Figure 3.
Figure 3. Effects of various MTS reagents on the single-channel conductance of colicin Ia cysteine mutants. Each MTS reaction event is highlighted by an asterisk. (A) Effects of trans MTSET on F588C. Before the start of the record, F588C was added to the cis compartment, and a total of 300 μg MTSET was added to the trans compartment on two occasions, with the latter addition occurring 16 min before the start of the record. The record shows single-channel currents at a constant membrane potential of +70 mV. First, a 59-pS channel opened. After 7 s, the conductance dropped to 43 pS (a 27% decrease), representing the channel's reaction with MTSET. Similarly, a second 60-pS channel opened and then dropped to 43 pS (a 28% decrease) 2 s after opening. In this analysis, we neglect the brief flickers to a higher conductance state (noted by Kienker et al., 2003) that are evident in this record, as well as in B and C. (B) Effects of trans MTSES on A585C. This mutant was particularly reactive, so we used less MTSES than usual, allowing the reactions to be observed on a convenient time scale. A585C was added to the cis compartment and, 15 min before the start of the record, 20 μg MTSES was added to the trans compartment. The record shows single-channel currents at +40 mV. First, a 48-pS channel opened and, 8 s later, increased in conductance to 64 pS, indicating reaction with MTSES. There was also a notable increase in current noise upon reaction. A second channel likewise showed a conductance increase from 47 to 62 pS, occurring 1 s after its opening. (The increases were 33 and 32%, respectively.) (C) Effect of trans MMTS on G609C. Before the start of the record, G609C was added to the cis compartment. The record begins with a channel opening at +70 mV. The membrane potential was then lowered to +50 mV, at which the single-channel conductance was 70 pS. (This conductance is a little higher than average, but not shockingly so.) Next, at the arrow, 200 μg MMTS was added to the trans compartment. After a delay of 28 s, the conductance dropped to 48 pS (a 31% decrease), reflecting reaction of G609C with MMTS. For this record, we were able to analyze the brief flickers to a higher conductance state, whose conductance also decreased upon reaction with MMTS, from 133 to 99 pS (a 26% decrease). (The conductance states were identified clearly by an all-points histogram analysis.) For cosmetic reasons, we suppressed the current trace at the time of MMTS addition, but the passage of time is represented accurately despite this break. The voltage trace applies only to C. For all three panels, the solution on both sides of the membrane was as in Fig. 1, except the KCl concentration was 1 M. The dashed line in each panel indicates the zero current level. The amounts of mutant colicin, octylglucoside, and DTT added to the cis compartment and DTT added to the trans compartment before the start of each record were, respectively, 24 ng, 0, 21 μM, and 50 μM in A; 2.1 ng, 10 μg, 10 μM, and 5 μM in B; and 3.4 ng, 0, 6 μM, and 5 μM in C.

Effects of various MTS reagents on the single-channel conductance of colicin Ia cysteine mutants. Each MTS reaction event is highlighted by an asterisk. (A) Effects of trans MTSET on F588C. Before the start of the record, F588C was added to the cis compartment, and a total of 300 μg MTSET was added to the trans compartment on two occasions, with the latter addition occurring 16 min before the start of the record. The record shows single-channel currents at a constant membrane potential of +70 mV. First, a 59-pS channel opened. After 7 s, the conductance dropped to 43 pS (a 27% decrease), representing the channel's reaction with MTSET. Similarly, a second 60-pS channel opened and then dropped to 43 pS (a 28% decrease) 2 s after opening. In this analysis, we neglect the brief flickers to a higher conductance state (noted by Kienker et al., 2003) that are evident in this record, as well as in B and C. (B) Effects of trans MTSES on A585C. This mutant was particularly reactive, so we used less MTSES than usual, allowing the reactions to be observed on a convenient time scale. A585C was added to the cis compartment and, 15 min before the start of the record, 20 μg MTSES was added to the trans compartment. The record shows single-channel currents at +40 mV. First, a 48-pS channel opened and, 8 s later, increased in conductance to 64 pS, indicating reaction with MTSES. There was also a notable increase in current noise upon reaction. A second channel likewise showed a conductance increase from 47 to 62 pS, occurring 1 s after its opening. (The increases were 33 and 32%, respectively.) (C) Effect of trans MMTS on G609C. Before the start of the record, G609C was added to the cis compartment. The record begins with a channel opening at +70 mV. The membrane potential was then lowered to +50 mV, at which the single-channel conductance was 70 pS. (This conductance is a little higher than average, but not shockingly so.) Next, at the arrow, 200 μg MMTS was added to the trans compartment. After a delay of 28 s, the conductance dropped to 48 pS (a 31% decrease), reflecting reaction of G609C with MMTS. For this record, we were able to analyze the brief flickers to a higher conductance state, whose conductance also decreased upon reaction with MMTS, from 133 to 99 pS (a 26% decrease). (The conductance states were identified clearly by an all-points histogram analysis.) For cosmetic reasons, we suppressed the current trace at the time of MMTS addition, but the passage of time is represented accurately despite this break. The voltage trace applies only to C. For all three panels, the solution on both sides of the membrane was as in Fig. 1, except the KCl concentration was 1 M. The dashed line in each panel indicates the zero current level. The amounts of mutant colicin, octylglucoside, and DTT added to the cis compartment and DTT added to the trans compartment before the start of each record were, respectively, 24 ng, 0, 21 μM, and 50 μM in A; 2.1 ng, 10 μg, 10 μM, and 5 μM in B; and 3.4 ng, 0, 6 μM, and 5 μM in C.

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