Hernandez Ciria C, Nascimento Jose H, Chaves Elen A, Costa Patricia C, Masuda Masako O, Kurtenbach Eleonora, Campos DE Carvalho Antonio C, Gimenez Luis E
Laboratorio de Cardiologia Celular e Molecular, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
J Recept Signal Transduct Res. 2008;28(4):375-401. doi: 10.1080/10799890802262319.
Chronic Chagasic patient immunoglobulins (CChP-IgGs) recognize an acidic amino acid cluster at the second extracellular loop (el2) of cardiac M(2)-muscarinic acetylcholine receptors (M(2)AChRs). These residues correspond to a common binding site for various allosteric agents. We characterized the nature of the M(2)AChR/CChP-IgG interaction in functional and radioligand binding experiments applying the same mainstream strategies previously used for the characterization of other allosteric agents. Dose-response curves of acetylcholine effect on heart rate were constructed with data from isolated heart experiments in the presence of CChP or normal blood donor (NBD) sera. In these experiments, CChP sera but not NBD sera increased the efficacy of agonist action by augmenting the onset of bradyarrhythmias and inducing a Hill slope of 2.5. This effect was blocked by gallamine, an M(2)AChR allosteric antagonist. Correspondingly, CChP-IgGs increased acetylcholine affinity twofold and showed negative cooperativity for [(3)H]-N-methyl scopolamine ([(3)H]-NMS) in allosterism binding assays. A peptide corresponding to the M(2)AChR-el2 blocked this effect. Furthermore, dissociation assays showed that the effect of gallamine on the [(3)H]-NMS off-rate was reverted by CChP-IgGs. Finally, concentration-effect curves for the allosteric delay of W84 on [(3)H]-NMS dissociation right shifted from an IC(50) of 33 nmol/L to 78 nmol/L, 992 nmol/L, and 1670 nmol/L in the presence of 6.7 x 10(- 8), 1.33 x 10(- 7), and 2.0 x 10(- 7) mol/L of anti-el2 affinity-purified CChP-IgGs. Taken together, these findings confirmed a competitive interplay of these ligands at the common allosteric site and revealed the novel allosteric nature of the interaction of CChP-IgGs at the M(2)AChRs as a positive cooperativity effect on acetylcholine action.
慢性恰加斯病患者免疫球蛋白（CChP-IgGs）可识别心脏M2型毒蕈碱型乙酰胆碱受体（M2AChRs）第二个细胞外环（el2）处的酸性氨基酸簇。这些残基对应于各种变构剂的共同结合位点。我们在功能和放射性配体结合实验中，采用与之前用于表征其他变构剂相同的主流策略，对M2AChR/CChP-IgG相互作用的性质进行了表征。利用在存在CChP或正常献血者（NBD）血清的情况下进行的离体心脏实验数据，构建了乙酰胆碱对心率影响的剂量反应曲线。在这些实验中，CChP血清而非NBD血清通过增强缓慢性心律失常的发作并诱导2.5的希尔斜率，提高了激动剂作用的效能。这种效应被M2AChR变构拮抗剂加拉明阻断。相应地，在变构结合试验中，CChP-IgGs使乙酰胆碱亲和力提高了两倍，并对[3H]-N-甲基东莨菪碱（[3H]-NMS）表现出负协同性。一个与M2AChR-el2对应的肽段可阻断这种效应。此外，解离试验表明，加拉明对[3H]-NMS解离速率的影响可被CChP-IgGs逆转。最后，在存在6.7×10-8、1.33×10-7和2.0×10-7mol/L抗el2亲和纯化的CChP-IgGs的情况下，W84对[3H]-NMS解离变构延迟的浓度效应曲线的IC50从33nmol/L右移至78nmol/L、992nmol/L和1670nmol/L。综上所述，这些发现证实了这些配体在共同变构位点的竞争性相互作用，并揭示了CChP-IgGs在M2AChRs上相互作用的新型变构性质，即对乙酰胆碱作用具有正协同效应。
