EC50 values for mutant α4β2 nAChRs
| Motivation | Mutant | EC50 (µm ACh) ± SEM | Hill coefficient ± SEM |
|---|---|---|---|
| WT-LS | (α4[wt])3(β2[wt])2 | 83.4 ± 4.67 | 0.905 ± 0.03 |
| WT-HS | (α4[wt])2(β2[wt])3 | 0.924 ± 0.0656 | 0.924 ± 0.1 |
| WT-α5 | α5[wt](α4[wt]β2[wt])2 | 0.409 ± 0.0418 | 1.06 ± 0.1 |
| 9´ residue | (α4[L9´A])2(β2[wt])3 | 0.499 ± 0.032 | 1.43 ± 0.1 |
| 9´ residue | (α4[L9´S])2(β2[wt])3 | 0.0356 ± 0.00145 | 1.29 ± 0.06 |
| 9´ residue | (α4[L9´I])2(β2[wt])3 | 960. ± 502 | 0.489 ± 0.03 |
| 9´ residue | (α4[L9´T])2(β2[wt])3 | 0.282 ± 0.0233 | 1.28 ± 0.1 |
| 9´ residue | (α4[L9´Q])2(β2[wt])3 | 0.530 ± 0.0136 | 1.284 ± 0.03 |
| 9´ residue | (α4[L9´C])2(β2[wt])3 | 2.68 ± 0.311 | 0.710 ± 0.1 |
| 9´ residue | α4[L9´F])2(β2[wt])3 | 0.547 ± 0.0209 | 1.26 ± 0.05 |
| 9´ residue | (α4[L9´W])2(β2[wt])3 | 0.240 ± 0.00906 | 1.19 ± 0.05 |
| GABA | (α4[L376W])3(β2[wt])2 | 247 ± 99.4 | 0.772 ± 0.1 |
| MD | (α4[V356A])3(β2[wt])2 | 244 ± 113 | 0.843 ± 0.2 |
| GABA | (α4[wt])3(β2[M305W])2 | 180. ± 38.2 | 0.938 ± 0.1 |
| MD | (α4[wt])3(β2[W355L])2 | 171 ± 28.0 | 1.04 ± 0.1 |
| MD | (α4[W363L])3(β2[wt])2 | 159 ± 55.2 | 0.941 ± 0.2 |
| GABA | (α4[wt])3(β2[G364L])2 | 153 ± 164 | 0.728 ± 0.3 |
| TRPM8 | (α4[L383A])3(β2[wt])2 | 150. ± 7.67 | 0.919 ± 0.03 |
| TRPM8 | (α4[wt])3(β2[R299A])2 | 140. ± 64.7 | 0.510 ± 0.05 |
| MD | (α4[I357A])3(β2[wt])2 | 124 ± 19.1 | 0.957 ± 0.09 |
| MD | (α4[W363L])3(β2[W355L])2 | 122 ± 11.6 | 1.31 ± 0.1 |
| GABA | (α4[wt])3(β2[F306W])2 | 106 ± 12.2 | 1.07 ± 0.1 |
| ADNFLE | (α4[S6'F])3(β2[wt])2 | 105 ± 47.5 | 1.27 ± 0.1 |
| MD | (α4[F299A])3(β2[wt])2 | 83.2 ± 7.35 | 1.22 ± 0.1 |
| 9´ residue | (α4[L9´O-Me-Thr])3(β2[wt])2 | 61.3 ± 4.64 | 1.07 ± 0.07 |
| GABA | (α4[wt])3(β2[L282W])2 | 77.5 ± 6.44 | 1.01 ± 0.06 |
| GABA | (α4[wt])3(β2[V360W])2 | 71.2 ± 5.79 | 1.24 ± 0.1 |
| GABA | (α4[M288W])3(β2[wt])2 | 70.8 ± 11.1 | 0.913 ± 0.1 |
| GABA | (α4[wt])3(β2[M280W])2 | 57.8 ± 6.94 | 1.29 ± 0.2 |
| ADNFLE | (α4[wt])3(β2[V287L])2 | 38.0 ± 23.8 | 0.493 ± 0.07 |
| Docking | (α4[G281A, V236A])3(β2[wt])2 | 28.1 ± 4.84 | 0.998 ± 0.1 |
| Docking | (α4[wt])3(β2[I218A,L257A])2 | 26.6 ± 3.65 | 0.862 ± 0.08 |
| Docking + GABA | (α4[G281A, V236A])3(β2[M368W])2 | 24.3 ± 5.55 | 0.629 ± 0.07 |
| GABA | (α4[wt])3(β2[Y275W])2 | 21.1 ± 6.75 | 0.988 ± 0.1 |
| GABA | (α4[wt])3(β2[M368W])2 | 1.58 ± 0.508 | 0.493 ± 0.09 |
| MD + GABA | (α4[V356W])3(β2[M368W])2 | 1.46 ± 0.219 | 1.56 ± 0.3 |
| # 9´A | α5[V9´A](α4[wt]β2[wt])2 | 0.818 ± 0.0178 | 0.818 ± 0.03 |
| ADNFLE | (α4[S10'L])3(β2[wt])2 | 0.671 ± 0.112 | 0.512 ± 0.04 |
| MD + GABA | (α4[F299W, L376W])3(β2[M368W])2 | 0.511 ± 0.0293 | 1.39 ± 0.09 |
| MD | (α4[F299W])3(β2[wt])2 | 0.508 ± 0.0309 | 1.20 ± 0.07 |
| MD + GABA | (α4[L376W])3(β2[M368W])2 | 0.504 ± 0.0711 | 1.41 ± 0.2 |
| 9´ residue | (α4[L9´M])3(β2[wt])2 | 0.503 ± 0.0663 | 0.585 ± 0.04 |
| MD + GABA | (α4[F299W])3(β2[M368W])2 | 0.486 ± 0.0434 | 1.25 ± 0.1 |
