Role of the Cys Loop and Transmembrane Domain in the Allosteric Modulation of α4β2 Nicotinic Acetylcholine Receptors

Allosteric modulators of pentameric ligand-gated ion channels are thought to act on elements of the pathways that couple agonist binding to channel gating. Using alpha 4 beta 2 nicotinic acetylcholine receptors and the alpha 4 beta 2-selective positive modulators 17 beta-estradiol (beta EST) and desformylflustrabromine (dFBr), we have identified pathways that link the binding sites for these modulators to the Cys loop, a region that is critical for channel gating in all pentameric ligand-gated ion channels. Previous studies have shown that the binding site for potentiating beta EST is in the C-terminal (post-M4) region of the alpha 4 subunit. Here, using homology modeling in combination with mutagenesis and electrophysiology, we identified the binding site for potentiating dFBr on the top half of a cavity between the third (M3) and fourth transmembrane (M4) alpha-helices of the alpha 4 subunit. We found that the binding sites for beta EST and dFBr communicate with the Cys loop, through interactions between the last residue of post-M4 and Phe(170) of the conserved FPF sequence of the Cys loop, and that these interactions affect potentiating efficacy. In addition, interactions between a residue in M3 (Tyr(309)) and Phe(167), a residue adjacent to the Cys loop FPF motif, also affect dFBr potentiating efficacy. Thus, the Cys loop acts as a key control element in the allosteric transduction pathway for potentiating beta EST and dFBr. Overall, we propose that positive allosteric modulators that bind the M3-M4 cavity or post-M4 region increase the efficacy of channel gating through interactions with the Cys loop.