Figure 1.
Acute responses of AChRs to application of saturating concentrations of ACh.
Fluorescent responses were measured using the FLEXStation with a membrane potential-sensitive indicator. The kinetics of responses of α4β2 (to 300 µM ACh) and α3β4 AChRs (to 1.0 mM ACh) were very similar, with a maximum response reached within 45 seconds of agonist application. The response of α7 AChRs (to 10 µM ACh) was more rapid, with a peak response within 5 seconds, followed by rapid desensitization. Each data point represents the average of 4 individual response curves. The absolute values of responses of saturating concentrations of ACh (expressed as relative fluorescence units) were similar for α4β2 (167,000+/−18,000) and α3β4 (161,000+/−14,000), but significantly lower for α7 AChRs (54,00+/−3000), probably as a result of rapid desensitization.
Figure 2.
Responses of human α4β2, α3β4 and α7-expressing cell lines to various concentrations of nicotinic agonists.
Responses were measured using the FLEXStation with an indicator sensitive to changes in membrane potential. Results are expressed as a percent of maximal fluorescence. Each data point is an average of the peak fluorescence of 4–8 individual dose-response curves. Nicotine and ACh are full agonists on α4β2, whereas varenicline, cytisine and sazetidine-A are partial agonists. All of the tested compounds are full agonists on α3β4 and α7 AChRs.
Table 1.
Agonist Efficacy and Sensitivity for Activation and Desensitization.
Figure 3.
Activation and Desensitization of α4β2 AChRs by Various Agonists.
Responses were measured using the FLEXStation with an indicator sensitive to changes in membrane potential. Results are expressed as a percentage of maximum fluorescence. Activity remaining after 16 hours desensitization by the indicated concentrations of agonist was assayed using 3 µM ACh (to assay function of the more sensitive stoichiometry (α4β2)2β2), and 100 µM ACh (to assay function of both stoichiometries). Each data point is the average of the peak fluorescence of 4–8 dose-response curves. The responses to acute application of agonists are the same as shown in Figure 2. The extent of smoldering activation (shaded area) was calculated by multiplying the extent of acute activation by the extent of sustained desensitization at each concentration. For nicotine, the area of overlap for the more sensitive (α4β2)2β2 stoichiometry was centered at 0.13 µM, which is a concentration typically found in smokers. Likewise for varenicline, the area of overlap for the more sensitive (α4β2)2β2 stoichiometry was centered at 0.16 µM, which corresponds to peak concentrations achieved in humans. Sazetidine-A was highly potent at activating as well as desensitizing α4β2 AChRs. The area of overlap for (α4β2)2β2 AChRs was centered at 1.5 nM. When 100 µM ACh was used for desensitization, there was a plateau on the dose response curve beginning at around 10 nM.
Figure 4.
Sazetidine-A desensitization of the more sensitive (α4β2)2β2 stoichiometry with 3 µM and 100 µM ACh.
Cells stably expressing α4β2 AChRs were further transfected with β2 subunits and cultured in nicotine as described, to enrich for the sensitive (α4β2)2β2 stoichiometry. Responses were measured with the FlexStation using a membrane potential sensitive indicator, and results were expressed as a percentage of maximum fluorescence. The responses to both 3 µM and 100 µM ACh overlapped, likely indicating that only the (α4β2)2β2 stoichiometry contributes to desensitization. The plateau on the desensitization curve with sazetidine-A on mixed stoichiometries of α4β2* (shown in Figure 3) indicates that the (α4β2)2α4 stoichiometry is not desensitized even at high concentrations of sazetidine-A.
Figure 5.
Activation and Desensitization of α3β4 AChRs by Various Agonists.
Responses were measured using the FLEXStation with an indicator sensitive to changes in membrane potential. Results were expressed as a percentage of maximum fluorescence. Activity remaining after 16 hours desensitization by the indicated concentrations of agonist was assayed using 1(shaded area) was calculated by multiplying the extent of acute activation by the extent of sustained desensitization at each concentration. For nicotine and varenicline, smoldering activation of α3β4 AChRs occurs at concentrations that are above levels that can be reached in humans.
Figure 6.
Activation and Desensitization of α7 AChRs by Various Agonists.
Responses were measured using the FLEXStation with an indicator sensitive to changes in membrane potential. Results were expressed as a percentage of maximum fluorescence. Activity remaining after 16 hours desensitization by the indicated concentrations of agonist was assayed using 10 µM ACh. The extent of smoldering activation (shaded area) was calculated by multiplying the extent of acute activation by the extent of sustained desensitization at each concentration. For nicotine, the intercept of the activation and desensitization curves was 1.7 µM (well above the clinically achievable range). However, for varenicline, the intercept of the activation and desensitization curves was 0.4 µM, a concentration which can be reached with therapeutic doses of this drug.
Figure 7.
Short Term Desensitization of α4β2, α3β4 and α7 AChRs by Various Agonists.
Responses of α4β2, α3β4 and α7 AChRs to the acute application of ACh (100 µM), nicotine (16 µM), varenicline (4 µM), cytisine (16 µM) and sazetidine-A (62.5 nM) were measured using the FlexStation as described. These drug concentrations were selected because they gave maximum sustained responses to these agonists. Results were expressed as a percentage of maximum response to ACh. Responses were monitored for 10 minutes, and then specific antagonists were added and responses recorded for another two minutes. The antagonists were dihydroβerythroidine (DHβE) (1 µM) for α4β2, mecamylamine (MCA (10 µM) for α3β4 and methyllycaconitine (MLA) (10 µM) for α7. These concentrations of antagonists were selected because they were sufficient to inhibit responses to the tested agonists without causing activation themselves.