Comparison of cis- and trans-side reaction rates of MTS-ET with T-domain mutant L307C. Records from two experiments are superimposed, showing normalized current versus time. About 7–10 min before the start of the record, 4–8 ng L307C was added to the cis compartment, and the channel activity was allowed to stabilize. At the arrow, 200 µg MTS-ET was added to the cis (cyan trace) or trans (red trace) compartment, resulting in a decrease in current of ∼19%. The pseudo-first order rate constants were estimated as k_1,cis = 0.0241 s⁻¹ and k_1,trans = 0.247 s⁻¹ for the cis- and trans-side experiments, respectively. Solutions were as in Fig. 2. The holding potential was 30 mV. The trans MTS-ET experiment was the same one depicted in Fig. 2. In the cis MTS-ET experiment, there was a further slow decrease in current after the end of the record, presumably because of channel gating or washout (see Data analysis in Materials and methods), but it was disregarded in the analysis.