Identification of P2X4 receptor transmembrane residues contributing to channel gating and interaction with ivermectin

Ivermectin (IVM), a large macrocyclic lactone, specifically enhances P2X 4 receptor-channel function by interacting with residues of transmembrane (TM) helices in the open conformation state. In this paper, we used cysteine-scanning mutagenesis of rat P2X 4 -TMs to identify and map residues of potential importance for channel gating and interaction with IVM. The receptor function was unchanged by mutations in 29 different residues, and among them, the IVM effects were altered in Gln 36 , Leu 40 , Val 43 , Val 47 , Trp 50 , Asn 338 , Gly 342 , Leu 346 , Ala 349 , and Ile 356 mutants. The substitution-sensitive Arg 33 and Cys 353 mutants could also be considered as IVM-sensitive hits. The pattern of these 12 residues was consistent with helical topology of both TMs, with every third or fourth amino acid affected by substitution. These predominantly hydrophobic-nonpolar residues are also present in the IVM-sensitive Schistosoma mansoni P2X subunit. They lie on the same side of their helices and could face lipids in the open conformation state and provide the binding pocket for IVM. In contrast, the IVM-independent hits Met 31 , Tyr 42 , Gly 45 , Val 49 , Gly 340 , Leu 343 , Ala 344 , Gly 347 , Thr 350 , Asp 354 , and Val 357 map on the opposite side of their helices, probably facing the pore of receptor or protein and playing important roles in gating.