EFFECTS OF ANTIBODY-BINDING ON STRUCTURAL TRANSITIONS OF THE NICOTINIC ACETYLCHOLINE-RECEPTOR

Patch-clamping and photoaffinity-labeling techniques were used to stud y the effects of binding of monoclonal antibodies (mAbs) on the functi on of Torpedo californica nicotinic acetylcholine receptor (nAChR). Th e rat anti-Torpedo nAChR mAbs examined here are known to inhibit ligan d binding to either the high-affinity (mAb 247) or both the high- and low-affinity binding sites (mAb 370 and mAb 387) [Mihovilovic, M. & Ri chman, 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 t he opening of the nAChR ion channel, although they have no effects on the structural transition from the resting to desensitized state as mo nitored by the extent of decreased labeling by the photoreactive probe (trifluoromethyl)-3-(m-[I-125]iodophenyl)diazirine ([I-125]-TID). In the presence of mAb 387, the nAChR single-channel amplitude was decrea sed by 20%, whereas Fab 387 completely inhibited channel opening. [I-1 25-TID]-labeling studies suggest that the mAb 387-nAChR and Fab 387-nA ChR complexes are able to undergo the transition between resting and d esensitized states. This result confirms that the nAChR can assume a d esensitized state without prior channel opening. In addition, mAb 35 a nd mAb 132, which recognize the main immunogenic region (MIR) of the n AChR, and mAb 370 do not alter either single-channel behavior or label ing patterns. Combining the results from characterization with respect to their epitopes and their effects on agonist (carbamylcholine) and antagonist [alpha-bungarotoxin (alpha-BTX) and curare] binding, these results indicate that mAbs could be used to map functional and structu ral domains.