IC50 values for mutant α4β2 nAChRs
| Motivation | Mutant | IC50 (µm (−)-menthol) ± SEM | Hill coefficient ± SEM | Maximum inhibition (%) |
|---|---|---|---|---|
| WT-LS | (α4[wt])3(β2[wt])2 | 33.7 ± 1.16 | 0.952 ± 0.04 | 95 |
| WT-HS | (α4[wt])2(β2[wt])3 | 33.9 ± 7.40 | 0.481 ± 0.1 | 71 |
| WT-α5 | α5[wt](α4[wt]β2[wt])2 | 51.0 ± 9.06 | 0.593 ± 0.2 | 53 |
| 9´ residue | (α4[L9´A])2(β2[wt])3 | 114 ± 152 | 0.779 ± 0.2 | 45 |
| 9´ residue | (α4[L9´S])2(β2[wt])3 | 514 ± 361 | 0.740 ± 0.1 | 65 |
| 9´ residue | (α4[L9´I])2(β2[wt])3 | 97.3 ± 38.5 | 0.836 ± 0.2 | 79 |
| 9´ residue | (α4[L9´T])2(β2[wt])3 | 717 ± 770. | 0.946 ± 0.3 | 59 |
| 9´ residue | (α4[L9´Q])2(β2[wt])3 | 165 ± 28.1 | 1.03 ± 0.1 | 81 |
| 9´ residue | (α4[L9´C])2(β2[wt])3 | 306 ± 173 | 0.744 ± 0.1 | 65 |
| 9´ residue | α4[L9´F])2(β2[wt])3 | 211 ± 75.6 | 1.01 ± 0.1 | 72 |
| 9´ residue | (α4[L9´W])2(β2[wt])3 | 28.8 ± 3.26 | 0.917 ± 0.1 | 87 |
| GABA | (α4[L376W])3(β2[wt])2 | 17.4 ± 43.2 | 2.38 ± 1.6 | 92 |
| MD | (α4[V356A])3(β2[wt])2 | 29.6 ± 11.0 | 0.631 ± 0.2 | 80 |
| GABA | (α4[wt])3(β2[M305W])2 | 70.6 ± 29.9 | 0.872 ± 0.2 | 90 |
| MD | (α4[wt])3(β2[W355L])2 | 28.8 ± 24.6 | 0.589 ± 0.4 | 80 |
| MD | (α4[W363L])3(β2[wt])2 | 42.7 ± 9.37 | 1.02 ± 0.2 | 96 |
| TRPM8 | (α4[L383A])3(β2[wt])2 | 27.9 ± 4.97 | 0.828 ± 0.1 | 96 |
| TRPM8 | (α4[wt])3(β2[R299A])2 | 52.0 ± 5.89 | 1.30 ± 0.2 | 96 |
| MD | (α4[I357A])3(β2[wt])2 | 31.4 ± 6.23 | 1.30 ± 0.3 | 94 |
| MD | (α4[W363L])3(β2[W355L])2 | 28.4 ± 6.96 | 0.866 ± 0.2 | 91 |
| ADNFLE | (α4[S6'F])3(β2[wt])2 | 32.3 ± 6.05 | 1.19 ± 0.3 | 95 |
| MD | (α4[F299A])3(β2[wt])2 | 20.4 ± 10.4 | 0.548 ± 0.2 | 93 |
| 9´ residue | (α4[L9´O-Me-Thr])3(β2[wt])2 | 135.9 ± 20.9 | 0.883 ± 0.7 | 79 |
| GABA | (α4[wt])3(β2[L282W])2 | 26.1 ± 5.40 | 0.883 ± 0.1 | 97 |
| GABA | (α4[wt])3(β2[V360W])2 | 16.7 ± 1.37 | 1.08 ± 0.09 | 96 |
| ADNFLE | (α4[wt])3(β2[V287L])2 | 17.0 ± 5.67 | 0.766 ± 0.2 | 89 |
| Docking | (α4[G281A, V236A])3(β2[wt])2 | 11.0 ± 0.767 | 0.958 ± 0.06 | 96 |
| Docking | (α4[wt])3(β2[I218A,L257A])2 | 23.0 ± 2.68 | 1.57 ± 0.3 | 99 |
| Docking + GABA | (α4[G281A, V236A])3(β2[M368W])2 | 59.5 ± 15.2 | 0.891 ± 0.2 | 95 |
