Figure 2.
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 = 6–12 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).

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 = 6–12 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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