Magnitude and rates of MTS effects in macroscopic experiments on TH8–TH9 segment mutant channels
| Mutant | cis pH | trans pH | MTS reagent | Conductance change (%) | kcis | ktrans |
| M−1 s−1 | M−1 s−1 | |||||
| Q331C | 7.0 | 7.2 | ET | −90 ± 5 (n = 4) | 45.0 ± 0.4 (n = 2) | 12.3 ± 0.8 (n = 2) |
| “ | 7.0 | 8.0 | Glucose | −74 ± 8 (n = 4) | 13 ± 1 (n = 2) | 5 ± 1 (n = 2) |
| A334C | 5.3 | 7.2 | ET | −83 ± 3 (n = 4) | 151 ± 20 (n = 3) | 44 ± 2 (n = 2) |
| L338C | 5.3 | 7.2 | ET | −25 ± 2 (n = 4) | 24 ± 6 (n = 2)a | 414 ± 212 (n = 4) |
| A341C | 5.3 | 7.2 | ET | −11 ± 2 (n = 4) | 7 ± 2 (n = 2) | 52 ± 13 (n = 2) |
| V347C | 5.3 | 7.2 | Glucose | −24 ± 4 (n = 6) | 11 ± 7 (n = 4)a | 1,776 ± 629 (n = 3) |
| Y358C | 5.3 | 7.2 | ET | −53 ± 16 (n = 3) | NE (n = 2) | 11 ± 7 (n = 4) |
| N359C | 6.0 | 8.0 | Glucose | −61 ± 7 (n = 4) | 34 ± 4 (n = 3) | 73 ± 9 (n = 3) |
| Mutant | cis pH | trans pH | MTS reagent | Conductance change (%) | kcis | ktrans |
| M−1 s−1 | M−1 s−1 | |||||
| Q331C | 7.0 | 7.2 | ET | −90 ± 5 (n = 4) | 45.0 ± 0.4 (n = 2) | 12.3 ± 0.8 (n = 2) |
| “ | 7.0 | 8.0 | Glucose | −74 ± 8 (n = 4) | 13 ± 1 (n = 2) | 5 ± 1 (n = 2) |
| A334C | 5.3 | 7.2 | ET | −83 ± 3 (n = 4) | 151 ± 20 (n = 3) | 44 ± 2 (n = 2) |
| L338C | 5.3 | 7.2 | ET | −25 ± 2 (n = 4) | 24 ± 6 (n = 2)a | 414 ± 212 (n = 4) |
| A341C | 5.3 | 7.2 | ET | −11 ± 2 (n = 4) | 7 ± 2 (n = 2) | 52 ± 13 (n = 2) |
| V347C | 5.3 | 7.2 | Glucose | −24 ± 4 (n = 6) | 11 ± 7 (n = 4)a | 1,776 ± 629 (n = 3) |
| Y358C | 5.3 | 7.2 | ET | −53 ± 16 (n = 3) | NE (n = 2) | 11 ± 7 (n = 4) |
| N359C | 6.0 | 8.0 | Glucose | −61 ± 7 (n = 4) | 34 ± 4 (n = 3) | 73 ± 9 (n = 3) |
The percent conductance change and rates are given as mean ± SD, with n being the number of experiments. Both cis and trans MTS results were used in calculating the conductance change, if they were generally in agreement. The percent conductance change and reaction rate could not always both be determined for a given experiment, so the n values for kcis and ktrans do not necessarily add up to the n value given for the conductance change. In the experiments with MTS-ET, kcis and ktrans were measured at 30 and −30 mV, respectively. Each of the mutant T-domains had its His6-tag removed, with the exception of V347C and N359C. NE, no effect.
Cis MTS reagent, even at high concentrations, produced a slow effect on the conductance. It is possible that this was caused by reaction in the cis solution, followed by slow washout of channel activity from the membrane; thus, the value for kcis should be taken as an upper limit.