NONEQUIVALENCE OF ALPHA-BUNGAROTOXIN BINDING-SITES IN THE NATIVE NICOTINIC RECEPTOR MOLECULE

In the native, membrane-bound form of the nicotinic acetylcholine receptor (M-AcChR) the two sites for the cholinergic antagonist α-bungarotoxin (α-BGT) have different binding properties. One site has high affinity, and the M-AcChR/α-BGT complexes thus formed dissociate very slowly, similar to the complexes formed with detergent-solubilized AcChR (S-AcChR). The second site has much lower affinity (KD ≈ 59 ± 35 nM) and forms quickly reversible complexes. The nondenaturing detergent Triton X-100 is known to solubilize the AcChR in a form unable, upon binding of cholinergic ligands, to open the ion channel and to become desensitized. Solubilization of the AcChR in Triton X-100 affects the binding properties of this second site and converts it to a high-affinity, slowly reversible site. Prolonged incubation of M-AcChR at 4 °C converts the low-affinity site to a high-affinity site similar to those observed in the presence of Triton X-100. Although the two sites have similar properties when the AcChR is solubilized in Triton X-100, their nonequivalence can be demonstrated by the effect on α-BGT binding of concanavalin A, which strongly reduces the association rate of one site only. The Bmax of α-BGT to either Triton-solubilized AcChR or M-AcChR is not affected by the presence of concanavalin A. Occupancy of the high-affinity, slowly reversible site in M-AcChR inhibits the Triton X-100 induced conversion to irreversibility of the second site. At difference with α-BGT, the long α-neurotoxin from Naja naja siamensis venom (α-NTX) binds with high affinity and in a very slowly reversible fashion to two sites in the M-AcChR (Conti-Tronconi & Raftery, 1986). We confirm here that Triton-solubilized AcChR or M-AcChR binds in a very slowly reversible fashion the same amount of α-NTX. In support of the contention that α-BGT binds “irreversibly” to one site only in the native M-AcChR, we found that when M-AcChR is saturated with radiolabeled α-BGT, addition of α-NTX markedly accelerates the dissociation of the bound α-BTX, presumably because occupancy of the second site by tightly bound α-NTX influences and decreases the affinity for α-BGT of the other site. The different properties of the two α-BGT binding sites in the native AcChR molecule support the possibility that these sites have different structural properties and that a sugar moiety is in close proximity to at least one such site. © 1990, American Chemical Society. All rights reserved.