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

Ivermectin is a macrocyclic lactone that acts as a positive allosteric modulator of alpha 7 nicotinic acetylcholine receptors (nAChRs) but has no modulatory activity on 5-hydroxytryptamine (5-HT) type 3 (5-HT3) receptors. By examining the influence of ivermectin on subunit chimeras containing domains from the nAChR alpha 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 alpha-helical transmembrane domains of the alpha 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 alpha 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 EC50 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 alpha 7 nAChRs are able to convert ivermectin from a positive to a negative allosteric modulator.