Neonicotinoids are synthetic, nicotine-derived insecticides used for agricultural and household pest control. While highly effective at activating insect nicotinic receptors, many neonicotinoids are also capable of directly activating and/or modulating the activation of vertebrate nicotinic receptors. In this study, we have investigated the actions of the neonicotinoids clothianidin (CTD) and imidacloprid (IMI) on human neuronal α4β2 nicotinic acetylcholine receptors. The data demonstrate that the compounds are weak agonists of the human receptors with relative peak currents of 1–4 % of the response to 1 mM acetylcholine (ACh). Coapplication of IMI strongly inhibited currents elicited by ACh. From Schild plot analysis, we estimate that the affinity of IMI to the human α4β2 receptor is 18 µM. The application of low concentrations of CTD potentiated responses to low concentrations of ACh, suggesting that receptors occupied by one ACh and one CTD molecule have a higher gating efficacy than receptors with one ACh bound. Interestingly, subunit stoichiometry affected inhibition by CTD, with (α4)2(β2)3 receptors significantly more strongly inhibited than the (α4)3(β2)2 receptors.

Potentiating neuroactive steroids are potent and efficacious modulators of the GABA A receptor that act by allosterically enhancing channel activation elicited by GABA. Steroids interact with the membrane-spanning domains of the ␣ subunits of the receptor, whereas GABA binds to pockets in the interfaces between ␤ and ␣ subunits. Steroid interaction with a single site is known to be sufficient to produce potentiation, but it is not clear whether effects within the same ␤-␣ pair mediate potentiation. Here, we have investigated whether the sites for GABA and steroids are functionally linked (i.e., whether the occupancy of a steroid site selectively affects activation elicited by GABA binding to the transmitter binding site within the same ␤-␣ pair). For that, we used receptors formed of mutated concatenated subunits to selectively eliminate one of the two GABA sites and one of the two steroid sites. The data demonstrate that receptors containing a single functional GABA site are potentiated by the neurosteroid allopregnanolone regardless of whether the steroid interacts with the ␣ subunit from the same or the other ␤-␣ pair. We conclude that steroids potentiate the opening of the GABA A receptor induced by either agonist binding site.