Potentiation of alpha7 nicotinic acetylcholine receptors via an allosteric transmembrane site

Abstract

Positive allosteric modulators of alpha7 nicotinic acetylcholine receptors (nAChRs) have attracted considerable interest as potential tools for the treatment of neurological and psychiatric disorders such as Alzheimer's disease and schizophrenia. However, despite the potential therapeutic usefulness of these compounds, little is known about their mechanism of action. Here, we have examined two allosteric potentiators of alpha7 nAChRs (PNU-120596 and LY-2087101). From studies with a series of subunit chimeras, we have identified the transmembrane regions of alpha7 as being critical in facilitating potentiation of agonist-evoked responses. Furthermore, we have identified five transmembrane amino acids that, when mutated, significantly reduce potentiation of alpha7 nAChRs. The amino acids we have identified are located within the alpha-helical transmembrane domains TM1 (S222 and A225), TM2 (M253), and TM4 (F455 and C459). Mutation of either A225 or M253 individually have particularly profound effects, reducing potentiation of EC(20) concentrations of acetylcholine to a tenth of the level seen with wild-type alpha7. Reference to homology models of the alpha7 nAChR, based on the 4A structure of the Torpedo nAChR, indicates that the side chains of all five amino acids point toward an intrasubunit cavity located between the four alpha-helical transmembrane domains. Computer docking simulations predict that the allosteric compounds such as PNU-120596 and LY-2087101 may bind within this intrasubunit cavity, much as neurosteroids and volatile anesthetics are thought to interact with GABA(A) and glycine receptors. Our findings suggest that this is a conserved modulatory allosteric site within neurotransmitter-gated ion channels.

Fig. 1.

Positive allosteric modulation by PNU-120596 examined by two-electrode voltage-clamp recording in Xenopus oocytes. Representative recordings are shown illustrating agonist-evoked responses obtained in the absence (Left) and presence (Right) of PNU-120596. Data are shown from oocytes expressing the rat α7 subunit (A), mouse 5-HT_3A subunit (B), α7^V201–5HT3A subunit chimera (C), α7^4TM-5HT3A chimera (D), and α7^3TM-5HT3A chimera (E). Data are shown for mouse 5-HT_3A subunit but similar data were also obtained with human 5-HT_3A. Traces illustrate the influence of PNU-120596 (1 μM) on responses evoked by maximally effective concentrations of agonist; either 1 mM acetylcholine (A and C–E) or 5 μM 1-(3-chlorophenyl)biguanide (B) in the absence (Left) and presence (Right) of PNU-120596. The duration of agonist and PNU-120596 application is illustrated by solid and hatched horizontal lines, respectively. Diagram representations of subunit topology illustrate domains derived from either the rat α7 subunit (black) or mouse 5-HT_3A subunit (gray). (Scale bars: vertical, 2 μA; horizontal, 2 s.)

Fig. 2.

The influence of point mutations in α7 transmembrane domains on potentiation by PNU-120596. Data represent amino acid mutations located within TM1 (A), TM2 (B), TM3 (C), and TM4 (D). In all cases, dose-response data are presented for a range of concentrations of PNU-120596 (0.1–30 μM) on responses evoked by a submaximal (EC₂₀) concentration of acetylcholine. For comparison, dose-response curves for wild-type α7 nAChRs are illustrated as a dotted line. Data shown are G211V (inverted filled triangles) (A), S222M (filled squares), L230Y (filled circles) and A225D (filled triangles) (A); M253L (filled circles) and M260L (filled squares) (B); Q272V (filled circles) and S276V (filled squares) (C); and F455A (filled circles), T456V (filled triangles), and C459Y (filled squares) (D). Data are means ± SEM of five to seven independent experiments, each from different oocytes.

Fig. 3.

Computer docking simulations. (A) Homology model of the α7 transmembrane domain based on the 4Å structure of the Torpedo nAChR α subunit (26), illustrating the location of mutated amino acids. The backbone of the four transmembrane α-helices (TM1-TM4) are colored gray. Side chains of amino acids that, when mutated, had a significant effect on potentiation by allosteric modulators are shaded red. Amino acids that had little or no effect when mutated are shaded blue. The model is shown from a side-on view (Right) and as viewed from above, looking down from the extracellular face of the lipid membrane (Left). In Right, part of the TM3 domain has been omitted, to avoid obscuring amino acid side chains. (B) The lowest energy (highest predicted binding affinity) docked position of PNU-120596 within the Cheng α7 homology model (25) is illustrated. The position of the five amino acids identified by site-directed mutagenesis as being important in potentiation of PNU-120596 are also shown.