Unraveling amino acid residues critical for allosteric potentiation of (α4)3(β2)2-type nicotinic acetylcholine receptor responses

Abstract

Neuronal nicotinic acetylcholine receptors (nAChRs) are promising drug targets to manage several neurological disorders and nicotine addiction. Growing evidence indicates that positive allosteric modulators of nAChRs improve pharmacological specificity by binding to unique sites present only in a subpopulation of nAChRs. Furthermore, nAChR positive allosteric modulators such as NS9283 and CMPI have been shown to potentiate responses of (α4)3(β2)2 but not (α4)2(β2)3 nAChR isoforms. This selective potentiation underlines that the α4:α4 interface, which is present only in the (α4)3(β2)2 nAChR, is an important and promising drug target. In this report we used site-directed mutagenesis to substitute specific amino acid residues and computational analyses to elucidate CMPI's binding mode at the α4:α4 subunit extracellular interface and identified a unique set of amino acid residues that determined its affinity. We found that amino acid residues α4Gly-41, α4Lys-64, and α4Thr-66 were critical for (α4)3(β2)2 nAChR potentiation by CMPI, but not by NS9283, whereas amino acid substitution at α4His-116, a known determinant of NS9283 and of agonist binding at the α4:α4 subunit interface, did not reduce CMPI potentiation. In contrast, substitutions at α4Gln-124 and α4Thr-126 reduced potentiation by CMPI and NS9283, indicating that their binding sites partially overlap. These results delineate the role of amino acid residues contributing to the α4:α4 subunit extracellular interface in nAChR potentiation. These findings also provide structural information that will facilitate the structure-based design of novel therapeutics that target selectively the (α4)3(β2)2 nAChR.

Keywords: CMPI; NS9283; Positive allosteric modulators; dFBr; drug design; drug development; electrophysiology; ion channel; nicotinic acetylcholine receptors (nAChR); pentameric ligand-gated ion channel.

Figure 1.

A, top view of the X-ray structure of human (α4)2(β2)3 nAChRs (PDB accession number 5KXI). B, top view of a homology model of an (α4)₃(β2)₂ nAChR based on the crystal structure of (α4)2(β2)3 nAChRs. C, chemical structure of nAChRs PAMs: NS9283 and CMPI.

Figure 2.

Representative traces showing the effects of CMPI, NS9283, and dFBr on ACh-induced current responses of (α4)3(β2)2 (A) and (α4)2(β2)3 (B) nAChRs, respectively. Xenopus oocytes expressing wild type human (α4)3(β2)2 or (α4)2(β2)3 nAChRs were voltage-clamped at −50 mV, and currents elicited by 10-s applications of 10 μm ACh were recorded in the absence or presence of 1 or 3 μm CMPI, NS9283, or dFBr.

Figure 3.

A, side view of a homology model of an (α4)3(β2)2 nAChR showing amino acid residues contributing to the α4(−) face of the α4:α4 extracellular interface. B, sequence alignments of amino acid contributing to the (−) face of the interface with corresponding regions in the β2 and α3 nAChR subunits. Amino acid numbering is for mature protein according to the X-ray structure of human (α4)2(β2)3 nAChRs (PDB accession number 5KXI). Add 26, 25, and 31 to α4, β2, and α3 sequence numbers, respectively, to obtain amino acid numbers starting from the translational N terminus. β-Strands in A are color-coded (β1, cyan; β2, red; β5, blue; β6, green) to match their sequence alignments in B.

Figure 4.

Effect of amino acid substitutions on CMPI, NS9283, and dFBr potentiation of (α4)3(β2)2 nAChRs. Representative two-electrode voltage clamp traces showing the effects of 1 μm CMPI, NS9283 or dFBr (A) and 10 μm CMPI or NS9283 (B) on ACh-induced current responses of Xenopus oocytes expressing WT or mutant (α4)3(β2)2 nAChRs containing an amino acid substitution at the (−) face of the α4 subunit. Quantification of CMPI, NS9283, and dFBr effects are presented in Table 1.

Figure 5.

