Figure 6. Further examination of the contribution of α5 to agonist-nAChR interactions: increased contribution of α5 nAChRs following α7 deletion.

The normalized peak amplitudes of the agonist-evoked responses from control and α7 antisense oligonucleotide-treated, and (α5 +α7) antisense-treated neurones are plotted against the concentration of ACh. A, ACh-elicited macroscopic currents in control neurones (○) are fitted by a single sigmoidal curve with an EC₅₀ of 76.6 μM, ACh-elicited responses in α7 minus neurones (▴) are fitted by a biphasic curve with apparent affinity for ACh of 26.6 μM and 443.3 μM, respectively. The apparent Hill coefficients of these components are 2.34 and 2.78, respectively. The calculated fits to the two components are indicated by the dotted lines. To test our hypothesis that low agonist affinity nAChRs include the α5 subunit, α5 was deleted from control (see Fig. 1) and α7 minus neurones. Concentration dependence of ACh-evoked responses in neurones treated with both α5 and α7 antisense oligonucleotides (×) is fitted by a single sigmoidal curve with an EC₅₀ of 37.8 μM and an apparent Hill coefficient of 2.58. Additional deletion of α5 from the α7 minus neurones removes all the low agonist affinity nAChRs. B, the concentration dependence of the nicotine-evoked response in α7 minus neurones also revealed the increased contribution of a low affinity component. Nicotine-elicited currents in control neurones are fitted by a single sigmoidal curve with an EC₅₀ of 24.4 μM and an apparent Hill coefficient of 1.44. The biphasic dose-response curve of nicotine-elicited currents from ‘α7 minus’ neurones indicates a high apparent affinity component with an EC₅₀ of 9.52 μM and a very low apparent affinity component with an EC₅₀ of 811.9 μM. The apparent Hill coefficients for these components are 1.49 and 2.58, respectively. Dotted curves indicate calculated fits to the two components.