Full and partial agonists evoke distinct structural changes in opening the muscle acetylcholine receptor channel

Abstract

The muscle acetylcholine (ACh) receptor transduces a chemical into an electrical signal, but the efficiency of transduction, or efficacy, depends on the particular agonist. It is often presumed that full and partial agonists elicit the same structural changes after occupancy of their binding sites but with differing speed and efficiency. In this study, we tested the alternative hypothesis that full and partial agonists elicit distinct structural changes. To probe structural changes, we substituted cysteines for pairs of residues that are juxtaposed in the three-dimensional structure and recorded agonist-elicited single-channel currents before and after the addition of an oxidizing reagent. The results revealed multiple cysteine pairs for which agonist-elicited channel opening changes after oxidative cross-linking. Moreover, we found that the identity of the agonist determined whether cross-linking affects channel opening. For the αD97C/αY127C pair at the principal face of the subunit, cross-linking markedly suppressed channel opening by full but not partial agonists. Conversely, for the αD97C/αK125C pair, cross-linking impaired channel opening by the weak agonist choline but not other full or partial agonists. For the αT51C/αK125C pair, cross-linking enhanced channel opening by the full agonist ACh but not other full or partial agonists. At the complementary face of the subunit, cross-linking between pairs within the same β hairpin suppressed channel opening by ACh, whereas cross-linking between pairs from adjacent β hairpins was without effect for all agonists. In each case, the effects of cross-linking were reversed after addition of a reducing reagent, and receptors with single cysteine substitutions remained unaltered after addition of either oxidizing or reducing reagents. These findings show that, in the course of opening the receptor channel, different agonists elicit distinct structural changes.

Figure 1.

Structural bases for generating pairwise cysteine substitutions. X-ray structure of the heteromeric α₄β₂ nicotinic AChR (Morales-Perez et al., 2016; PDB accession no. 5KXI) with one of the α₄ subunits shown in secondary structure representation (purple). Insets show residues subjected to cysteine substitution from the principal (right) and complementary (left) faces of the α₁ subunit (Dellisanti et al., 2007; PDB accession no. 2QCI).

Figure 2.

Oxidative cross-linking of AChRs with cysteine substituted for αD97 and αY127 suppresses channel opening by ACh. (A) Single-channel currents from the αD97C/αY127C mutant receptor were recorded in the presence of the indicated concentration of ACh under control conditions (top trace), after pretreatment with H₂O₂ (middle trace), or after successive treatment with H₂O₂ and DTT (bottom trace); the middle and bottom traces were obtained from the same membrane patch. See Materials and methods for the procedure to pretreat and remove H₂O₂ and apply DTT. Individual clusters of channel openings, all from the same AChR channel, are shown at a bandwidth of 5 kHz (Gaussian response). To the right of each trace is a histogram of open probability of individual clusters collected over the entire recording; clusters were defined by using an intercluster closed interval determined from a histogram of all closed intervals (Materials and methods). (B) Recordings and histograms of cluster open probability for the wild-type adult human receptor, as in A.

Figure 3.

Receptors in which cysteine was substituted for a single residue are unaffected by H₂O₂ or DTT. (A) Single-channel currents from the αD97C mutant receptor were recorded in the presence of the indicated concentration of ACh under control conditions (top trace), after pretreatment with H₂O₂ (middle trace), or after successive treatment with H₂O₂ and DTT (bottom trace). (B) Single-channel currents from the αY127C mutant receptor were recorded in the presence of the indicated concentration of ACh under control conditions (top trace), after pretreatment with H₂O₂ (middle trace), or after successive treatment with H₂O₂ and DTT (bottom trace). To the right of each trace is a histogram of open probability of individual clusters collected over the entire recording, as in Fig. 2.

Figure 4.

Preincubation with ACh prevents oxidative cross-linking of the αD97C/αY127C mutant. (A) Single-channel currents from the αD97C/αY127C mutant receptor were recorded under control conditions (top trace), or after preincubation with H₂O₂ (bottom trace). (B) Single-channel currents were recorded in the presence of ACh under control conditions (top trace), or after successive preincubation with ACh and then H₂O₂ in the continued presence of ACh (bottom trace). The concentration of ACh was 10 µM during the preincubation and subsequent single-channel recording. To the right of each trace is a histogram of cluster open probability, as in Fig. 2.

Figure 5.

Oxidative cross-linking of AChRs with cysteine substituted for αD97 and αY127 suppresses channel opening by SubCho but not PIP. (A) Single-channel currents from the αD97C/αY127C mutant receptor in the presence of SubCho were recorded under control conditions (top trace), after pretreatment with H₂O₂ (middle trace), or after successive treatment with H₂O₂ and DTT (bottom trace); the middle and bottom traces were obtained from the same membrane patch. Histograms of cluster open probability were generated as in Fig. 2. (B) Same as in A, but with PIP as the agonist.

Figure 6.

Oxidative cross-linking of AChRs with cysteine substituted for αD97 and αK125 suppresses channel opening by Cho. (A) Single-channel currents from the αD97C/αK125C mutant receptor were recorded in the presence of Cho under control conditions (top trace), after pretreatment with H₂O₂ (middle trace), or after successive treatment with H₂O₂ and DTT (bottom trace); the middle and bottom traces were obtained from the same membrane patch. Individual clusters of channel openings, all from the same AChR channel, are shown at a bandwidth of 5 kHz (Gaussian response). To the right of each trace is a histogram of cluster open probability as in Fig. 2. (B) Recordings and analyses for the wild-type adult human AChR activated by Cho, as in A.

Figure 7.

Oxidative cross-linking of AChRs with cysteine substituted for αT51 and αK125 enhances channel opening by ACh but not other agonists. (A) Single-channel currents from the αD97C/αK125C mutant receptor were recorded in the presence of ACh under control conditions (top trace), after pretreatment with H₂O₂ (middle trace), or after successive treatment with H₂O₂ and DTT (bottom trace); the middle and bottom traces were obtained from the same membrane patch. Individual clusters of channel openings, all from the same AChR channel, are shown at a bandwidth of 5 kHz (Gaussian response). To the right of each trace is a histogram of cluster open probability as in Fig. 2. (B) Same as in A, but with PIP as the agonist.

Figure 8.

Oxidative cross-linking of AChRs with cysteine substituted for αL109 and αT117 suppresses channel opening by ACh but not the partial agonist PIP. (A) Single-channel currents from the αL109/αT117 mutant receptor were recorded in the presence of ACh under control conditions (top trace), after pretreatment with H₂O₂ (middle trace), or after successive treatment with H₂O₂ and DTT (bottom trace); the middle and bottom traces were obtained from the same membrane patch. Individual clusters of channel openings, all from the same AChR channel, are shown at a bandwidth of 5 kHz (Gaussian response). To the right of each trace is a histogram of cluster open probability as in Fig. 2. (B) Same as in A, but with PIP as the agonist.

Figure 9.

Oxidative cross-linking of AChRs with cysteine substituted for αT32 and αQ59 selectively suppresses channel opening by ACh but not other agonists. (A) Single-channel currents from the αT32/αQ59 mutant receptor were recorded in the presence of ACh under control conditions (top trace), after pretreatment with H₂O₂ (middle trace), or after successive treatment with H₂O₂ and DTT (bottom trace); the middle and bottom traces were obtained from the same membrane patch. Individual clusters of channel openings, all from the same AChR channel, are shown at a bandwidth of 5 kHz (Gaussian response). To the right of each trace is a histogram of cluster open probability as in Fig. 2. (B) Same as in A, but with SubCho as the agonist.