Protection of a single S305C channel from reaction with cis MTS-ACE by His6-tag blockade. Two additions of S305C totaling 50 ng were made to the cis compartment 7–11 min before the start of the record. After a 45-pS channel opened at 60 mV (3 min before the start of the record), the potential was pulsed between −30 and 60 mV to establish normal His6-tag blocking and unblocking behavior. After blocking again at −30 mV, 400 µg MTS-ACE was added to the cis compartment (at the arrow), and the potential continued to be held at −30 mV for 2 min of MTS-ACE exposure. (During this period, there were a few brief unblocking events of ∼1 s total duration, but no reaction.) Upon switching back to 60 mV, the conductance increased rapidly to its normal (unreacted) level as the channel unblocked, followed 2 s later by a drop to 10 pS (marked by an asterisk), representing reaction of the channel. Solutions were as in Fig. 2, except that the buffers were 20 mM MES, pH 6.0 (cis), versus 5 mM HEPES, pH 8.0 (trans).
