MTSET effect on the ionic selectivity of colicin Ia mutant G577C channels. Vrs were measured with a gradient of 100 mM KCl (cis)/1 M KCl (trans), with the other pH 7.2 buffer ingredients as in Fig. 1. The top and bottom traces, respectively, show the current and voltage versus time. Because of space constraints, some of the numbers giving the voltage (in mV) are placed near the current trace. Approximately 4 min before the start of the record, 135 ng G577C (as well as 0.9 μg octylglucoside and DTT to 6 μM) was added to the cis compartment. About 1,500 channels were opened at +50 mV, and the Vr (+30 mV) was determined as the voltage needed to zero the current. This is the normal Vr for colicin Ia channels under these conditions, indicating preferential selectivity for K+ over Cl−. The potential was then held at +35 mV. The addition of 200 μg MTSET (marked by the arrow) to the cis compartment caused a pronounced increase in the current. This was due not to a conductance increase (the conductance actually decreased), but to a change in the Vr to a negative voltage. By the end of the record, the conductance was too low (because of channel closure at these voltages) to provide a precise value for the Vr, but the measurement of tail currents in subsequent pulses (not depicted) showed it to be Vr = −18 mV. Thus, reaction of G577C with MTSET changed the channel selectivity from favoring K+ permeation to preferring Cl−.
