Figure 4.

Trans addition of the membrane-impermeant disulfide reducing agent TCEP eliminates rapid turn-off by protease-nicked, amino-terminal H6-tagged DT(C+T) channels. Before the start of the record, nicked H6-tagged DT(C+T) was added to the cis solution to a concentration of 110 ng/ml, and the voltage was held at +30 mV. The solutions on both sides of the membrane contained 1 M KCl, 2 mM CaCl₂, and 1 mM EDTA; the cis solution contained 5 mM Mes (pH 5.3), and the trans solution contained 5 mM Hepes (pH 7.2). After channel activity appeared, the pHs of the cis and trans solutions were brought to 6.0 with Mes (final concentrations 35 mM cis, 60 mM trans). Channel conductance turned off rapidly at −70 mV. Four minutes before the start of the record, TCEP (pH 6.2) was added to the cis solution to a concentration of 100 mM, with no effect on channel conductance at both +30 and −70 mV. At the arrow, TCEP (pH 6.2) was added to the trans solution to a concentration of 100 mM. Subsequently, channel conductance failed to turn off at −70 mV. (TCEP addition to the trans solution, without prior addition to the cis solution, gave the same results as in this experiment.) The record was filtered at 10 Hz by the chart recorder.