Ivermectin (IVM), a large macrocyclic lactone, specifically enhances P2X₄ 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 P2X₄-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 Gln³⁶, Leu⁴⁰, Val⁴³, Val⁴⁷, Trp⁵⁰, Asn³³⁸, Gly³⁴², Leu³⁴⁶, Ala³⁴⁹, and Ile³⁵⁶ mutants. The substitution-sensitive Arg³³ and Cys³⁵³ 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 Met³¹, Tyr⁴², Gly⁴⁵, Val⁴⁹, Gly³⁴⁰, Leu³⁴³, Ala³⁴⁴, Gly³⁴⁷, Thr³⁵⁰, Asp³⁵⁴, and Val³⁵⁷ map on the opposite side of their helices, probably facing the pore of receptor or protein and playing important roles in gating.