Nicotinic Acetylcholine Receptor Transmembrane Mutations Convert Ivermectin from a Positive to a Negative Allosteric Modulator

Abstract

Ivermectin is a macrocyclic lactone that acts as a positive allosteric modulator of ␣7 nicotinic acetylcholine receptors (nAChRs) but has no modulatory activity on 5-hydroxytryptamine (5-HT) type 3 (5-HT 3 ) receptors. By examining the influence of ivermectin on subunit chimeras containing domains from the nAChR ␣7 subunit and the 5-HT3A subunit, we have concluded that the transmembrane domains play a critical role in influencing allosteric modulation by ivermectin. A series of mutations located within the ␣-helical transmembrane domains of the ␣7 subunit were examined, and seven were found to have significant effects on allosteric modulation by ivermectin. Four mutations (A225D, Q272V, T456Y, and C459Y) caused a significant reduction in the potency of ivermectin as an allosteric potentiator. Compared with wild-type ␣7 nAChRs, potentiation by ivermectin was reduced dramatically (by 89 -97%) by these mutations. Somewhat unexpectedly, three mutations (S222M, M253L, and S276V located in TM1, TM2, and TM3) converted ivermectin from a positive allosteric modulator into an antagonist. Levels of inhibition of 56, 84, and 89% were observed on M253L, S276V, and S222M, respectively. Antagonism by ivermectin was insurmountable and had no effect on EC 50 of acetylcholine, indicating that it is acting noncompetitively. The seven mutations that influence allosteric modulation by ivermectin are located near a predicted intrasubunit transmembrane cavity. Computer docking simulations provide support for the hypothesis that ivermectin binds in close proximity to this cavity. We conclude that transmembrane mutations in ␣7 nAChRs are able to convert ivermectin from a positive to a negative allosteric modulator.

Agonist activation of α7 nicotinic acetylcholine receptors via an allosteric transmembrane site

“…As has been demonstrated previously, MLA causes a surmountable block of acetylcholine-evoked responses (22,26). Here, MLA was found to act as a reversible antagonist of responses evoked by 4BP-TQS (Fig.…”

“…In previous studies (21) we have identified an intrasubunit cavity, located between the four transmembrane α-helices, as the allosteric binding site for both type I and type II PAMs acting on α7 nAChRs (21,22). Here, we provide evidence that ligand binding at this transmembrane allosteric site is able to activate nAChRs in the absence of agonist binding at the orthosteric site.…”

“…S1A). However, in contrast to its effects on acetylcholine (22,26), antagonism of 4BP-TQS responses by MLA was not surmountable (Fig. 5), a feature that is characteristic of noncompetitive antagonism.…”

“…We have shown previously that mutations located within a proposed intrasubunit transmembrane cavity of α7 nAChRs reduce levels of potentiation by both type I and type II PAMs (21,22). For example, a single-point mutation (M253L) located in the second transmembrane domain (TM2), reduced levels of potentiation by PNU-120596 to about 10% of that observed with wild-type α7 nAChRs (21).…”

“…MLA is a competitive antagonist of acetylcholine acting on α7 nAChRs (22,26), but acts as a noncompetitive antagonist of 4BP-TQS. When acting as an antagonist of acetylcholine, MLA binds competitively at the conventional (orthosteric) binding site.…”