Autoantibodies enhance agonist action and binding to cardiac muscarinic receptors in chronic Chagas' disease

Abstract

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.

Figure 1

Anti-M₂AChR specific antibody response in the sera from CChPs and NBD. (A) The relative titers of anti-M₂AChR el2 IgGs were characterized by serial limiting dilutions of the sera from the CChP and NBD individuals. The data were fitted with an empiric four-parameter sigmoidal function (see Materials and Methods section for further details). The values of the relative titers expressed as dilutions for the CChP and NBD sera are tabulated on the right. The means of the log₁₀ transforms of the relative titers from each group were compared with the unpaired t-test. (B) Recognition of M₂AChRs from porcine membrane preparations by specific anti M₂AChR-el2 affinity-purified CChP-IgGs. Lane 1: Specific anti M₂AChR-el2 monoclonal antibody. Lanes 2 and 4: Specific IgGs from samples CChP-02 and CChP-06, respectively. Lanes 3 and 5: Unbound fractions from the samples for CChP-02 and CChP-06. Lanes 6 and 7: Recovered and unbound fractions from NBD-03, respectively.

Figure 2

Electrocardiographic tracings showing the enhancement of the ACh-induced negative chronotropic effect by CChP sera in isolated rabbit hearts. ACh (1 × 10⁻⁷ to 1 × 10⁻⁵ mol/L in this figure) produced a dose-dependent reduction of the heart rate in isolated rabbit hearts (left column). The responses to 1 × 10⁻⁶ to 1 × 10⁻⁵ mol/L ACh were significantly augmented by the presence of 1:100 CChP sera (central column). The arrowheads on the record corresponding to an ACh concentration of 3 × 10⁻⁶ mol/L and CChP sera highlight nonconducting P waves characterizing an AV block. The effect of CChP sera on ACh response was completely blocked when the allosteric antagonist gallamine (100 μmol/L) was added (right column). This experiment was performed with the CChP-10 sera and this set of data is included in the graphs in Figure 3. The protocol used in the course of the experiments is shown in the bottom left-hand corner. The horizontal bar represents 20 s.

Figure 3

Enhancement of the negative chronotropic response to ACh by human chagasic antibodies in isolated rabbit hearts. (A) Dose-response curves of varying concentrations of ACh (1 × 10⁻⁸ to 1 × 10⁻⁴ mol/L) in the absence (control condition: open squares and continuous line) or presence (filled circles and dashed line) of 1:100 v/v of CChP or NBD sera (open circles and dashed line). (B) Dose-response curves of varying concentrations of ACh (1 × 10⁻⁸ to 1 × 10⁻⁴ mol/L) in the absence (control condition: open squares and continuous line; same set of experiments as in panel A) or presence of gallamine 100 μmol/L (open triangles and dashed line) or the combination of gallamine 100 μmol/L and 1:100 v/v CChP sera (filled triangles and dashed line). Each data point from the curves represents the means ± SEM from three to ten independent experiments with each of the sera from all the CChP or NBD individuals used in the study. The curves were generated from raw data as the shown in Figure 2. pEC₅₀ and Hill slopes were derived from nonlinear regression analysis (see Materials and Methods section for more details).

Figure 4

Effect of Chagasic antibodies on the ratio of the apparent affinities of [³H]-NMS and ACh at the cardiac M₂AChRs. Affinity ratio plots (A and B) were derived from quadruplicate observations of the binding of [³H]-NMS 400 pmol/L to cardiac M₂AChRs (raw data presented in panels C and D). These experiments were carried out in the absence (filled circles) or the presence (open circles) of ACh and in the presence of various concentrations of CChP (A and C) and NBD purified IgGs (B and D). (A, inset): A 25-residue peptide corresponding to the M₂AChR el2 was preincubated with the CChP-IgGs prior to the semiquantitative allosterism assay. Guanosine-5′-triphosphate (GTP) was added at a concentration of 0.2 mmol/L to assays performed in the presence of ACh. Data represent the means ± SEM of five independent experiments. CChP-02, CChP-03, CChP-06, CChP-07, and CChP-10 IgGs, and all NBD-IgGs were used in the distinct experiments. The estimated affinity ratios of acetylcholine (rACh) and [³H]-NMS (rNMS) in the presence of the highest concentration of CChP- or NBD-IgGs used in these assays were summarized in Table 1. The pK_D, log affinities of the antibodies for the free unbound receptors estimated from these assays are mentioned in the text.

Figure 5

Effect of human chagasic antibodies and gallamine on the dissociation rate of [³H]-NMS. (A) The effect of 8 × 10⁻⁷ mol/L CChP-IgG on the apparent rate constant K_off of the dissociation of [³H]-NMS 200 pmol/L was determined alone (filled circles and dashed line) and in combination (filled triangles and dashed line) with gallamine 1 μmol/L. The dissociation rate of [³H]-NMS (control condition, open squares, and continuous line) and the allosteric effect of gallamine 1 μmol/L alone (open triangles and continuous line) were also determined. The dissociation curves for the [³H]-NMS specific binding are expressed as percentages of the radioligand specific binding at the equilibrium. (B): The effects of CChP-IgG and gallamine on the [³H]-NMS dissociation from the experiments depicted in (A) are presented as percentages of [³H]-NMS K_off in the presence of these agents as compared to the control value (K_off in the absence of modulators = 0.01215 ± 0.003 min⁻¹). The data correspond to the means ± SEM of a representative experiment derived from four independent dissociation experiments in quadruplicates. The purified IgG fractions from CChP-03, CChP-05, CChP-07, and CChP-09 were used in these experiments. n.s., difference between the means not significant (P > 0.05), Gal, gallamine, ^*P< 0.05; one way ANOVA and Bonferroni post-test.

Figure 6

Competitive behavior of M₂AChRs-el2 affinity-purified human chagasic IgGs on the allosteric delay of W84 on [³H]-NMS dissociation rate. (A) Concentration effect curves for the W84 action on the K_off of the dissociation of [³H]-NMS 200 pmol/L in the absence or presence of 6.7 × 10⁻⁸, 1.3 × 10⁻⁷, and 2.0 × 10⁻⁷ mol/l of affinity-purified anti-M₂ CChP-IgGs. The K_off values of the [³H]-NMS dissociations in the presence of the different W84 concentrations were expressed as percentages of the K_off of the control assays. Data points represent the means ± SEM of K_off values derived from two to six dissociation curves performed in quadruplicates. Anti-M₂ CChP-IgGs purified from CChP 01, CChP-02, CChP-05, CChP-07, CChP-08, and CChP-10 were used in these experiments. (B) Schild plot derived from the data shown in (A) of the competitive effect of the affinity-purified CChP-IgGs on the W84 allosteric delay of [³H]-NMS dissociation. The line was fitted by linear regression giving a slope of 1 and the pK_D (affinity of the anti-M₂AChR el2 affinity-purified IgGs for the allosteric binding site) was estimated as 7.194. The K_off value under control conditions for this set of experiments was 0.01104 ± 0.003 min⁻¹. (C), Representative dissociation curves of the specific binding of [³H]-NMS 200 pmol/L expressed as a percentage of the radioligand binding at equilibrium in the absence or presence of either affinity-purified anti-M₂ CChP-IgG 2 × 10⁻⁷ mol/L or W84 3 μmol/L (refer to Materials and Methods section for more details).