Tryptophan 46 is a site for ethanol and ivermectin action in P2X4 receptors

Abstract

ATP-gated purinergic P2X4 receptors (P2X4Rs) are the most alcohol-sensitive P2XR subtype. We recently reported that ivermectin (IVM), an antiparasitic used in animals and humans, antagonized ethanol inhibition of P2X4Rs. Furthermore, IVM reduced ethanol intake in mice. The first molecular model of the rat P2X4R, built onto the Xray crystal structure of zebrafish P2X4R, revealed an action pocket for both ethanol and IVM formed by Asp331, Met336 in TM2 and Trp46, and Trp50 in TM1 segments. The role of Asp331 and Met336 was experimentally confirmed. The present study tested the hypothesis that Trp46 plays a role in ethanol and IVM modulation of P2X4Rs. Trp46 was mutated to residues with different physicochemical properties and the resultant mutants tested for ethanol and IVM responses using Xenopus oocyte expression system and twoelectrode voltage clamp. Nonaromatic substitutions at position 46 reduced ethanol inhibition at higher concentrations and switched IVM potentiation to inhibition. Simultaneous substitution of alanine at positions Trp46 and Met336 also resulted in similar changes in ethanol and IVM responses. Furthermore, a new molecular model based on the open pore conformation of zebrafish P2X4R suggested a role for Tyr42 that was further supported experimentally. Our previous and current findings, combined with our preliminary evidence of increased ethanol consumption in P2X4R knockout mice, suggest that the ethanol and IVM action pocket in P2X4Rs formed by positions 42, 46, 331, and 336 presents a potential target for medication development for alcohol use disorders.

Molecular structure and function of P2X receptors

“…In contrast, chimeras of the two transmembrane segments of rP2X4 receptor into rP2X2 receptor are sufficient to confer IVM sensitivity (Silberberg et al, 2007), indicating a major role of both transmembrane segments of P2X4 receptor in the positive allosteric effect of the modulator. Consistent with a transmembrane location, site-directed mutagenesis analysis of residues located in the putative TM domains (Jelinkova et al, 2008;Popova et al, 2013;Silberberg et al, 2007) indicated that V28, Q36, I39, L40, Y42, V43, V47, W50 (TM1) and N338, G340, G342, L345, L346, V348, A349, I356 (TM2) in rP2X4 are involved in IVM sensitivity of the channel (Jelinkova et al, 2008;Silberberg et al, 2007). A recent study confirmed the importance of some of these residues (L40, V43, V47 and W50) in the effect of IVM on deactivation in rP2X4, and further identified new ones (W46, G53, F330, D331, I332 and I337) (Zemkova et al, 2015).…”

“…In support of this hypothesis, mutations of the amino acid W46 to other aromatic residues (tyrosine or phenylalanine) in heterologously expressed rP2X4 receptor did not modify IVM-induced potentiation of ATP-dependent currents, whereas substitution with nonaromatic residues (leucine, valine, alanine, asparagine, lysine or aspartate) switched the potentiation to inhibition (Popova et al, 2013). Therefore, it has been suggested that the presence of an aromatic group at position 46 is a critical determinant of IVM-induced allosteric effects, most probably by means of a cation-π interaction (Popova et al, 2013).…”

“…It was previously suggested that the interaction between W46 and M336 participates to a binding pocket for IVM (Asatryan et al, 2010). However, whilst both individual W46A and M336A mutations as well as the double mutation significantly reduced the inhibitory effect of ethanol, only the mutation W46A (or the combined mutation) modified the positive allosteric effect of IVM (Popova et al, 2013). Finally, the analysis of a new molecular model based on the ATP-bound structure of zfP2X4 suggested a prominent role of the amino acid Y42 of rP2X4 receptor, confirmed by the mutation Y42F which also switched the potentiating allosteric effect of IVM to inhibition (Popova et al, 2013).…”

“…Therefore, it has been suggested that the presence of an aromatic group at position 46 is a critical determinant of IVM-induced allosteric effects, most probably by means of a cation-π interaction (Popova et al, 2013). It was previously suggested that the interaction between W46 and M336 participates to a binding pocket for IVM (Asatryan et al, 2010).…”

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

“…NF770, a suramin derivative that competitively inhibits the P2X2 receptor at nanomolar concentrations, acts on G72, E167 and R290 (rP2X2 numbering), which are also important for ATP binding [112,113] . Interestingly, nearly all of those identified sites are located in or around the ATP binding pocket except for IVM, a P2X4 positive modulator, which has been identified to act on the TM domains [114][115][116][117] .…”