Fig. 9.
Pharmacological analysis of [3H]cytisine and [125I]α-bungarotoxin binding in layers 8 and 9 of the adult optic tectum. A: Saturation binding analysis of [3H]cytisine binding. The apparent dissociation constant (KD) was 0.47 ± 0.04 nM with a maximum binding capacity (Bmax) of 40.4 ± 0.71 fmol/mg. The data were best fit by an equation for binding to a single site. The inset is a Scatchard plot of the same data that yielded similar binding constants. B: Competition binding analysis of [3H]cytisine (5 nM) binding. Dihydro-β-erythroidine (DHBE), which is a competitive antagonist at non-α7 nicotinic acetylcholine receptors (nAChRs) in mammalian systems, competed with [3H]cytisine at two sites. The high affinity DHBE site demonstrated an inhibition constant (Ki) of 40.3 pM, whereas the low-affinity site had a Ki of 24.5 nM. Methyllycaconitine (MLA) and unlabeled α-bungarotoxin (BtX) are both high-affinity, competitive antagonists of the α7 nAChR in mammalian systems and did not compete with [3H]cytisine binding until micromolar concentrations of competitor were present. MLA had a Ki of 3.29 μM. Unlabeled BtX was unable to reduce [3H]cytisine binding to background levels and demonstrated a Ki of 12.5 μM. C: Saturation analysis of [125I]α-bungarotoxin binding demonstrated a KD = 0.81 ± 0.19 nM and a Bmax = 1.86 ± 0.10 fmol/mg. The data fit best to an equation for single-site binding, and Scatchard analysis (inset) again produced similar binding values. D: Competition of MLA, unlabeled cytisine, and DHBE against [125I]α-bungarotoxin (2.5 nM) binding. The competition curves fit to equations that produced the following inhibition constants: MLA = 2.3 pM and 2.6 nM, unlabeled cytisine = 17.6 nM, DHBE = 21.1 μ M. All points are the mean ± S.E.M. of specific binding (n = 4 or 5) for all graphs.