A consensus phosphoserine within the large cytoplasmic loop of insect nAChR?8 subunits modulated interaction between 14-3-3? and nAChRs to regulate neonicotinoid efficacy

Neonicotinoids are insect-selective nicotinic acetylcholine receptors (nAChRs) agonists that are used extensively for plant protection and animal health care. Some chaperone proteins, such as 14-3-3 proteins, importantly modulate nAChRs to display the physiological and pharmacological properties. Here we found that there is a 14-3-3 binding motif RSPSTH within the cytoplasmic loop of most insect alpha 8 subunits. In the motif, a potential phosphorylated serine residue, serine 337, was a putative protein kinase A (PKA) substrate. Using Locusta migratoria alpha 8 subunit as a representative, here we demonstrated that Loc14-3-3 epsilon interacted with the unique phosphoserine (alpha 8S337) of Loc alpha 8 subunit to regulate agonist efficacy on hybrid Loc alpha 8/beta 2 nAChRs in Xenopus oocytes. Co-expression of Loc14-3-3 epsilon caused a dramatic rise of maximal inward currents (Imax) of Loc alpha 8/beta 2 for acetylcholine and imidacloprid to 2.9-fold and 3.1-fold of that of Loc alpha 8/beta 2 alone. The S337A substitution of Loc alpha 8 reduced the Imax rise when Loc alpha 8S337A/beta 2 and Loc14-3-3 epsilon were co-expressed. The increased agonist currents by exogenous Loc14-3-3 epsilon on Loc alpha 8/beta 2 could be almost abolished by either PKA inhibitor KT5720 or 14-3-3 inhibitor difopein. The findings revealed that serine 337 within motif RSPSTH was important for the interaction between insect nAChRs and 14-3-3 epsilon, and inhibiting the interaction would change the pharmaco-logical property of insect nAChRs to agonist such as neonicotinoids which may provide insights to develop new targets for insecticide design.

Automated summary

This study demonstrates the interaction between insect nAChRs and 14-3-3 epsilon, which is mediated by the phosphorylated serine 337 residue within the RSPSTH motif of the cytoplasmic loop of insect alpha 8 subunits. The co-expression of 14-3-3 epsilon with alpha 8/beta 2 nAChRs significantly increases the agonist efficacy, while the substitution of serine 337 reduces this effect. The increased agonist currents can be blocked by PKA inhibitor or 14-3-3 inhibitor. These findings provide insights into the design of new insecticides by targeting the interaction between nAChRs and 14-3-3 proteins.