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

Abstract

P2X3 receptors (P2X3R) are trimeric ATP-gated cation channels involved in sensory neurotransmission and inflammatory pain. We used homology modeling and molecular dynamic simulations of the hP2X3R to identify intersubunit interactions of residues that are instrumental to elucidate conformational changes associated with gating of the hPX3R. We identified an ionic interaction between E112 and R198 of the head domain and dorsal fin domain, respectively, and E57 and T263 of the lower body domains of adjacent subunits and detected a marked rearrangement of these domains during gating of the hP3X3R. Double-mutant cycle analysis of the inter-subunit residue pairs E112/R198 and E57/ T263 revealed significant interaction-free energies. Disulfide locking of the hP2X3R E112C/R198C or the E57C/T263C double cysteine mutants markedly reduced the ATP-induced current responses. The decreased current amplitude following inter-subunit disulfide cross-linking indicates that disulfide locking of the head and dorsal fin domains or at the level of the lower body domains of the hP2X3R prevents the gatinginduced conformational rearrangement of the subunits with respect to each other. The distinct reorganization of the subunit interfaces during gating of the hP2X3R is generally consistent with the gating mechanism of other P2XRs. Chargereversal mutagenesis and methanethiosulfonate (MTS)-modification of substituted cysteines demonstrated that E112 and R198 interact electrostatically. Both disulfide locking and salt bridge breaking of the E112/R198 interaction reduced the hP2X3R function. We conclude that the inter-subunit salt bridge between E112 and R198 of the head and dorsal fin domains, respectively, serves to control the mobility of these domains during agonist-activation of the hP2X3R.

Acupuncture-Induced Analgesia

“…The agonist binding pouches are located at the border of two neighboring subunits at the upper part of the receptor. Modified from Stephan and others (2016). (B) Schematic presentation of the effect of acupuncture against neuropathic pain symptoms; the “mechanical withdrawal threshold” is a measure of hyperalgesia and the “thermal withdrawal threshold” is a measure of allodynia.…”

Conformational changes during human P2X7 receptor activation examined by structural modelling and cysteine-based cross-linking studies

“…This technique has been successfully applied in previous studies of ion channel gating (e.g. P2X2R [ 16 ], GABA A R [ 17 ] and K V [ 18 ]), subunit stoichiometry of the P2X2/3R [ 19 ] and the inter-subunit ATP-binding site in the P2XR [ 20 , 21 ] and in recent studies of agonist-induced conformational changes in the P2X1R [ 22 ], P2X2R [ 23 ] and P2X3R [ 24 ]. Here, we used this approach to examine conformational changes in the extracellular and transmembrane domains that are predicted, based on the structural models, to occur during the hP2X7R activation.…”