Conformational flexibility of the agonist binding jaw of the human P2X3 receptor is a prerequisite for channel opening

Abstract

BACKGROUND AND PURPOSEIt is assumed that ATP induces closure of the binding jaw of ligand-gated P2X receptors, which eventually results in the opening of the membrane channel and the flux of cations. Immobilization by cysteine mutagenesis of the binding jaw inhibited ATP-induced current responses, but did not allow discrimination between disturbances of binding, gating, subunit assembly or trafficking to the plasma membrane. EXPERIMENTAL APPROACHA molecular model of the pain-relevant human (h)P2X3 receptor was used to identify amino acid pairs, which were located at the lips of the binding jaw and did not participate in agonist binding but strongly approached each other even in the absence of ATP. KEY RESULTSA series of cysteine double mutant hP2X3 receptors, expressed in HEK293 cells or Xenopus laevis oocytes, exhibited depressed current responses to α,β-methylene ATP (α,β-meATP) due to the formation of spontaneous inter-subunit disulfide bonds. Reducing these bonds with dithiothreitol reversed the blockade of the α,β-meATP transmembrane current. Amino-reactive fluorescence labelling of the His-tagged hP2X3 receptor and its mutants expressed in HEK293 or X. laevis oocytes demonstrated the formation of inter-subunit cross links in cysteine double mutants and, in addition, confirmed their correct trimeric assembly and cell surface expression. CONCLUSIONS AND IMPLICATIONSIn conclusion, spontaneous tightening of the binding jaw of the hP2X3 receptor by inter-subunit cross-linking of cysteine residues substituted at positions not directly involved in agonist binding inhibited agonist-evoked currents without interfering with binding, subunit assembly or trafficking.

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

“…1A) (16,17). It has been demonstrated that channel activation was associated with the downward motion of the head domain that is coordinated with ATP binding (20,25). As revealed in MD simulations, we observed alterations of movement in the head domain after losing this salt bridge (Figs.…”

Insights into the channel gating of P2X receptors from structures, dynamics and small molecules

“…Although the sequence of the head domain is not highly www.nature.com/aps Wang J et al Acta Pharmacologica Sinica npg conserved throughout the P2X family, the architecture of this domain in different subtypes shares certain similarity due to three conserved disulfide bonds that contribute to the folding of P2X receptors [38][39][40][41][42] (Figure 2A and 2C). The architecture of head domain of the zfP2X4 receptor was determined by X-ray diffraction and showed a high similarity in folding pattern with rat P2X4 (rP2X4) resolved by nuclear magnetic resonance, suggesting the conservation of the P2X4 head domain in different species [40] .…”

Inter-subunit disulfide locking of the human P2X3 receptor elucidates ectodomain movements associated with channel gating

“…In the hP2X3R, we have recently shown by disulfide trapping analysis that the conformational mobility of domains constituting the inter-subunit ATP-binding site is essential for ATP-induced channel opening [21]. By using homology modeling and functional analysis of alanine mutants, the residues K63, K65, T172, K176, N279, F280, R281, and K299 of the hP2X3R were identified to be important for ATP-binding [22].…”

“…In a preceding paper, we simulated molecular dynamics of the hP2X3R, based upon its closed homology model, and thereby identified regions of the receptor ectodomain approaching each other around the agonist binding pocket [21]. Then, we generated potential cysteine double mutants in order to check the possibility of disulfide bond formation and subsequent immobilization/inactivation of the receptor.…”

“…The apo closed-state and ATP-bound open-state models of the hP2X3 were generated by the Modeller 9v8 program (http://salilab.org/modeller) using the pdb entries 3H9V [5] and 4DW1 [4], respectively, as previously described [21,31]. To determine the probability of electrostatic or hydrogen bond interactions of amino acid residues of the hP2X3 closed state throughout the protein dynamics in absence of the agonist, we simulated molecular dynamics with NAMD [32] (http://www.ks.uiuc.edu/Research/namd) based on the closed-state hP2X3 homology model, visualized the receptor structure and dynamics with visual molecular dynamics (VMD) [33] (http://www.ks.uiuc.edu/Research/ vmd) and used the Salt Bridges plugin (version 1.1) or HBonds plugin (version 1.2) of VMD, respectively, with default settings for the prediction of salt bridges or hydrogen bond interactions throughout the MD trajectories.…”