Fig. 2C) and D266A (IC 50 > 30 μM; Fig.…”

“…Whole-cell recordings were performed on HEK-293 cells 24 h to 48 h after transfection (27). Homology modeling was carried out using MODELLER (23,38). MD and metadynamics were performed using DESMOND (39).…”

Mapping the Allosteric Action of Antagonists A740003 and A438079 Reveals a Role for the Left Flipper in Ligand Sensitivity at P2X7 Receptors

“…Interestingly, this chimera increased sensitivity to ATP ∼30-fold ( Allsopp et al, 2017 ) and swapping with the equivalent region from the P2X4R increased potency ∼15-fold (this study). Movement of the left flipper has been proposed to be coupled to channel gating upon ATP binding ( Zhao et al, 2014 ; Karasawa and Kawate, 2016 ; Wang et al, 2017 ). Investigating the effect of individual residues in the region 279–285 reveals that the D280A mutation renders the P2X7R 15-times more sensitive to ATP.…”

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].…”