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Figure 1.
α4β2 nAChR stoichiometry and functional effects of ACh and NS9283. X. laevis oocytes were injected with cRNA mixtures of α4 and β2 or α4VFL and β2 subunits in 10:1 ratios and subjected to two-electrode voltage-clamp electrophysiology as described in Materials and methods. The 10:1 cRNA ratios were used to ensure uniform populations of (α4)3(β2)2 and (α4VFL)3(β2)2 receptors. Data for α4 and β2 injected in a 1:4 ratio ((α4)2(β2)3 receptor) are from Harpsøe et al. (2011). (A) Functional α4β2 nAChRs can express in 2α:3β or 3α:2β stoichiometries (left and middle, respectively). The stoichiometry affects the total number of ACh-binding sites, as the 3α:2β stoichiometry contains an additional site in the α4âα4 interface. Furthermore, NS9283 binds with high selectivity in the α4âα4 site, where it behaves as an agonist. Upon mutating three amino acids in the complementary face of the α4 subunit to give α4VFL, ACh sensitivity is increased in the α4VFLâα4VFL site, and NS9283 binding is lost (right). (B) ACh CRRs. Baseline-subtracted, ACh-evoked peak current amplitudes (I) for the indicated receptors were fitted to the Hill equation by nonlinear regression and normalized to the maximal fitted values (Imax fit ACh). Normalized responses are depicted as means ± SEM as a function of the ACh concentrations, and they are fitted to biphasic equations with a fixed bottom of 0 and a Hill slope of 1. Data were obtained from n = 9â14 experiments, and regression results are presented in Table 1. Data for the (α4)2(β2)3 receptor are from Harpsøe et al. (2011). (C) NS9283 CRRs. NS9283 enhancement of ACh-evoked currents was evaluated for (α4)3(β2)2 and (α4VFL)3(β2)2 receptors by coapplication with a submaximal control concentration of ACh (10 µM). Baseline-subtracted peak current amplitudes (I) were expressed as percent change from IACh_control and are depicted as means ± SEM as a function of the NS9283 concentration. Data points were fitted by nonlinear regression to the Hill equation with a fixed bottom of 0 and a Hill slope of 1. Data were obtained from n = 13â16 experiments, and regression results are presented in Table 1. Data for the (α4)2(β2)3 receptor are from Timmermann et al. (2012). (D) Hypothetically, injection of a cRNA mixture of α4, α4VFL, and β2 into oocytes could yield eight different receptors in the 3α:2β stoichiometry. Using NS9283 as a marker, these can be divided into those that are sensitive and those that are insensitive, depending on whether the α4VFL subunit is participating in the complementary position of the α4âα4 interface.
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Figure 2.
ACh and NS9283 sensitivity and potential stoichiometry of receptors from the concatenated β-6-α construct. X. laevis oocytes were subjected to two-electrode voltage-clamp electrophysiology as described in Materials and methods. (A and B) ACh (A) and NS9283 (B) CRRs were obtained from oocytes injected with the β-6-α dimer construct alone or coinjected with monomeric α4 or α4VFL subunits in a 1:1 ratio. The linker sequence is shown in Table 3. Electrophysiological data were evaluated as described in Materials and methods; also see Fig. 1. Data from n = 6â12 experiments are depicted as means ± SEM as a function of the ACh or NS9283 concentration, and regression results are presented in Table 1. Data for wild-type receptors from monomeric subunits in Fig. 1 are indicated as dashed lines. (C) Representative traces illustrating NS9283 responses at oocytes injected with β-6-α, β-6-α and α4, or β-6-α and α4VFL. Bars above the traces indicate the 30-s application time and concentrations of applied compounds. (D) The simplest way in which a dimeric β-6-α construct could lead to functional 3α:2β stoichiometry receptors is three sets of linked dimers assembling with a dangling β2 subunit. This, again, could be envisioned to lead to three different assemblies because the two dimers in each receptor can be oriented in the clockwise, the counterclockwise, or both orientations when viewed from the synaptic cleft. Note that other, more complex assemblies cannot be excluded. (E) When coinjecting β-6-α and α4VFL, four different possible assemblies involving two dimer constructs and one monomeric subunit could arise. Of the four possibilities, one can likely be considered nonfunctional, given that all three α subunits are placed consecutively (right). If one or both dimers assemble in the clockwise orientation, the receptor will mimic wild-type 3α:2β receptors with respect to NS9283 sensitivity (middle). However, if both dimer constructs assemble in the counterclockwise orientation, the receptor will mimic wild-type 2α:3β receptors (left).
