Competing ligand | IC50 for competition with [3H]epibatidine | |||
---|---|---|---|---|
Brain mAb 35 AChRs | Brain mAb 289 AChRs | HEK 293 α4/β2/α5 AChRs | HEK 293 α4/β2 AChRs | |
μm | ||||
ACh | 0.31 ± 0.01 | 0.20 ± 0.08 | 0.55 ± 0.07 | 0.15 ± 0.02 |
Nicotine | 0.19 ± 0.06 | 0.07 ± 0.02 | 0.22 ± 0.02 | 0.06 ± 0.01 |
Cytisine | 0.06 ± 0.05 | 0.05 ± 0.04 | 0.02 ± 0.01 | 0.004 ± 0.003 |
Lobeline | 4.1 ± 1.7 | 1.5 ± 1.0 | N.D. | N.D. |
DHBE | 37.8 ± 4.2 | 20.2 ± 1.3 | 38.8 ± 5.5 | 9.0 ± 3.1 |
AChRs from brain and HEK 293 cell extracts were immunotethered with subunit-specific mAbs in solid-phase immunoprecipitation assays and subjected to competition binding with [3H]epibatidine and the indicated ligands as described in Fig. 4. Brain mAb 35 AChRs were isolated from brain extracts with mAb 35; brain mAb 289 AChRS were isolated from brain extracts with mAb 289 after depletion with mAb 35; HEK 293 α4/β2/α5 AChRs were isolated from HEK 293 cells transfected with the α4, β2, and α5 genes and using mAb 35; and HEK 293 α4/β2 AChRs were isolated from HEK 293 cells transfected with the α4 and β2 genes and using mAb 270. Values are mean ± standard error of three or four separate determinations. Few differences exist among them; HEK 293 α4/β2/α5 AChRs seem to be indistinguishable from brain mAb 35 AChRs, whereas HEK 293 α4/β2 AChRs are very similar to brain mAb 289 AChRs.
DHBE, dihydro-β-erythroidine.