TABLE 1.
Functional properties of chimeras and mutants
The pH0.5 of activation (pH0.5 act) was modified for most chimeras and mutants (see Fig. 2 for curves). However, activation was always shifted toward more acidic pH compared with wild-type ASIC1a, which means that channels have a more stable closed state. The mambalgin effect is largely supported by a shift of the activation curves toward more acidic pH; i.e. it stabilizes the closed state of the channel (21). One would therefore expect, if the effects are supported by gating modifications, a more potent inhibition of these chimeras and mutants by the toxin, which was not the case. The pH0.5 of inactivation (pH0.5 inact) was also modified in some channels, but at holding pH 7.4, which was used in our experimental conditions, all chimeras and mutants were in the closed state before test with pH 5.0 (curves shown in Fig. 2). In addition, the effect of mambalgin-2 (Mamb-2) was not significantly different from a holding pH of 7.4 or 8.0, excluding an effect due to a modification of the pH-dependent inactivation in the presence of toxin (note that only the double mutant 1aD349G,F350L was tested and not the related single mutants 1aD349G and 1aF350L). Changes of the initial biophysical properties of the different chimeras and mutants are thus not able to explain the effects we observed, supporting a direct alteration of the contact between the chimeric (or mutated) channels and the toxin. ND, not determined.
| Channels | pH dependence (n = 4–7) |
Current after incubation with 400 nm Mamb-2 |
||
|---|---|---|---|---|
| pH0.5 act | pH0.5 inact | From holding pH 7.4 (n = 7–24) | From holding pH 8.0 (n = 5–13) | |
| % of control current at pH 5.0 | ||||
| 1a | 6.36 ± 0.03 | 7.10 ± 0.01 | 7 ± 1 | 7 ± 1 |
| 1a/2aT | 5.00 ± 0.03 | 6.76 ± 0.01 | 97 ± 1 | 97 ± 2 |
| 1a/2aP | 5.31 ± 0.02 | 7.17 ± 0.01 | 20 ± 2 | 20 ± 4 |
| 1a/2aβ | 5.42 ± 0.07 | 6.71 ± 0.02 | 57 ± 5 | 60 ± 7 |
| 1aRDQ-KQE | 5.50 ± 0.07 | 6.90 ± 0.01 | 46 ± 6 | 46 ± 10 |
| 1a/2aP+RDQ-KQE | 4.47 ± 0.01 | 7.01 ± 0.01 | 585 ± 60 | 666 ± 67 |
| 1aD349G | 5.96 ± 0.07 | 7.11 ± 0.01 | 16 ± 5 | ND |
| 1aF350L | 5.63 ± 0.04 | 7.05 ± 0.01 | 54 ± 4 | ND |
| 1aDF/GL | 5.08 ± 0.07 | 7.08 ± 0.01 | 86 ± 2 | 85 ± 2 |
| 2a | 4.48 ± 0.09 | 5.05 ± 0.04 | 100 ± 1 | 99 ± 1 |
| 2a/1aT | 4.50 ± 0.12 | 5.92 ± 0.02 | 195 ± 9 | 174 ± 7 |
| 2a/1aT+P | 4.46 ± 0.05 | 5.99 ± 0.02 | 69 ± 5 | 75 ± 2 |
| 2a/1aT+β | 4.70 ± 0.06 | 6.33 ± 0.01 | 195 ± 8 | 199 ± 14 |