Table 1

Ligand binding properties of AChRs immunoisolated from brain and transfected HEK 293 cells

Competing ligandIC50 for competition with [3H]epibatidine
Brain mAb 35 AChRsBrain mAb 289 AChRsHEK 293 α4/β2/α5 AChRsHEK 293 α4/β2 AChRs
μm
ACh0.31  ± 0.010.20  ± 0.080.55  ± 0.070.15  ± 0.02
Nicotine0.19  ± 0.060.07  ± 0.020.22  ± 0.020.06  ± 0.01
Cytisine0.06  ± 0.050.05  ± 0.040.02  ± 0.010.004  ± 0.003
Lobeline4.1  ± 1.71.5  ± 1.0N.D.N.D.
DHBE37.8  ± 4.220.2  ± 1.338.8  ± 5.59.0  ± 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.