Percent conductance change upon MTS-ET reaction with TH6–TH7 segment mutant channels
| Mutant | cis MTS-ET macroscopic | trans MTS-ET macroscopic | cis MTS-ET single channel | trans MTS-ET single channel |
| T300Ca | −98 ± 2 (n = 4) | NE (n = 2) | −100 ± 0 (n = 3) | ND |
| T301Cb | −64 ± 4 (n = 4) | NE (n = 2) | −60 ± 3 (n = 3) | −47 ± 3 (n = 6) |
| A302C | −82 ± 2 (n = 2)c | NE (n = 2) | −73 ± 4 (n = 6) | ND |
| A303Ca | −100 ± 0 (n = 3) | NE (n = 3) | ND | ND |
| L304Cd | −98 ± 2 (n = 3) | NE (n = 2) | −100 ± 0 (n = 3) | ND |
| S305C | −71 ± 4 (n = 6) | −56 ± 6 (n = 5) | ND | −79 ± 2 (n = 7) |
| I306Cd | −40 ± 18 (n = 2) | −49 ± 0 (n = 2) | −59 ± 1 (n = 3) | −63 ± 2 (n = 3) |
| L307C | −28 ± 4 (n = 2) | −36 ± 10 (n = 6) | ND | −70 ± 3 (n = 12)e |
| P308Cf | Increase | Increase | ND | −16 ± 2 (n = 3) |
| G309C | −16 ± 6 (n = 3) | −14 ± 2 (n = 5) | ND | −31 ± 3 (n = 19)e |
| I310C | −50 ± 8 (n = 3)b | NE (n = 2)b | Flickers (n = 4)g | Flickers (n = 2)g |
| S312C | −75 ± 13 (n = 7) | −67 ± 18 (n = 8) | −81 ± 4 (n = 9) | −76 ± 3 (n = 3) |
| V313Cb | −96 ± 4 (n = 4) | NE (n = 2) | −100 ± 0 (n = 5) | ND |
| M314Cb | −96 ± 4 (n = 6) | NE (n = 4) | −39 ± 1 (n = 2)h,i | ND |
| G315Cj | −85 ± 5 (n = 7) | NE (n = 8) | −84 ± 3 (n = 5) | −81 ± 3 (n = 3) |
| I316C | −98 ± 2 (n = 3) | NE (n = 2) | −100 ± 0 (n = 12) | ND |
| A317Ca | −97 ± 3 (n = 3) | NE (n = 2) | −100 ± 0 (n = 4)h | ND |
| Mutant | cis MTS-ET macroscopic | trans MTS-ET macroscopic | cis MTS-ET single channel | trans MTS-ET single channel |
| T300Ca | −98 ± 2 (n = 4) | NE (n = 2) | −100 ± 0 (n = 3) | ND |
| T301Cb | −64 ± 4 (n = 4) | NE (n = 2) | −60 ± 3 (n = 3) | −47 ± 3 (n = 6) |
| A302C | −82 ± 2 (n = 2)c | NE (n = 2) | −73 ± 4 (n = 6) | ND |
| A303Ca | −100 ± 0 (n = 3) | NE (n = 3) | ND | ND |
| L304Cd | −98 ± 2 (n = 3) | NE (n = 2) | −100 ± 0 (n = 3) | ND |
| S305C | −71 ± 4 (n = 6) | −56 ± 6 (n = 5) | ND | −79 ± 2 (n = 7) |
| I306Cd | −40 ± 18 (n = 2) | −49 ± 0 (n = 2) | −59 ± 1 (n = 3) | −63 ± 2 (n = 3) |
| L307C | −28 ± 4 (n = 2) | −36 ± 10 (n = 6) | ND | −70 ± 3 (n = 12)e |
| P308Cf | Increase | Increase | ND | −16 ± 2 (n = 3) |
| G309C | −16 ± 6 (n = 3) | −14 ± 2 (n = 5) | ND | −31 ± 3 (n = 19)e |
| I310C | −50 ± 8 (n = 3)b | NE (n = 2)b | Flickers (n = 4)g | Flickers (n = 2)g |
| S312C | −75 ± 13 (n = 7) | −67 ± 18 (n = 8) | −81 ± 4 (n = 9) | −76 ± 3 (n = 3) |
| V313Cb | −96 ± 4 (n = 4) | NE (n = 2) | −100 ± 0 (n = 5) | ND |
| M314Cb | −96 ± 4 (n = 6) | NE (n = 4) | −39 ± 1 (n = 2)h,i | ND |
| G315Cj | −85 ± 5 (n = 7) | NE (n = 8) | −84 ± 3 (n = 5) | −81 ± 3 (n = 3) |
| I316C | −98 ± 2 (n = 3) | NE (n = 2) | −100 ± 0 (n = 12) | ND |
| A317Ca | −97 ± 3 (n = 3) | NE (n = 2) | −100 ± 0 (n = 4)h | ND |
The percent conductance change is given as mean ± SD, with n being the number of macroscopic experiments or the number of single-channel reactions. All mutant channels were tested with pH 5.3 in the cis compartment and 7.2 in the trans compartment and at positive voltage (20–120 mV), except where stated otherwise. Mutants that showed no effect or a very slow effect of MTS-ET at pH 5.3/7.2 were also tested with the cis pH raised, as indicated in the table. Solutions were as in Fig. 2, except that 5 mM MES buffer was used when the cis pH was to be raised (by the addition of concentrated HEPES buffer). Mutant G311C was excluded because its noisy macroscopic conductance made it difficult to assess MTS-ET effects. NE, no effect.
The cis pH was raised to 7.0–7.3 before MTS-ET addition.
The cis pH was raised to 6.2–6.3 before MTS-ET addition.
In macroscopic experiments with A302C at positive voltage, cis MTS-ET caused a decrease, but the conductance became noisy before the decrease had completed. The reported value is from experiments at −20 mV using A302C with the His6-tag removed.
The cis pH was raised to 6.5–6.7 before MTS-ET addition.
For L307C and G309C, the single-channel conductance decrease caused by reaction with MTS-ET was larger than the corresponding macroscopic effect. This was apparently because only a fraction of the channels reacted.
In macroscopic experiments with P308C (with the His6-tag removed), both cis and trans MTS-ET caused large, gradual conductance increases on the order of 10–30-fold. In single-channel experiments, in addition to the initial small conductance decrease seen after MTS-ET addition (as shown in the table), larger conductance channels (∼75 pS) were also observed.
Single-channel experiments with I310C were performed with the cis pH raised to 6.2–6.7. The effect of MTS-ET was to increase the amount of channel flickering to a short-lived closed state without changing the open-channel conductance.
Cis MTS-ET reaction with M314C and A317C single channels caused a large decrease in the occupancy probability of the open state.
The decrease was measured with 1,000-Hz filtering.
The conductance of single channels formed by G315C was abnormally high, at ∼75 pS.