| 9´ residue | (α4[L9´M])2(β2[wt])3 | 0.380 ± 0.0307 | 1.22 ± 0.1 |
| MD | (α4[V356W])3(β2[wt])2 | 0.360 ± 0.0188 | 1.65 ± 0.1 |
| 9´ residue + GABA | (α4[L9´A])3(β2[M368W])2 | 0.0230 ± 0.00200 | 0.900 ± 0.06 |
| # 9´A | (α4[wt])3(β2[L9´A])2 | 0.0695 ± 0.00353 | 1.32 ± 0.07 |
| 9´ residue | (α4[L9´T])3(β2[wt])2 | 0.0981 ± 0.00729 | 0.995 ± 0.06 |
| 9´ residue | (α4[L9´I])3(β2[wt])2 | 160. ± 50.3 | 0.866 ± 0.1 |
| 9´ residue | (α4[wt])2(β2[L9´A])3 | 0.0197 ± 0.00163 | 1.12 ± 0.09 |
| 9´ residue | (α4[L9´A])3(β2[wt])2 | 0.0159 ± 0.000691 | 1.16 ± 0.05 |
| 9´ residue + MD | (α4[L9´A])3(β2[V360W])2 | 0.00352 ± 0.000290 | 1.19 ± 0.1 |
| 9´ residue + MD | (α4[L9´A])3(β2[F328W])2 | 0.00318 ± 0.000237 | 1.13 ± 0.08 |
| ACh Binding Site | (α4[H116V,Q124F,T126L])3(β2[wt])2 | 76.3 ± 1.85 | 1.63 ± 0.06 |
| Intra-subunit | (α4[C233A])3(β2[wt])2 | 143 ± 17.2 | 0.99 ± 0.08 |
| Intra-subunit | (α4[T235A])3(β2[wt])2 | 43.6 ± 3.89 | 1.10 ± 0.09 |
| Intra-subunit | (α4[V236A])3(β2[wt])2 | 76.2 ± 14.2 | 1.31 ± 0.3 |
| Intra-subunit | (α4[G281F])3(β2[wt])2 | 33.5 ± 9.87 | 1.49 ± 0.6 |
| Intra-subunit | (α4[S232A])3(β2[wt])2 | 162 ± 20.6 | 0.993 ± 0.08 |
| Intra-subunit | (α4[G281A])3(β2[wt])2 | 19.9 ± 1.47 | 1.49 ± 0.2 |
| Near Cys loop | (α4[E52L])3(β2[wt])2 | 33.3 ± 2.71 | 1.27 ± 0.1 |
| Near Cys loop | (α4[E182Q])3(β2[wt])2 | 63.5 ± 4.54 | 1.66 ± 0.2 |
| Near Cys loop | (α4[wt])3(β2[S44A])2 | 86.1 ± 8.21 | 1.70 ± 0.3 |
| Channel pore | (α4[L265A])3(β2[wt])2 | 26.5 ± 6.58 | 0.864 ± 0.2 |
| Channel pore | (α4[wt])3(β2[L257A])2 | 0.316 ± 0.101 | 1.31 ± 0.4 |
| Interfacial | (α4[F286A])3(β2[wt])2 | 46.5 ± 8.45 | 0.962 ± 0.1 |
| Interfacial | (α4[wt])3(β2[I218A])2 | 119 ± 11.7 | 1.21 ± 0.1 |
| Interfacial | (α4[G281A])3(β2[I218A])2 | 23.8 ± 2.28 | 0.868 ± 0.5 |
WT-LS, Wild-type α4β2 receptor with the low sensitivity stoichiometry; WT-HS, wild-type α4β2 receptor with the high sensitivity stoichiometry; WT-α5, wild-type α4β2 receptor with α5 in the auxiliary position; 9´ residue, a mutant at the 9´ position to probe the effects of mutations at this site; GABA, analogous site for the menthol binding site in the GABA receptor; MD, molecular dynamics predicted position; TRPM8, analogous site for the menthol binding site on the TRPM8 receptor; ANDFLE, a mutant that is found in ADNFLE patients; Docking, position predicted by preliminary docking studies; #L9´A, mutant made to probe the effects that the number of 9´A mutations has on the effects of menthol; ACh binding site, the putative ACh binding site; Intra-subunit, site between subunits in the transmembrane domain of α4β2; Near Cys loop, site that is near the Cys loop of α4β2; Channel pore, site that is in the channel pore of α4β2; Interfacial, a site on the transmembrane domain of a single subunit that faces toward the other transmembrane domain helices on the same subunit.