Autoantibodies enhance agonist action and binding to cardiac muscarinic receptors in chronic Chagas' disease.
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.
Hernandez
Ciria C
CC
Laboratorio de Cardiologia Celular e Molecular, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.
Nascimento
Jose H
JH
Chaves
Elen A
EA
Costa
Patricia C
PC
Masuda
Masako O
MO
Kurtenbach
Eleonora
E
Campos DE Carvalho
Antonio C
AC
Gimenez
Luis E
LE
D000109:Acetylcholine / Q000378:metabolism / Q000494:pharmacology*; D000495:Allosteric Site; D000818:Animals; D001323:Autoantibodies / Q000276:immunology / Q000378:metabolism*; D014355:Chagas Disease / Q000276:immunology* / Q000378:metabolism; D018678:Cholinergic Agents / Q000494:pharmacology; D005703:Gallamine Triethiodide / Q000494:pharmacology; D006321:Heart / Q000187:drug effects; D006339:Heart Rate / Q000187:drug effects; D006801:Humans; D008024:Ligands; D009206:Myocardium / Q000378:metabolism*; D003473:Neuromuscular Nondepolarizing Agents / Q000494:pharmacology; D018733:Nicotinic Antagonists / Q000494:pharmacology; D011817:Rabbits; D043585:Receptor, Muscarinic M2 / Q000819:agonists* / Q000187:drug effects / Q000378:metabolism; D013552:Swine
D001323:Autoantibodies; D018678:Cholinergic Agents; D008024:Ligands; D003473:Neuromuscular Nondepolarizing Agents; D018733:Nicotinic Antagonists; D043585:Receptor, Muscarinic M2; D000109:Acetylcholine; D005703:Gallamine Triethiodide