TY - JOUR T1 - The E loop of the transmitter-binding site is a key determinant of the modulatory effects of physostigmine on neuronal nicotinic α4β2 receptors JF - Molecular Pharmacology JO - Mol Pharmacol DO - 10.1124/mol.116.106484 SP - mol.116.106484 AU - Xiaochun Jin AU - Megan M McCollum AU - Allison L Germann AU - Gustav Akk AU - Joseph H. Steinbach Y1 - 2016/01/01 UR - http://molpharm.aspetjournals.org/content/early/2016/11/28/mol.116.106484.abstract N2 - Physostigmine is a well-known inhibitor of acetylcholinesterase, which can also activate, potentiate and inhibit acetylcholine receptors, including neuronal nicotinic receptors comprising α4 and β2 subunits. We have found that the two stoichiometric forms of this receptor differ in the effects of physostigmine. The form containing 3 copies of α4 and 2 of β2 was potentiated at low concentrations of ACh and physostigmine, while the form containing 2 copies of α4 and 3 of β2 was inhibited. Chimeric constructs of subunits indicated that the presence of inhibition or potentiation depended on the source of the extracellular ligand-binding domain of the subunit. Further sets of chimeric constructs demonstrated that a portion of the ACh-binding domain, the E loop, is a key determinant. Transferring the E loop from the β2 subunit to the α4 subunit resulted in strong inhibition, while the reciprocal transfer reduced inhibition. We expressed chimeric constructs with subunit dimers, to control the number and position of the incorporated chimeric subunits. Surprisingly, incorporation of a subunit with an altered E loop had similar effects whether it contributed either to an intersubunit interface containing a canonical ACh-binding site or to an alternative interface. The observation that the α4 E loop is involved suggests that physostigmine interacts with regions of subunits that contribute to the ACh-binding site, while the lack of interface specificity indicates that interaction with a particular ACh-binding site is not the critical factor. ER -