Effects of Antibody Binding on Structural Transitions of the Nicotinic Acetylcholine Receptor

Abstract

Patch-clamping and photoaffinity-labeling techniques were used to study the effects of binding of monoclonal antibodies (mAbs) on the function of Torpedo californica nicotinic acetylcholine receptor (nAChR). The rat anti-Torpedo nAChR mAbs examined here are known to inhibit ligand binding to either the high-affinity (mAb 247) or both the high- and low−affinity binding sites (mAb 370 and mAb 387) [Mihovilovic, M. & Richman, D. P. (1984) J. Biol. Chem. 259, 15051−15059; Mihovilovic, M., & Richman, D. P. (1987) J. Biol. Chem. 262, 4978−4986]. Single-channel analysis shows that mAb 247 and the Fab fragment of mAb 247 inhibit the opening of the nAChR ion channel, although they have no effects on the structural transition from the resting to desensitized state as monitored by the extent of decreased labeling by the photoreactive probe 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine ([125I]TID). In the presence of mAb 387, the nAChR single-channel amplitude was decreased by 20%, whereas Fab 387 completely inhibited channel opening. [125I-TID]-labeling studies suggest that the mAb 387−nAChR and Fab 387−nAChR complexes are able to undergo the transition between resting and desensitized states. This result confirms that the nAChR can assume a desensitized state without prior channel opening. In addition, mAb 35 and mAb 132, which recognize the main immunogenic region (MIR) of the nAChR, and mAb 370 do not alter either single-channel behavior or labeling patterns. Combining the results from characterization with respect to their epitopes and their effects on agonist (carbamylcholine) and antagonist [α-bungarotoxin (α-BTX) and curare] binding, these results indicate that mAbs could be used to map functional and structural domains.

Structural insights into the molecular mechanisms of myasthenia gravis and their therapeutic implications

“…This model suggests that Fab35, by binding to the extruding tip of an α1 subunit, makes no direct contact to the neighboring subunits. Moreover, Fab35 binds nAChR α1 at a site that is far away from the ligand-binding site, consistent with the observation that MIR-directed antibodies generally do not affect the channel function (Tzartos et al, 1981; Gomez et al, 1981; Tamamizu et al, 1996).

10.7554/eLife.23043.013Figure 6.Modeling the binding of Fab35 to a nAChR pentamer.( a ) Superposition of the Fab35/nAChR α1 ECD/α-Btx ternary complex on one subunit of the α7/AChBP chimera pentamer (blue) (PDB ID, 3SQ9) (Li et al, 2011) using the C α backbone of ECDs as the reference.

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Real Time Ligand-Induced Motion Mappings of AChBP and nAChR Using X-ray Single Molecule Tracking

“…To monitor the internal motion of the proteins from multiple orientations, we controlled the position of the nanocrystal labels on the two protein molecules. The nanocrystal was immobilised on the C-terminus of AChBP via a Met-tag (Figure 1a) and on the F(ab')2 fragment of the monoclonal antibody (mAb) 35, which recognises the extracellular side of the nAChR α-subunit18 (Figure 1b). The antibody used has been shown to have no adverse effect on the ligand binding or channel gating of nAChR.…”

Localization of agonist and competitive antagonist binding sites on nicotinic acetylcholine receptors

“…(3) The dierences between neuronal AChR subtypes (for a review see Lindstrom, 1986); (4) AChR ion channel activation (Bu¯er et al, 1996(Bu¯er et al, , 1998. ( 5) The AChR conformational changes elicited by the same antibodies (Tamamizu et al, 1996;Valenzuela et al, 1994b). For example, monoclonal antibodies raised against native Torpedo (Conti-Tronconi et al, 1990;Watters and Maelicke, 1983) and mouse (Goldberg et al, 1983;Mochly-Rosen and Fuchs, 1981) AChRs, as well as polyclonal antibodies raised against native human AChRs, are mutually exclusive with a-BTx and cholinergic ligand binding.…”