Unraveling the high- and low-sensitivity agonist responses of nicotinic acetylcholine receptors

Abstract

The neuronal α4β2 nicotinic acetylcholine receptors exist as two distinct subtypes, (α4)(2)(β2)(3) and (α4)(3)(β2)(2), and biphasic responses to acetylcholine and other agonists have been ascribed previously to coexistence of these two receptor subtypes. We offer a novel and radical explanation for the observation of two distinct agonist sensitivities. Using different expression ratios of mammalian α4 and β2 subunits and concatenated constructs, we demonstrate that a biphasic response is an intrinsic functional property of the (α4)(3)(β2)(2) receptor. In addition to two high-sensitivity sites at α4β2 interfaces, the (α4)(3)(β2)(2) receptor contains a third low-sensitivity agonist binding site in the α4α4 interface. Occupation of this site is required for full activation and is responsible for the widened dynamic response range of this receptor subtype. By site-directed mutagenesis, we show that three residues, which differ between the α4β2 and α4α4 sites, control agonist sensitivity. The results presented here provide a basic insight into the function of pentameric ligand-gated ion channels, which enables modulation of the receptors with hitherto unseen precision; it becomes possible to rationally design therapeutics targeting subpopulations of specific receptor subtypes.

Figure 1.

The α4β2 nAChR subtypes and the agonists used. A, Diagram of the (α4)₂(β2)₃ and (α4)₃(β2)₂ subtypes showing arrangement of subunits and location of agonist binding sites. The black arrows show the position of the confirmed orthosteric binding sites, and the gray arrow show the position of the hypothesized third agonist site of the (α4)₃(β2)₂ subtype. (+) and (−) refers to the principal and complementary components of the binding sites. B, Chemical structures of the endogenous ligand for α4β2 receptors, ACh, and a partial agonist, NS3573.

Figure 2.

Sequence alignment used for homology modeling. The multi-template sequence alignment of α4 and β2 to the three templates used to construct the dimeric α4α4 and α4β2 homology models. The residues shown in bold were used as templates for modeling both α4 and β2, whereas framed residues were used only for α4. Residues that were not modeled on a template or not used as template are shown in gray. Numbering follows the PDB files for 1UW6 (AChBP with nicotine bound), 2BYQ (AChBP with epibatidine bound), and 2QC1 (mouse α1 nAChR ligand binding domain with toxin bound) and is according to P43681 and P17787 at www.uniprot.org for α4 and β2, respectively. The mutated residues are on a gray background, the secondary structure is indicated above the sequences, and the classical notation of the binding site regions (loops A–F) is given below the sequences.

Figure 3.

Structural comparison of the α4β2 and α4α4 sites. Superimposition of the homology models of the α4β2 and α4α4 dimers with the protein backbone represented as cartoon, whereas nicotine and the three residues that differ between the sites are shown as sticks. From the top, these residues are His142, Gln150, and Thr152 in α4 (green carbon atoms) and Val136, Phe144, and Leu146 in β2 (slate carbon atoms). The identical (+)-side of the binding sites and nicotine are colored with white carbons and for clarity reasons only displayed from the α4α4 homology model.

Figure 4.

Representative current traces for ACh and NS3573 in two-electrode voltage-clamp electrophysiological experiments in X. laevis oocytes. A, ACh concentration–response experiment in an oocyte injected with α4 and β2 nAChR subunits in a 1:4 ratio (representing a low-current amplitude experiments). B, NS3573 concentration–response experiment in an oocyte injected with α4 and β2 nAChR subunits in a 4:1 ratio (representing a high-current amplitude experiments). Application of compound is indicated by a bar above each trace, and “C” denotes an ACh control concentration of 1 μm (1:4 ratio) or 10 μm (4:1 ratio), “M” denotes a 3 mm ACh_max concentration, “B” denotes a buffer application, and the numbers 1–12 denote increasing concentrations of ACh or NS3573 in half-log unit increments with minimal concentration of 316 pm in the first application and maximal concentration of 100 μm in the last application.

Figure 5.

Functional concentration–response profiles of ACh and NS3573 on α4β2 wild-type and mutant receptors. A, ACh concentration–response curves from wild-type α4β2 receptors expressed in 1:4 and 4:1 ratios. B, ACh concentration–response curves on α4^mβ2 mutant receptors expressed in 1:4 and 4:1 ratios and α4β2^m mutant receptors expressed in a 4:1 ratio. C, NS3573 concentration–response curves from wild-type α4β2 receptors expressed in 1:4 and 4:1 ratios. D, NS3573 concentration–response curves on α4^mβ2 mutant receptors expressed in 1:4 and 4:1 ratios and α4β2^m mutant receptors expressed in a 4:1 ratio. Data points are presented as mean ± SEM. Potencies, fractions of the high-sensitivity component on biphasic curves, number of experiments, and statistics are presented in Table 1.

Figure 6.

Functional concentration–response profiles for ACh and NS3573 on α4β2 receptors with different subunit ratios or concatenated subunits. A, B, ACh concentration–response curves from α4β2 receptors expressed in 20:1 and 100:1 ratios. C, ACh concentration–response curve from α4β2 receptors expressed from the dimeric concatenated β2-6-α4 construct and α4 in a 1:1 ratio. D, ACh concentration–response curve from α4β2 receptors expressed from the tetrameric concatenated β2-6-α4-9-β2-6-α4 construct and α4 in a 1:1 ratio. E, F, NS3573 concentration–response curves from α4β2 receptors expressed in 20:1 and 100:1 ratios. G, NS3573 concentration–response curve from α4β2 receptors expressed from the dimeric concatenated β2-6-α4 construct and α4 in a 1:1 ratio. H, NS3573 concentration–response curve from α4β2 receptors expressed from the tetrameric concatenated β2-6-α4-9-β2-6-α4 construct and α4 in a 1:1 ratio. Theoretical sigmoidal curves using the obtained fitted values are plotted as dotted lines to visualize the two fractions. Data points are presented as mean ± SEM. Potencies, fractions of the high-sensitivity component, number of experiments, and statistics are presented in Table 1.