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Figure 3.
ACh and NS9283 sensitivity and potential stoichiometry of receptors from concatenated tetrameric or pentameric constructs. X. laevis oocytes were subjected to two-electrode voltage-clamp electrophysiology. Electrophysiological data were evaluated as described in Materials and methods; also see Fig. 1. (A and B) ACh (A) and NS9283 (B) CRRs were obtained from oocytes injected with the tetrameric β-6-α-9-β-6-α (T) construct alone or coinjected with monomeric α4 or α4VFL subunits in a 1:1 ratio. Data from n = 5â13 experiments are depicted as means ± SEM as a function of the ACh or NS9283 concentration, and regression results are presented in Table 2. Data for wild-type receptors from monomeric subunits in Fig. 1 are indicated as dashed lines. (C) Functional 3α:2β stoichiometry receptors arising from coinjections of β-6-α-9-β-6-α and α4VFL could originate from assembly of the concatenated construct in either a clockwise or a counterclockwise orientation when viewed from the synaptic cleft. With respect to NS9283 sensitivity, receptors with the tetramer in the clockwise orientation will resemble wild-type 3α:2β receptors, whereas receptors with the tetramer in the counterclockwise orientation will resemble wild-type 2α:3β receptors. (D and E) ACh (D) and NS9283 (E) CRRs were obtained from oocytes injected with the indicated pentameric constructs, in which x indicates either the α4 or the α4VFL subunit in the fifth construct position. Data from n = 12â19 experiments are depicted as means ± SEM as a function of the ACh or NS9283 concentration, and regression results are presented in Table 2. (F) When injecting the pentameric construct including the α4VFL subunit, clockwise and counterclockwise assemblies lead to different receptors, and NS9283 behaves differentially as described in C. Note that in the receptor illustrations, the first construct linkers are indicated with bold purple font, and specific linker sequences are shown in Table 3.
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Figure 4.
ACh and NS9283 sensitivity and potential stoichiometry of receptors from concatenated pentameric constructs with the α4âα4 site in the second to third construct positions. X. laevis oocytes were subjected to two-electrode voltage-clamp electrophysiology. Electrophysiological data were evaluated as described in Materials and methods; also see Fig. 1. (A and B) ACh (A) and NS9283 (B) CRRs were obtained from oocytes injected with the indicated pentameric constructs, where x and y denote an α4 or an α4VFL subunit in the second and third construct positions. Data from n = 9â23 experiments are depicted as means ± SEM as a function of the ACh or NS9283 concentration, and regression results are presented in Table 2. Data for wild-type receptors from monomeric subunits in Fig. 1 are indicated as dashed lines. (C) Representative traces illustrating NS9283 responses at oocytes injected with β-21a-α-α-β-α, β-21a-α-αVFL-β-α, or β-21a-αVFL-α-β-α. Bars above the traces indicate the 30-s application time and concentrations of applied compounds. (D) For the β-21a-α-αVFL-β-α receptor (α4VFL subunit in the third construct position), NS9283-sensitive receptors originate from assembly of the linkers in a clockwise orientation (top right). However, for the β-21a-αVFL-α-β-α receptor (α4VFL subunit in the second construct position), NS9283-sensitive receptors are assembled with the linkers in a counterclockwise orientation (bottom left). Note that in the receptor illustrations, the first construct linkers are indicated with bold purple font, and specific linker sequences are shown in Table 3.
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Figure 5.
