TABLE 1

Agonist stimulation parameters: ACh activation of unmodified versus mutant LSP α4β2-nAChRs

All values are the mean ± S.E.M.

ConcatemerMutation SiteACh OnlyACh Post-Saz-A
Log (M) EC50 (HS)HS FractionLog (M) EC50 (LS)nLog (M) EC50 (LS)n
β2-α4-β2-α4-α4 (LSP)−6.44 ± 0.260.21 ± 0.03−4.39 ± 0.0512−3.94 ± 0.038
β2-α4-β2-α4W88A-α4Loop D−6.39 ± 0.080.51 ± 0.02***−4.13 ± 0.069−3.77 ± 0.056
β2-α4-β2-α4-Y126Aα4Loop A−5.87 ± 0.090.40 ± 0.03**−4.21 ± 0.059−3.83 ± 0.046
β2-α4-β2-α4-W182Aα4Loop B−6.75 ± 0.080.65 ± 0.02***−4.44 ± 0.089−3.76 ± 0.05*6
β2-α4-β2-α4-Y184Aα4Loop B−6.68 ± 0.180.29 ± 0.03−4.92 ± 0.05***9−4.05 ± 0.046
β2-α4-β2-α4-Y223Aα4Loop C−5.62 ± 0.14*0.42 ± 0.04***−3.74 ± 0.08***9−3.42 ± 0.05***6
β2-α4-β2-α4-Y230Aα4Loop C−5.77 ± 0.210.29 ± 0.05−4.06 ± 0.07*9−3.56 ± 0.05***6
β2-α4-β2-α4-β2 (HSP)−5.56 ± 0.0415−5.36 ± 0.058
  • Unmodified and mutant LSP construct mRNAs were expressed in X. laevis oocytes. ACh CRCs were performed either in the absence or presence of Saz-A preincubation (3.16 nM, 5 minutes). Oocytes were also injected with HSP mRNA for comparison with the LSP variants. Details are provided in the legend for Fig. 5 and in Materials and Methods. HS- and LS-phase log10(EC50/M), and fraction of HS-phase function were determined by least-squares curve fitting where possible (Saz-A preincubation rendered HS-phase function unmeasurable for all constructs). Numbers of individual oocytes tested are shown in the table (n = 6–12; derived from three separate experiments). Initially, two-way ANOVA was performed to determine the effect of mutation and treatment on the LS-phase EC50 values of the LSP construct ACh CRCs. Both factors significantly affected the observed EC50 values (F6,28 = 53.8, P < 0.001; and F1,28 = 300, P < 0.001, respectively), and a significant mutation × treatment interaction was also seen (F6,28 = 6.54, P < 0.001). For LS-phase EC50 values determined only with acute ACh exposure, ACh potency at the loop B Y184A mutant was significantly increased, whereas potency at the two C-loop mutants (Y223A and Y230A) was significantly reduced compared with the unmodified LSP control [one-way ANOVA with Dunnett’s post hoc test (F6,20 = 33.1, P < 0.001)]. For residual LS-like responses measured after Saz-A preincubation, statistically significant decreases in ACh potency compared with the unmodified LSP control construct were observed for three mutants (one-way ANOVA with Dunnett’s post hoc test F6,20 = 24.0, P < 0.001). The fraction of HS-phase function and ACh EC50 values for this function were determined before pretreatment with Saz-A (HS-phase responses were undetectable after pretreatment). A highly significant overall increase in the HS fraction was seen across the set of mutations (one-way ANOVA, F6,20 = 22.8, P < 0.001). Again, ACh potency was reduced compared with the unmodified LSP control construct by one of the C-loop mutants (Y223A; one-way ANOVA F6,20 = 7.83). *P < 0.05; **P < 0.01; ***P < 0.001 denote individual mutant differences from the control.