Protection of S312C channels from reaction with MTS-ET by His6-tag blockade. Records from three experiments are superimposed in each panel; the top traces are normalized current, and the bottom traces are voltage, with all plotted against time (relative to the moment of MTS-ET addition). The protection experiments (blue and green traces) used S312C with an amino-terminal His6-tag, whereas the unblocked control experiments (red traces) used S312C from which the His6-tag had been removed by thrombin treatment. About 4–10 min before the start of each record, 8–9 ng S312C was added to the cis compartment, and the channel activity was allowed to stabilize. At the arrow, while holding at −30 mV, 2.6 mg MTS-ET was added to the cis (A) or trans (B) compartment. In the unblocked control experiments (red traces), reaction with MTS-ET at −30 mV caused a decrease in conductance that was largely complete within 1–2 min. In the His6-tag protection experiments (blue and green traces), the potential was held at −30 mV for 1–4 min of exposure to MTS-ET. Upon switching to 60 mV, there was a rapid increase in current, representing unblocking of the channels by the His6-tag, followed by a slower decay of the current, representing reaction with MTS-ET. (No such decay was seen without MTS addition.) The relatively large peak currents after unblocking show that His6-tag blockade protected Cys-312 from reaction. Note that the reaction rate of cis MTS-ET with S312C channels is voltage dependent (see Fig. 6 B); the relevant rate here is that at −30 mV (seen in the unblocked control experiment), rather than the faster rate seen at 60 mV. Solutions were as in Fig. 2. Some of the voltage traces are shown as dashed lines so that all the superimposed traces can be seen. The time scale bar applies to both panels.
