Comparison of cis- and trans-side reaction rates of MTS-ET with T-domain TH8 segment mutants A334C and L338C. Records from two experiments are superimposed in each panel, showing normalized current versus time. (Both negative and positive initial currents were normalized to +1.) T-domain with the His₆-tag removed was used in all four experiments. (A) Multiple additions of A334C totaling 0.1–1 ng were made to the cis compartment 7–18 min before the start of the record, and the channel activity was allowed to stabilize. While holding, respectively, at 30 or −30 mV, 200 µg MTS-ET was added (at the arrow) to the cis or trans compartment (cyan or red trace), causing a conductance decrease of 87 or 85%. Using the 85% value in both cases, Eq. 1a gives k_1,cis = 0.107 s⁻¹ and k_1,trans = 0.0297 s⁻¹. (The erratic jumps in current occurred frequently for this mutant after MTS-ET addition.) (B) 1–8 ng L338C was added to the cis compartment 8–9 min before the start of the record. While holding, respectively, at 30 or −30 mV, 200 µg MTS-ET was added (at the arrow) to the cis or trans compartment (cyan or red trace), causing a conductance decrease of ∼21 or 23%. (For the cis MTS-ET experiment, 21% was the amount of the fast component of the decrease; there was also a slower component extending ∼25 min beyond the end of the record.) Using the 23% value in both cases, Eq. 1a gives k_1,cis = 0.0235 s⁻¹ and k_1,trans = 0.179 s⁻¹. For both panels, solutions were as in Fig. 2.