CMPI concentration-dependent potentiation of WT and mutant (α4)3(β2)2 nAChRs. A, representative two-electrode voltage clamp traces showing the effect of increasing concentrations of CMPI on ACh-induced current responses of Xenopus oocytes expressing WT or mutant (α4)3(β2)2 nAChRs containing amino acid substitutions at the (−)face of α4 subunit. B, for each recording run, peak currents were normalized to the peak current elicited by 10 μm ACh alone, replicas from individual oocytes were averaged, and the average ± S.E. of data from several oocytes were plotted and fit (when possible) to a single-site model using Equation 1. Parameters (potentiation EC₅₀ and I_max) are shown in Table 2.

Figure 6.

Effect of CMPI on the ACh dose-response curve of WT and mutant (α4)3(β2)2 nAChRs. Currents elicited by Xenopus oocytes expressing WT and mutant (α4)3(β2)2 nAChRs in response to 10-s applications of increasing concentrations of ACh (alone (○) or +1 μm CMPI (●)) were recorded and normalized to peak currents elicited by 1 mm ACh alone. Replicas from the same oocyte were averaged, and the average ± S.E. of data from several oocytes were plotted and fit to a single-site model using Equation 1. Parameters (ACh EC₅₀ in the presence and absence of CMPI) are shown in Table 3.

Figure 7.

PAM-binding sites at the α4:α4 extracellular interface in a model based on the X-ray structure of agonist-bound AChBP. Top (A) and side (B–F) views show ACh (cyan), CMPI (magenta), and NS9283 (green) docked at the α4:α4 extracellular interface of an (α4)3(β2)2 nAChR homology model based on the structure of Lymnaea AChBP crystallized in the presence of carbamylcholine (PDB accession number 1UV6). Secondary structure of α4 (brown and yellow) and β2 (cherry red) subunits is shown with α-helices as cylinders and β-strands as ribbons (β1, β2, β5, and β6 are labeled in blue). A, Connolly surfaces are included for ACh and a superimposition of ACh/CMPI/NS9283 docked at the α4:β2 and α4:α4 extracellular interfaces, respectively. ACh, CMPI, and NS9283 were individually docked to the α4:α4 interface alone (B–D), and CMPI and NS9283 were docked in the presence of ACh (E and F). The lowest energy docking solutions for each ligand are shown in stick and ball representation, and amino acid side-chains are shown in stick representation colored by atom type (C, gray; O, red; N, blue). For each ligand, conserved amino acid residues that make up the agonist site aromatic box and amino acid residues that significantly reduced its effects when substituted to the corresponding residue from the β2 subunit are shown in Connolly surface representation colored by atom charge (positive, blue; negative, red). Residues are labeled using the single letter code with those from the α4 (+) face underlined.

Figure 8.

PAM-binding sites at the α4:α4 extracellular interface in a model based on the X-ray structure of agonist-bound (α4)2(β2)3 nAChR. Side views showing CMPI (magenta and brown) and NS9283 (green and cherry red) docked at the α4:α4 extracellular interface of (α4)3(β2)2 nAChR homology model based on the X-ray structure of (α4)2(β2)3 nAChR (PDB accession number 5KXI). This model was generated by replacing a β2 subunit with an α4 subunit from the same crystal structure as described under “Experimental Procedures.” A–C, side views showing secondary structure of two α4 subunits (brown and yellow) with α-helices as cylinders and β-strands as ribbons. Amino acid residues contributing to the α4:α4 extracellular interface are shown in stick representation colored by atom type (C, gray; O, red; N, blue), and the four transmembrane helices of each α4 subunit are labeled M1–M4. Connolly surfaces of a superimposition of the two lowest energy docking solutions for CMPI and NS9283 are shown in B and C, respectively. D–G, close-up side views of the α4:α4 extracellular interface showing the lowest energy docking solutions for CMPI (D and E) and NS9283 (F and G) are in stick and ball representation. For each ligand, conserved amino acid residues that make up the agonist site aromatic box and amino acid residues that significantly reduced its effects when substituted to the corresponding residue from the β2 subunit are shown in Connolly surface representation colored by atom charge (positive, blue; negative, red). Residues are labeled using the single letter code with those from the α4 (+) face underlined.