Table 1.
Inhibition of current by internal MTSET
| Amino-terminal mutants
|
Carboxyl-terminal mutants
|
||||||
|---|---|---|---|---|---|---|---|
| Channel | % inhibition
|
k, M−1·s−1 | Channel | % inhibition
|
k, M−1·s−1 | ||
| 2 min | 5 min | 2 min | 5 min | ||||
| WT | 93 ± 3 | 19 ± 1 | WT | 93 ± 3 | 19 ± 1 | ||
| V54C | 44 ± 12 | 91 ± 3 | 4.3 ± 1.9 | G215C | 74 ± 8.6 | 94 ± 3 | 6.6 ± 1.4 |
| F58C | 77 ± 4 | 94 ± 2 | 6.3 ± 1.2 | N216C* | 31 ± 5 | ND | 16 ± 8 |
| V61C | 99 ± 0.6 | ND | 2,600 ± 608 | L217C | 93 ± 3 | ND | 9.3 ± 1.5 |
| Q66C* | 21 ± 2 | ND | 5 ± 2 | R218C* | 26 ± 1 | 37 ± 6 | 6.6 ± 2.2 |
| R67C* | 16 ± 6 | 26 ± 5 | 3.8 ± 1.4 | S220C* | 35 ± 6 | ND | 28 ± 2 |
| Y68C* | 21 ± 3 | 28 ± 4 | 4.5 ± 0.6 | H221C | 77 ± 11 | 93 ± 3 | 6.8 ± 2.0 |
| L69C | 38 ± 5 | 60 ± 5 | 3.2 ± 0.3 | L222C | 76 ± 10 | 95 ± 3 | 8.3 ± 1.5 |
| D71C* | 20 ± 4 | ND | 22 ± 12 | V223C | 43 ± 5 | 77 ± 3 | 2.4 ± 1.0 |
| T74C* | 29 ± 5 | 41 ± 7 | 3.3 ± 0.3 | E224C | 95 ± 2 | ND | 2,217 ± 189 |
| T75C* | 41 ± 1 | 46 ± 3 | 6.5 ± 1.3 | H226C | 91 ± 1 | ND | 25 ± 1 |
| V76C | 19 ± 4 | 34 ± 9 | 2.0 ± 0.7 | R228C | 38 ± 2 | ND | 35 ± 3 |
| D78C* | 46 ± 3 | ND | 17 ± 6 | ||||
| I79C | 92 ± 1 | ND | 58 ± 19 | ||||
| W81C | 64 ± 7 | 92 ± 1.3 | 7.3 ± 4.5 | ||||
| W83C | 32 ± 3 | 56 ± 3 | 2.9 ± 0.5 | ||||
MTSET was applied at 2 mM to the intracellular side of giant inside-out patches containing the wild-type or mutant channels for 2 or 5 min, depending on the reaction rate to reach the steady-state effect. For V61C and E224C, 20 μM MTSET was used in experiments to determine the reaction rate (the right-hand column). Only reactive channels are shown. Percentage inhibition and the apparent second-order rate constant k were calculated as described in Materials and Methods. ND, not determined, because steady-state inhibition was achieved in 2 min. Data are presented as mean ± SD (n = 3–6).
*
Mutants rescued by tandum tetramers.