Determination of the Residues in the Extracellular Domain of the Nicotinic α Subunit Required for the Actions of Physostigmine on Neuronal Nicotinic Receptors.

Physostigmine can potentiate and inhibit neuronal nicotinic receptors, in addition to inhibiting the activity of acetylcholinesterase. We found that receptors containing three copies of the α 2 subunit are inhibited by low concentrations of physostigmine in contrast to receptors containing three copies of the α 4 subunit that are potentiated. We exploited this observation to determine the regions required for the actions of physostigmine. Chimeric constructs of the α 2 and α 4 subunits located two regions in the extracellular amino-terminal domain of the subunit: the E loop (a loop of the transmitter-binding domain) and a region closer to the amino-terminus that collectively could completely determine the different effects of physostigmine. Point mutations then identified a single residue, α 2(I92) versus α 4(R92), that, when combined with transfer of the E loop, could convert the inhibition seen with α 2 subunits to potentiation and the potentiation seen with α 4 subunits to inhibition. In addition, other point mutations could affect the extent of potentiation or inhibition, indicating that a more extensive set of interactions in the amino-terminal domain plays some role in the actions of physostigmine.