Tamamizu S, Butler D H, Lasalde J A, McNamee M G
Section of Molecular and Cellular Biology, University of California, Davis 95616, USA.
Biochemistry. 1996 Sep 10;35(36):11773-81. doi: 10.1021/bi960369u.
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 [alpha-bungarotoxin (alpha-BTX) and curare] binding, these results indicate that mAbs could be used to map functional and structural domains.
采用膜片钳技术和光亲和标记技术，研究单克隆抗体（mAb）与加州电鳐烟碱型乙酰胆碱受体（nAChR）结合对其功能的影响。本文检测的大鼠抗加州电鳐nAChR单克隆抗体已知可抑制配体与高亲和力结合位点（单克隆抗体247）或高、低亲和力结合位点（单克隆抗体370和单克隆抗体387）的结合[米霍维洛维奇，M.和里奇曼，D.P.（1984年）《生物化学杂志》259，15051 - 15059；米霍维洛维奇，M.和里奇曼，D.P.（1987年）《生物化学杂志》262，4978 - 4986]。单通道分析表明，单克隆抗体247及其Fab片段可抑制nAChR离子通道的开放，尽管它们对通过光反应性探针3 -（三氟甲基）- 3 -（间-[125I]碘苯基）重氮甲烷（[125I]-TID）标记减少程度监测的从静息态到脱敏态的结构转变没有影响。在单克隆抗体387存在的情况下，nAChR单通道幅度降低了20%，而Fab 387完全抑制通道开放。[125I - TID]标记研究表明，单克隆抗体387 - nAChR和Fab 387 - nAChR复合物能够经历静息态和脱敏态之间的转变。这一结果证实，nAChR在没有预先通道开放的情况下也能呈现脱敏态。此外，识别nAChR主要免疫原性区域（MIR）的单克隆抗体35和单克隆抗体132以及单克隆抗体37对单通道行为或标记模式均无影响。结合关于它们的表位及其对激动剂（氨甲酰胆碱）和拮抗剂[α - 银环蛇毒素（α - BTX）和箭毒]结合的影响的表征结果，这些结果表明单克隆抗体可用于绘制功能和结构域图谱。
