TABLE 2.
Effects of α4(−)/(+)α4 agonist binding site mutations on LSP construct function and cell-surface expression
All values are the mean ± S.E.M.
| Concatemer | Mutation Site | ACh Imax | Specific [125I]mAb 295 Binding | n | HS Phase | LS Phase |
|---|---|---|---|---|---|---|
| nA | fmol/oocyte | µA/fmol | µA/fmol | |||
| β2-α4-β2-α4-α4 (LSP) | — | 3100 ± 530 | 0.036 ± 0.005 | 5 | 19.9 ± 5.8 | 75.7 ± 19.8 |
| β2-α4-β2-α4W88A-α4 | Loop D | 2700 ± 920 | 0.030 ± 0.003 | 3 | 50.2 ± 12.1** | 48.1 ± 11.4 |
| β2-α4-β2-α4-Y126Aα4 | Loop A | 300 ± 50** | 0.017 ± 0.001 | 3 | 7.2 ± 1.6 | 10.9 ± 2.3* |
| β2-α4-β2-α4-W182Aα4 | Loop B | 3700 ± 670 | 0.053 ± 0.018 | 3 | 65.8 ± 9.0*** | 36.2 ± 4.8 |
| β2-α4-β2-α4-Y184Aα4 | Loop B | 1900 ± 170 | 0.032 ± 0.005 | 3 | 19.7 ± 4.5 | 49.1 ± 10.2 |
| β2-α4-β2-α4-Y223Aα4 | Loop C | 220 ± 30** | 0.021 ± 0.003 | 3 | 4.6 ± 0.9 | 6.4 ± 1.1* |
| β2-α4-β2-α4-Y230Aα4 | Loop C | 120 ± 30** | 0.016 ± 0.001 | 3 | 2.3 ± 0.8 | 5.5 ± 1.7** |
| β2-α4-β2-α4-β2 (HSP) | — | 710 ± 60* | 0.039 ± 0.003 | 3 | 18.4 ± 2.8 | Not applicable |
Unmodified and mutant LSP, and HSP, nAChR construct mRNAs were expressed in X. laevis oocytes. ACh CRCs were performed in the absence of Saz-A preincubation. Maximum function (Imax) and the fraction of HS-phase function were determined by least-squares curve fitting. Surface expression of nAChR was then determined for the same oocytes using [125I]mAb295 binding (see Materials and Methods). Data were collected from three to five individual experiments, with six oocytes tested per condition in each experiment. One-way ANOVA was used to test within each measure for differences from control (unmodified LSP) parameters, with Dunnett’s post hoc test used to identify individual constructs that differed from control. Overall ANOVA results showed significant differences in overall Imax (sum of HS- and LS-phase function; F7,17 = 8.43, P = 0.0002), and also in surface expression levels (F7,17 = 3.101, P = 0.025, although post hoc testing failed to identify differences versus the unmodified LSP control for the HSP or for any of the mutant LSP constructs). Overall (Imax) function was subdivided into HS- and LS-phase responses for each construct where possible (no LS-phase function was produced by HSP nAChRs), and normalized to nAChR surface expression levels. Significant differences were seen in both normalized HS-phase function (F7,17 = 12.6, P < 0.0001; two constructs showed increased HS-phase function) and in normalized LS-phase function (F6,16 = 4.57, P = 0.0069; all constructs showed a trend to decreased LS-phase function, with three reaching statistical significance). *P < 0.05; **P < 0.01; ***P < 0.001.