3-D structure of the human α4β2 nAChR (from Protein Data Bank accession no. 5KXI; Morales-Perez et al., 2016) showing direction of N-terminal α-helix and modeled linkers. The α4 subunit is colored green, and β2 is blue. (A) Top view of the (α4)2(β2)3 receptor with the N-terminal helixes shown in red. (B) Top view of the α4β2 dimer with the N-terminal LLxxLF motif shown as red spheres. The surface of the protein constituting hydrophobic residues on the top of the α4 and β2 subunits is colored yellow. The LLxxLF motif interacts with the hydrophobic surface to form a hydrophobic patch. (C) α4β2 dimer with modeled long clockwise and short counterclockwise linkers. The shortest possible number of AGS repeats required to connect the first residue after TM4 of the β2 subunit (Leu479) to the mature N terminus of α4 (Ala34) is shown and had lengths of 10 and 8 repeats, respectively. (D) Illustration of total linker length calculations for new concatenated β-xa-α dimer constructs. Note that only parts of the cDNA sequences close to the linkers are shown.
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Figure 6.
ACh and NS9283 sensitivity and potential stoichiometry of receptors from concatenated β-xa-α dimer constructs. X. laevis oocytes were subjected to two-electrode voltage-clamp electrophysiology. Electrophysiological data were evaluated as described in Materials and methods; also see Fig. 1. (A and B) ACh (A) and NS9283 (B) CRRs were obtained from oocytes coinjected with β-xa-α and the monomeric α4VFL subunit in a 1:1 ratio. X represents the number of amino acids in the linker, and specific linker sequences are shown in Table 3. Data from n = 5â24 experiments are depicted as means ± SEM as a function of the ACh or NS9283 concentration, and regression results are presented in Table 1. Data for the receptor obtained from monomeric α4VFL and β2 subunits in Fig. 1 are indicated with dashed lines. (C) Representative traces illustrating NS9283 responses at oocytes injected with β-9a-α and α4VFL, β-6a-α and α4VFL, β-3a-α and α4VFL, or β-0a-α and α4VFL. Bars above the traces indicate the 30-s application time and concentrations of applied compounds. (D) Based on the lack of NS9283 efficacy observed in B, receptors originating from coinjection of β-0a-α with the monomeric α4VFL subunit have the concatenated construct assembled exclusively in the counterclockwise orientation when viewed from the synaptic cleft.
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Figure 7.
ACh and NS9283 sensitivity and potential stoichiometry of receptors from concatenated pentameric constructs with short first linkers. X. laevis oocytes were subjected to two-electrode voltage-clamp electrophysiology. Electrophysiological data were evaluated as described in Materials and methods; also see Fig. 1. (A and B) ACh (A) and NS9283 (B) CRRs were obtained from oocytes injected with the indicated pentameric constructs in which x denotes an α4 or an α4VFL subunit in the fifth construct position. Data from n = 5â15 experiments are depicted as means ± SEM as a function of the ACh or NS9283 concentration, and regression results are presented in Table 2. Data for wild-type receptors from monomeric subunits in Fig. 1 are indicated as dashed lines. (C) Representative traces illustrating NS9283 responses at oocytes injected with β-3a-α-β-α-α (top) or β-3a-α-β-α-αVFL (bottom). Bars above the traces indicate the 30-s application time and concentrations of applied compounds. (D) Based on the NS9283 responses observed with x = α4VFL in B, the receptor pool consists mainly of pentamers with the linkers assembled in the counterclockwise orientation when viewed from the synaptic cleft. Note that in the receptor illustrations, the first construct linkers are indicated with bold purple font, and specific linker sequences are shown in Table 3.
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Figure 8.
ACh and NS9283 sensitivity of receptors from α4VFL subunit containing concatenated pentameric constructs with short first linkers. X. laevis oocytes were subjected to two-electrode voltage-clamp electrophysiology. Electrophysiological data were evaluated as described in Materials and methods; also see Fig. 1. (A and B) ACh (A) and NS9283 (B) CRRs were obtained from oocytes injected with the indicated pentameric constructs. Data from n = 9â15 experiments are depicted as means ± SEM as a function of the ACh or NS9283 concentration, and regression results are presented in Table 2. Data for the β-3a-α-β-α-αVFL construct are from Fig. 7, and data for wild-type receptors from monomeric subunits in Fig. 1 are indicated as dashed lines. The preferred expression orientation of each pentamer is indicated for the NS9283 data. (C) Representative traces illustrating NS9283 responses at oocytes injected with β-3a-αVFL-β-α-α or β-3a-αVFL-α-β-α. Bars above the traces indicate the 30-s application time and concentrations of applied compounds.
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