Figure 5.

(A) Standing inward currents and ACh responses in oocytes expressing nAChRs with Tyr-O3Q incorporated at α149 and a Leu-9′ → Ser mutation in M2 of the β subunit. The initial current of almost −3 μA, the standing current as a result of the mutant channels, is substantially eliminated by channel blockers such as QX-314 and TMB-8 or the cholinergic antagonist dTC. Application of ACh leads to increased currents. (B) ACh-induced desensitization affects the standing current. During the first application of TMB-8 (5 μM), the standing outward current was reduced reversibly to nearly zero. ACh (25 μM) was then added, inducing an inward current that desensitized. During the desensitization phase, the standing current was reduced. TMB-8 was added again during the desensitization phase. The TMB-8 deflection reached the former plateau level rather than producing a net outward current. These interactions show that ACh desensitizes the conductance mechanism that produces the standing current; therefore, the standing current is produced by functional nAChR. (C) Single-channel records and open-time distribution from receptors containing Tyr-O3Q at position α149. The traces show selected openings from cell-attached patches recorded at −80 mV. The open-time distribution from a typical patch is fitted (least-squares) to a single exponential decay with a time constant of 1.4 ms. Openings less than 0.6 ms were excluded from the analysis to eliminate contributions from the spontaneous openings of βLeu-9′ → Ser receptors, which have a time constant of 0.3 ms (H. Zhang et al., personal communication).