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    • Given that there are substitutions between Anc and the

    2023-10-13

    Given that there are 132 substitutions between βAnc and the human β subunit, we wondered whether βAnc could replace the human β subunit in hybrid AChRs formed from ancestral and human subunits. We first confirmed that the β subunit is required for expression of human AChRs in HEK293 latrunculin by co-transfecting cells with a mixture of plasmids encoding the various subunits, and then measuring cell-surface binding of radiolabeled α-bungarotoxin (α-Btx). Derived from the venom of Bungarus multicinctus (Chang and Lee, 1963), α-Btx is a competitive antagonist that binds to the AChR agonist sites located at the interfaces between the α-δ and α-ɛ subunits. When cells are co-transfected with plasmids encoding all four human AChR subunits, robust cell-surface binding of α-Btx is detected (Figure 1D). However, when the plasmid encoding the human β subunit is omitted from the transfection mixture, negligible α-Btx binding is observed, confirming that the human β subunit is required for cell-surface expression of intact α-Btx sites and, presumably, pentameric AChRs. We then tested whether βAnc could rescue expression of α-Btx sites by co-transfecting a mixture of plasmids encoding the human α, δ, and ɛ subunits, along with the plasmid encoding βAnc. Co-expression of the human α, δ, and ɛ subunits with βAnc leads to robust α-Btx binding on the cell surface, demonstrating that βAnc can act as a surrogate for the human β subunit, and rescue cell-surface expression of intact α-Btx sites. In contrast, under the same conditions, co-expression of the human α, δ, and ɛ subunits with the Torpedo β subunit fails to rescue cell-surface expression of α-Btx sites in HEK293 cells. The simplest explanation for these combined observations is that βAnc is able to form hybrid AChRs when co-expressed with human α, δ, and ɛ subunits, while the Torpedo β subunit cannot. Intrigued by the ability of βAnc to rescue expression of human α, δ, and ɛ subunits, we set out to characterize the activity of hybrid AChRs incorporating βAnc. We began by looking at macroscopic currents from cells expressing either wild-type or βAnc-containing AChRs (Figure 2). For both types of cells, the magnitude of peak whole-cell currents increases with increasing concentrations of acetylcholine in a dose-dependent manner. Cells transfected with βAnc alone, or with the human α, δ, and ɛ subunits, show no currents over the same acetylcholine concentration range. On average, the maximum currents for cells expressing βAnc-containing AChRs plateaued at a lower value, and the EC50 for maximal response was shifted to 3-fold higher acetylcholine concentrations (Figure 2B and Table 1). In addition, the response of βAnc-containing AChRs to increasing concentrations of acetylcholine appeared less steep, indicating a reduced apparent cooperativity. Despite expression in a different cell line and the use of an automated device, our wild-type measurements are in the same range as those reported previously (Shao et al., 1998). Given similar levels of expression of wild-type and βAnc-containing AChRs (see Figure 1D), these data show that in comparison with wild-type, acetylcholine is not as potent an agonist for βAnc-containing AChRs. To identify the basis for these macroscopic differences in function, we examined the activity of βAnc-containing AChRs at the single-channel level. We began by comparing the single-channel conductance of βAnc-containing AChRs with that of human wild-type AChRs (Figure 3). Under the same conditions, hybrid AChRs containing βAnc had a mean amplitude of 10.01 ± 0.16 pA, while the mean amplitude of wild-type AChRs was 12.89 ± 0.65 pA. This amplitude for wild-type AChRs matches well with previously reported values recorded under the same conditions (Mukhtasimova et al., 2016). At these transmembrane voltages (−120 mV), these amplitudes correspond to single-channel conductances of 83.42 ± 1.36 pS and 107.46 ± 5.36 pS for βAnc-containing and wild-type AChRs, respectively. These single-channel data demonstrate that the unitary conductance of βAnc-containing AChRs is reduced in comparison with wild-type AChRs.