The activity of GAT107, an allosteric activator and positive modulator of {alpha}7 nAChR, is regulated by aromatic amino acids that span the subunit interface [Neurobiology]

December 20th, 2013 by Papke, R. L., Horenstein, N. A., Kulkarni, A. R., Stokes, C., Corrie, L. W., Maeng, C.-Y., Thakur, G. A.

GAT107, the (+)-enantiomer of racemic 4BPTQS, is a strong positive allosteric modulator (PAM) of α7 nAChR activation by orthosteric agonists with intrinsic allosteric agonist activity. The direct activation produced by GAT107 in electrophysiological studies is observed only as long as GAT107 is freely diffusible in solution, while the potentiating activity primed by GAT107 can persist for over 30 minutes after drug washout. Direct activation is sensitive to α7 nAChR antagonist methyllycaconitine, while the primed potentiation is not. The data are consistent with GAT107 activity arising from two different sites. We show that the coupling between PAMs and the binding of orthosteric ligands requires tryptophan 55 (W55), which is located at the subunit interface on the complementary surface of the orthosteric binding site. Mutations of W55 increase the direct activation produced by GAT107 and reduce or prevent the synergy between allosteric and orthosteric binding sites, such that these mutants can also be directly activated by other PAMs such as PNU-120596 and TQS, which do not activate wild-type α7 in the absence of orthosteric agonists. We identify Y93 as an essential element for orthosteric activation, such that Y93C mutants are insensitive to orthosteric agonists but respond to GAT107. Our data show that both orthosteric and allosteric activation of α7 nAChR require cooperative activity at the interface between the subunits in the extracellular domain. These cooperative effects rely on key aromatic residues, and while mutations of W55 reduce the restraints placed on the requirement for orthosteric agonists, Y93 can conduct both orthosteric activation and desensitization among the subunits.
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