| GABA | (α4[wt])3(β2[Y275W])2 | 21.7 ± 5.21 | 1.06 ± 0.2 | 97 |
| GABA | (α4[wt])3(β2[M368W])2 | 57.8 ± 8.07 | 0.865 ± 0.09 | 93 |
| # 9´A | α5[V9´A](α4[wt]β2[wt])2 | 1750 ± 3360 | 0.907 ± 0.3 | 36 |
| ADNFLE | (α4[S10'L])3(β2[wt])2 | 29.4 ± 4.32 | 1.39 ± 0.2 | 97 |
| MD + GABA | (α4[F299W, L376W])3(β2[M368W])2 | 107 ± 7.07 | 1.07 ± 0.06 | 94 |
| MD | (α4[F299W])3(β2[wt])2 | 32.5 ± 9.35 | 0.604 ± 0.09 | 87 |
| MD + GABA | (α4[L376W])3(β2[M368W])2 | 107 ± 29.6 | 0.890 ± 0.2 | 87 |
| 9´ residue | (α4[L9´M])3(β2[wt])2 | 39.2 ± 7.98 | 0.848 ± 0.09 | 83 |
| MD + GABA | (α4[F299W])3(β2[M368W])2 | 92.5 ± 36.8 | 0.777 ± 0.2 | 79 |
| 9´ residue | (α4[L9´M])2(β2[wt])3 | 26.6 ± 1.90 | 1.04 ± 0.07 | 91 |
| MD | (α4[V356W])3(β2[wt])2 | 52.6 ± 43.6 | 0.599 ± 0.2 | 70 |
| # 9´A | (α4[wt])3(β2[L9´A])2 | 1920 ± 9620 | 0.807 ± 0.6 | 38 |
| 9´ residue | (α4[L9´I])3(β2[wt])2 | 127 ± 29.1 | 0.947 ± 0.1 | 84 |
| 9´ residue | (α4[wt])2(β2[L9´A])3 | 3030 ± 2480 | 0.832 ± 0.8 | 31 |
| ACh binding site | (α4[H116V,Q124F,T126L])3(β2[wt])2 | 64.4 ± 2.09 | 1.20 ± 0.05 | 90 |
| Intra-subunit | (α4[C233A])3(β2[wt])2 | 46.1 ± 6.43 | 1.53 ± 0.3 | 98 |
| Intra-subunit | (α4[T235A])3(β2[wt])2 | 66.6 ± 8.03 | 1.60 ± 0.3 | 97 |
| Intra-subunit | (α4[V236A])3(β2[wt])2 | 58.9 ± 10.0 | 1.79 ± 0.4 | 91 |
| Intra-subunit | (α4[G281F])3(β2[wt])2 | 108 ± 19.1 | 1.14 ± 0.2 | 99 |
| Intra-subunit | (α4[S232A])3(β2[wt])2 | 92.0 ± 26.7 | 1.64 ± 0.7 | 92 |
| Intra-subunit | (α4[G281A])3(β2[wt])2 | 25.6 ± 1.43 | 1.49 ± 0.1 | 99 |
| Near Cys Loop | (α4[E52L])3(β2[wt])2 | 22.7 ± 1.67 | 0.969 ± 0.07 | 96 |
| Near Cys loop | (α4[E182Q])3(β2[wt])2 | 24.3 ± 0.593 | 1.23 ± 0.04 | 81 |
| Near Cys loop | (α4[wt])3(β2[S44A])2 | 59.9 ± 5.09 | 2.74 ± 0.6 | 94 |
| Channel pore | (α4[L265A])3(β2[wt])2 | 21.1 ± 3.06 | 1.16 ± 0.2 | 97 |
| Channel pore | (α4[wt])3(β2[L257A])2 | 74.8 ± 15.6 | 1.36 ± 0.3 | 98 |
| Interfacial | (α4[F286A])3(β2[wt])2 | 54.2 ± 19.9 | 0.927 ± 0.3 | 87 |
| Interfacial | (α4[wt])3(β2[I218A])2 | 60.9 ± 7.63 | 1.12 ± 0.1 | 99 |
| Interfacial | (α4[G281A])3(β2[I218A])2 | 23.8 ± 1.58 | 2.25 ± 0.3 | 99 |
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.