Effect of trans MTSET on the macroscopic current through colicin Ia mutant N578C channels. The top trace shows the membrane current and the bottom trace shows the voltage, each as a function of time. Before the start of the record, DTT was added to the trans compartment to a final concentration of 5 μM and 1.0 μg N578C (along with 4.5 μg octylglucoside and DTT to 5 μM) was added to the cis compartment. We quickly opened on the order of 1,000 channels by stepping the membrane potential to +70 mV, and then switched it to +50 mV to establish a slower channel-opening rate. At the arrow, 200 μg MTSET was added to the trans compartment. This caused a decrease in current of ∼25%, demonstrating that residue N578C was accessible for reaction. Finally, we confirmed that the channels closed normally when the membrane potential was switched to −50 mV. The solution on both sides of the membrane was 100 mM KCl, 5 mM CaCl2, 1 mM EDTA, 20 mM HEPES, pH 7.2. (The membrane broke after colicin addition and was reformed before the start of the record; similar results were obtained with membranes that had not broken.)
