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

  • Ion Channels, Receptors and Transporters
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Abstract

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.

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Acknowledgments

This study was supported by the Intramural Research Program of the NICHD, NIH, the Grant Agency of the Czech Republic (305/07/0681 and 303/07/0915), the Internal Grant Agency of Academy of Sciences (IAA5011408 and IAA500110702, Research Project AVOZ 50110509), and the Centrum for Neuroscience (Research Project LC554).

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Correspondence to Hana Zemkova.

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Supplemental Fig. 1

The time required for solution exchange was measured by recording K+ current using standard patch pipette filled with intracellular solution positioned about 500 μm from the common outlet of the application system. Altered KCl concentrations generated a current (gray trace) with the rates of 130 and 220 ms (black lines) for the wash-in and washout periods, respectively. (PDF 78.2 KB)

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Jelínkova, I., Vávra, V., Jindrichova, M. et al. Identification of P2X4 receptor transmembrane residues contributing to channel gating and interaction with ivermectin. Pflugers Arch - Eur J Physiol 456, 939–950 (2008). https://doi.org/10.1007/s00424-008-0450-4

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  • DOI: https://doi.org/10.1007/s00424-008-0450-4

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