Table II. Affinity constants and pairwise interaction energies of α7/5HT3 mutants for ACh, nicotine and epibatidine.
| K (µM) | nH,Y | ΔK | ΔΔG (kcal/mol) | Ω | ΔΔGINT (kcal/mol) | |
|---|---|---|---|---|---|---|
| ACh |
|
|
|
|
|
|
| α7/5HT3 | 27.4 ± 1.3a | 1.57 ± 0.13 | 1.0 | – | – | – |
| G152K | 0.37 ± 0.03a | 1.07 ± 0.08 | 74 | –2.51 | – | – |
| P193I | 2.5 ± 0.4a | 2.00 ± 0.10 | 11 | –1.40 | – | – |
| G152K/P193I | 3.4 ± 1.2b | 0.94 ± 0.06 | 8.1 | –1.22 | 101 | 2.69 |
| P195L |
74.2 ± 11.6a |
2.09 ± 0.67 |
0.37 |
– |
– |
– |
| Nicotine |
|
|
|
|
|
|
| α7/5HT3 | 1.43 ± 0.10a | 1.25 ± 0.10 | 1.0 | – | – | – |
| G152K | 0.014 ± 0.002a | 0.72 ± 0.04 | 102 | –2.70 | – | – |
| P193I | 0.22 ± 0.02a | 1.45 ± 0.15 | 7.0 | –1.09 | – | – |
| G152K/P193I | 0.113 ± 0.009b | 0.94 ± 0.05 | 13 | –1.48 | 52 | 2.31 |
| P195L |
6.93 ± 0.95a |
1.30 ± 0.22 |
0.21 |
– |
– |
– |
| Epibatidine |
|
|
|
|
|
|
| α7/5HT3 | 0.126 ± 0.011a | 1.25 ± 0.11 | 1.0 | – | – | – |
| G152K | 0.0068 ± 0.0033a | 0.95 ± 0.15 | 19 | –1.70 | – | – |
| P193I | 0.0111 ± 0.0030a | 1.53 ± 0.29 | 11 | –1.42 | – | – |
| G152K/P193I | 0.0082 ± 0.0016b | 0.92 ± 0.05 | 15 | –1.59 | 13 | 1.53 |
| P195L | 0.603 ± 0.135a | 0.91 ± 0.18 | 0.21 | – | – | – |
aProtection constants Kp (mean ± SE) were obtained from at least two different independent experiments performed in duplicate and were determined from initial velocities of [125I]α-BgTx binding (Corringer et al., 1995).
bInhibition constants Ki (mean ± SE) were obtained from at least two different independent experiments performed in duplicate and were calculated using the measured IC50 at equilibrium from [3H]epibatidine binding (Cheng and Prusoff, 1973).
ΔK = Kp(α7/5HT3)/Kp(mut) or Kp(α7/5HT3)/Ki(G152K/P193I).
ΔΔG = –RT ln(ΔK) where R = 1.99 cal mol–1K–1 and T = 293 K.
ΔΔGINT = RT lnΩ where Ω = K(mut1/mut2)K(WT1/WT2)/K(mut1/WT2)K(WT1/mut2) and K is Kp or Ki, mut1 is G152K, mut2 is P193I, WT1 is G152 and WT2 is P193.