Ligand | Ki at nAChR Subtype, nM (Ratio to Ki at α4β2) | |||
---|---|---|---|---|
α4β2 | α3β4x | α7 | Muscle | |
(±)-IPH | 0.027 ± 0.004 (1) | 0.11 ± 0.04 (4) | 30 ± 3 (1,100) | 6.5 ± 0.1 (240) |
(±)-Epibatidine | 0.008 ± 0.001 (1) | 0.049 ± 0.02 (6) | 4.0 ± 0.5 (500) | 7.5 ± 0.5 (900) |
(−)-Nicotine | 0.84 ± 0.13 (1) | 100 ± 20 (120) | 130 ± 10 (150) | 1000 ± 100 (1,200) |
(−)-Cytisine | 0.18 ± 0.02 (1) | 54 ± 9 (300) | 260 ± 20 (1,400) | 190 ± 20 (1,100) |
A-85380 | 0.017 ± 0.002 (1) | 14 ± 2 (800) | 17 ± 2 (1,000) | 320 ± 20 (19,000) |
5-Iodo-A-85380 | 0.010 ± 0.001 (1) | 51 ± 5 (5,000) | 250 ± 20 (25,000) | 1400 ± 200 (140,000) |
In assays of the α4β2 subtype, rat brain P2 membrane fractions (200 μg of protein) were incubated in a total volume of 0.5 ml with 0.5 nM (±)-[3H]epibatidine and 9 to 11 concentrations of competitors for 1.5 h at 22°C. In assays of the nAChRs containing α3 and β4 subunits (α3β4x), total membrane fractions of rat adrenal glands (250 μg of protein) were incubated in a total volume of 1 ml with 0.4 nM (±)-[3H]epibatidine and five to nine concentrations of competitors for 1.5 h at 22°C. In assays of the α7 subtype, rat brain P2 membrane fractions (100 μg of protein) were incubated in a total volume of 0.1 ml with 2 nM125I-α-bungarotoxin and 9 to 11 concentrations of competitors for 3 h at 22°C. In assays of the muscle subtype, total membrane fractions from T. californica electric organ (0.1 μg of protein) were incubated in a total volume of 0.1 ml with 2 nM125I-α-bungarotoxin and 9 to 11 concentrations of competitors for 1.5 h at 22°C. The inhibition constants (K i values) were calculated by the Cheng-Prusoff equation from measured IC50 values using the followingK d values: 10 and 55 pM for (±)-[3H]epibatidine binding in rat forebrain (α4β2) and in rat adrenal glands (α3β4x), respectively; and 1.5 and 2.3 nM for125I-α-bungarotoxin binding in rat forebrain (α7) and inT. californica electric organ (α1β1δɛ), respectively. These K d values were obtained from three to six separate saturation assays per constant. Data represent means ± S.E. obtained from three to seven competition assays per constant.