Figure 3.

Effects of quinacrine on the reactions of MTSEA with the mutant receptors αT244C and αV218C in the open state. Oocytes were treated alternately with ACh alone and with a mixture of ACh and MTSEA or with a mixture of ACh, MTSEA, and quinacrine. Following each of these additions, the oocytes were washed with CFFR for at least 180 s. The extent of reaction of MTSEA was taken as proportional to its effect on the response to ACh. The additions to the oocytes are indicated by the italicized letters above the current recording. (A) αT244C was treated alternately with 100 μM ACh alone (“A”) applied for 10 s and with 100 μM ACh + 2 μM MTSEA (“B”) for 10 s. The values taken as the test responses to ACh are marked with circles. (B) αT244C was treated alternately with 100 μM ACh alone (“A”) for 10 s and with 10 μM quinacrine alone for 15 s, immediately followed by 100 μM ACh + 2 μM MTSEA + 10 μM quinacrine (“B”) for 10 s. The currents taken as the test responses to ACh are marked with squares. (C) αV218C was treated alternately with 10 μM ACh (EC₅₀) alone (“A”) for 15 s and with 100 μM ACh + 100 μM MTSEA (“B”) for 10 s (four cycles) and then with 10 μM ACh alone for 15 s (“A”) and with 20 μM quinacrine alone for 15 s immediately followed by 100 μM ACh + 100 μM MTSEA + 20 μM quinacrine (“C”) for 10 s (four cycles). The currents taken as the test responses are marked with triangles. (D) ACh-evoked current (I) as a function of the duration (t) of the MTSEA reaction. The peak test currents in A (○), B (□), and C (▿) were fitted by an exponential function (see Materials and Methods). The test currents in C corresponding to reaction times 0–40 s, in the absence of quinacrine, and those for reaction times 40–80 s, in the presence of quinacrine, were fitted separately. The fit yields the second-order rate constants 9,500 ± 300 M⁻¹·s⁻¹ for A, 2,450 ± 180 M⁻¹·s⁻¹ for B, and 23.7 ± 1.5 M⁻¹·s⁻¹ (absence of quinacrine) and 220 ± 76 M⁻¹·s⁻¹ (presence of quinacrine) for C.