Effect of trans MTS-ET on T-domain mutant L307C. The top trace is current and the bottom trace is voltage, with both plotted against time. After the addition of 4 ng L307C to the cis compartment (first arrow), the current at 30 mV gradually increased as channels formed in the membrane. Upon switching to negative voltage, the magnitude of the current rapidly decreased toward zero, indicating blocking by the amino-terminal His₆-tag (Senzel et al., 1998); the current rapidly recovered upon returning to positive voltage. At the second arrow, after the channel activity had stabilized, 200 µg MTS-ET was added to the trans compartment, resulting in a decrease in current of ∼19%. The solutions were 1 M KCl, 2 mM CaCl₂, 1 mM EDTA, with 20 mM malic acid, pH 5.3, in the cis compartment and 20 mM HEPES, pH 7.2, in the trans compartment. The break in the record was 3.6 min.