Fig. 4.

Effect of pH. (A and B) Stretches of single-channel currents recorded in the outside-out configuration (Xenopus oocyte membranes) at −80 mV, in the continued presence of cysteamine. The pH of the patch-pipette solution was 7.4. The pH of the external solution was adjusted to 6.5 using 25 mM N-(2-acetamido)iminodiacetic acid (ADA) as the proton buffer in an attempt to mimic the conditions in the crystallization buffer (SI Materials and Methods). Channel openings are downward deflections. Display f_c ∼5 kHz. Clusters were defined as series of openings separated by shuttings shorter than 200 ms. The intracluster open probability of the L9′A + F16′L double mutant increases from ∼0.44 at external pH 7.4 (Fig. 3E) to ∼0.93 at external pH 6.5. (C) Normalized time courses of currents elicited by the application of cysteamine for 5 s. The pH of the two external solutions flowing from the θ-type tubing was adjusted to 6.5 using 25 mM ADA as the proton buffer. The pH of the patch-pipette solution was 7.4. (D) Extent of desensitization (mean ± SE).