Table I. Binding properties of α7/5HT3 mutants for [3H]epibatidine and α-bungarotoxin.
| Construction | [3H]epibatidine binding |
α-Bgtx binding |
N | ||
|---|---|---|---|---|---|
| KD (nM) | nH | IC50 (nM) | nH | ||
| α7/5HT3 | NQ | NQ | 5.7 ± 1.4a | 1.05 ± 0.16 | 3 |
| α4β2 | 1.0 ± 0.1 | 0.87 ± 0.06 | >100 000 | – | 1 |
| ChimB | 11.2 ± 3.5 | 0.93 ± 0.08 | 5.3 ± 1.3 | 0.61 ± 0.05 | 4 |
| 4.6 ± 1.1a | 1.49 ± 0.23 | 2 | |||
| P193I | 5.6 ± 0.3 | 0.93 ± 0.07 | 22.5 ± 4.4 | 1.14 ± 0.01 | 2 |
| G152S | 14.3 ± 4.2 | 0.97 ± 0.05 | 38 ± 25 | 0.71 ± 0.05 | 1 |
| G152D | 4.5 ± 1.5 | 1.08 ± 0.10 | 25 ± 9 | 0.89 ± 0.67 | 2 |
| G152E | 76 ± 31 | 0.98 ± 0.03 | 53 ± 30 | 0.98 ± 0.12 | 3 |
| G152K | 10.7 ± 0.5 | 1.00 ± 0.01 | 39 ± 12 | 0.83 ± 0.01 | 2 |
| ChimB/P193I | 9.3 ± 2.1 | 0.98 ± 0.02 | 202 ± 65 | 1.21 ± 0.63 | 2 |
| G151D/P193I | 4.7 ± 0.4 | 1.01 ± 0.01 | 7.9 ± 0.3 | 0.63 ± 0.08 | 2 |
| G152K/P193I | 6.4 ± 1.0 | 0.97 ± 0.02 | 600 ± 132* | 1.03 ± 0.06 | 4 |
| W153A/P193I | 7.1 ± 1.9 | 0.99 ± 0.01 | 30 ± 1 | 1.02 ± 0.88 | 2 |
| L155I/P193I | 9.4 ± 2.7 | 1.00 ± 0.02 | 19.3 ± 4.8 | 0.92 ± 0.19 | 2 |
| G152S/P193I | 6.7 ± 1.7 | 0.96 ± 0.05 | 40 ± 9 | 0.85 ± 0.13 | 1 |
| G152D/P193I | 27 ± 11 | 0.98 ± 0.01 | 109 ± 14 | 0.97 ± 0.21 | 2 |
| G152N/P193I | 50 ± 20 | 0.97 ± 0.04 | >100 000 | – | 2 |
| G152N/S154A/P193Ib | 21 ± 6 | 0.99 ± 0.03 | 502 ± 128 | 0.92 ± 0.12 | 2 |
| G152E/P193I | 13.1 ± 4.2 | 1.01 ± 0.02 | 184 ± 67 | 0.84 ± 0.12 | 2 |
IC50, concentration of α-BgTx that yields 50% inhibition of [3H]epibatidine binding (mean ± SE); KD, dissociation constant (mean ± SE); nH, Hill coefficient (mean ± SE); N, number of independent experiments, each performed at least in duplicate; NQ, not quantifiable.
*Statistically different from α7/5HT3 using an unpaired Student’s t-test (p < 0.025).
aProtection constant Kp determined from initial velocities of [125I]α-BgTx binding (mean ± SE) (Corringer et al., 1995).
bMutant in which the putative N-glycosylation site NWS (created by G152N mutation) was suppressed by introducing the mutation S154A (sequence NWA).