Ivermectin Antagonizes Ethanol Inhibition in Purinergic P2X4 Receptors

Abstract

ATP-gated purinergic P2X4 receptors (P2X4Rs) are expressed in the central nervous system and are sensitive to ethanol at intoxicating concentrations. P2XRs are trimeric; each subunit consists of two transmembrane (TM) ␣-helical segments, a large extracellular domain, and intracellular amino and carboxyl terminals. Recent work indicates that position 336 (Met336) in the TM2 segment is critical for ethanol modulation of P2X4Rs. The anthelmintic medication ivermectin (IVM) positively modulates P2X4Rs and is believed to act in the same region as ethanol. The present study tested the hypothesis that IVM can antagonize ethanol action. We investigated IVM and ethanol effects in wild-type and mutant P2X4Rs expressed in Xenopus oocytes by using a two-electrode voltage clamp. IVM antagonized ethanol-induced inhibition of P2X4Rs in a concentrationdependent manner. The size and charge of substitutions at position 336 affected P2X4R sensitivity to both ethanol and IVM. The first molecular model of the rat P2X4R, built onto the X-ray crystal structure of zebrafish P2X4R, revealed a pocket formed by Asp331, Met336, Trp46, and Trp50 that may play a role in the actions of ethanol and IVM. These findings provide the first evidence for IVM antagonism of ethanol effects in P2X4Rs and suggest that the antagonism results from the ability of IVM to interfere with ethanol action on the putative pocket at or near position 336. Taken with the building evidence supporting a role for P2X4Rs in ethanol intake, the present findings suggest that the newly identified alcohol pocket is a potential site for development of medication for alcohol use disorders.

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

“…We have shown that IVM antagonizes ethanolmediated inhibition of P2X4Rs in vitro [12]. Moreover, IVM significantly reduced ethanol intake and preference in mouse models of ethanol self-administration [22].…”

“…We have identified residues Asp331 and Met336 in the ectodomain-TM2 segment interface to be important for ethanol modulation, whereas residues Asn338, Ser341, Gly342, Leu346, Gly347, Ala349, and Ile356 in the TM2 segment were found to play a role in IVM modulation of the receptor [25,26]. Furthermore, we found that Met336 at the ectodomain-TM2 interface is a common site of action for both ethanol and IVM [12]. Interestingly, these sites are closely located to lateral fenestrations found in P2X4Rs that were shown to be sites for allosteric modulation of the receptor [27], including by IVM [28].…”

“…Ethanol and/or IVM were co-applied with ATP for 20 s. Submaximal ATP concentrations that ranged from EC 5 to EC 10 were used in these studies that are designated as EC 10 throughout the manuscript. We have previously shown that ethanol inhibition of ATP function on P2X4Rs is more robust and reliable when tested in the presence of low concentrations of ATP (typically EC [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] ) [11,13]. ATPactivated EC 10 responses were measured before and after each ethanol application to take into account possible shifts in the baseline current values.…”

“…We built a homology model of rat P2X4R by threading the primary sequence of rat P2X4R onto the recent X-ray structure of the zebrafish P2X4R [12]. Briefly, we used the crystal structure of the open conformation of zebrafish P2X4R-A (PDB ID 4DW1) [6] as a template for the homology models of the rat P2X4R.…”

“…P2X4Rs are the most alcohol-sensitive P2XR subtype known [11]. In vitro, they are inhibited by a broad range of ethanol concentrations beginning well below the concentrations considered legally intoxicating in the United States (0.08 % or 17 mM) [11][12][13][14][15][16][17]. Evidence from genetic and genomic studies [18,19] suggests that these receptors play a role in regulating ethanol intake.…”