Article Figures & Data
Figures
- 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 AChmax 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 α4mβ2 mutant receptors expressed in 1:4 and 4:1 ratios and α4β2m 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 α4mβ2 mutant receptors expressed in 1:4 and 4:1 ratios and α4β2m 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.
Tables
- Table 1.
Concentration–response data for ACh and NS3573 on wild-type, mutant, and concatenated α4β2 receptors expressed with different subunit ratios
Subunits Ratio Imax (nA)a ACh NS3573 EC50_1 (μm)b EC50_2 (μm)b Frac.c nd F value (DFn, DFd)e EC50_1 (nm)b EC50_2 (μm)b Frac.c nd F value (DFn, DFd)e α4:β2 1:4 920 1.6 6 23 20 α4:β2 4:1 3800 0.95 83 0.16 17 38 (2,197) 9.8 7.9 0.24 24 31 (2,279) α4m:β2 1:4 360 0.97 7 20 7 α4m:β2 4:1 1400 3.2 8 22 8 α4m:β2 100:1 1500 3.8 9 27 10 α4:β2m 4:1 6600 140 7 23 11 α4:β2 20:1 2900 1.4 141 0.14 20 58 (2,216) 4.4 10 0.26 14 63 (2,131) α4:β2 100:1 2700 1.9 170 0.15 24 80 (2,284) 4.0 4.0 0.24 12 42 (2,138) α4:β2-6-α4 1:1 2400 1.3 130 0.21 10 47 (2,116) 6.3 6.3 0.23 5 12 (2,56) α4:β2-6-α-9-β2-6-α4 1:1 3300 2.5 130 0.24 8 42 (2,80) 5.0 7.9 0.26 9 64 (2,103) β2-6-α-9-β2-6-α4:β2 1:1 290 1.3 12 18 13 ↵aAverage of maximal ACh-evoked current amplitudes in X. laevis oocyte experiments. Oocytes were clamped at holding potentials ranging from −40 to −100 mV. Peak AChmax-evoked current amplitudes from each experiment were recorded and baseline subtracted. Average current levels for all experiments on a given receptor population were rounded off to two significant digits, and SEM values were typically 10%.
↵bEC50_1 and EC50_2 correspond to the high- and low sensitivity potencies observed from the wild-type α4β2 in a 4:1 ratio, respectively.
↵cFraction of the high-sensitivity component on receptors resulting in biphasic activation profiles.
↵dNumber of experiments behind the data.
↵eFor the experiments in which data were best fitted to biphasic Hill equations, the results from the best-model F test with p < 0.0001 are given as follows: F value (degrees of freedom for the numerator, degrees of freedom for the denominator).
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