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Nicotinic receptor interloop proline anchors beta1-beta2 and Cys loops in coupling agonist binding to channel gating.

Lee WY, Free CR, Sine SM - J. Gen. Physiol. (2008)

Bottom Line: We find that in contributing to channel gating, alphaVal 46 and alphaVal 132 of the signature Cys loop couple energetically to alphaPro 272.Furthermore, these residues are optimized in both their size and hydrophobicity to mediate rapid and efficient channel gating, suggesting naturally occurring substitutions at these positions enable a diverse range of gating rate constants among the Cys-loop receptor superfamily.The overall results indicate that alphaPro 272 functionally couples to flanking Val residues extending from the beta1-beta2 and Cys loops within the ACh binding to channel opening transduction pathway.

View Article: PubMed Central - PubMed

Affiliation: Receptor Biology Laboratory, Department of Physiology and Biomedical Engineering, Mayo Clinic College of Medicine, Rochester, MN 55905, USA.

ABSTRACT
Nicotinic acetylcholine receptors (AChRs) mediate rapid excitatory synaptic transmission throughout the peripheral and central nervous systems. They transduce binding of nerve-released ACh into opening of an intrinsic channel, yet the structural basis underlying transduction is not fully understood. Previous studies revealed a principal transduction pathway in which alphaArg 209 of the pre-M1 domain and alphaGlu 45 of the beta1-beta2 loop functionally link the two regions, positioning alphaVal 46 of the beta1-beta2 loop in a cavity formed by alphaPro 272 through alphaSer 269 of the M2-M3 loop. Here we investigate contributions of residues within and proximal to this pathway using single-channel kinetic analysis, site-directed mutagenesis, and thermodynamic mutant cycle analysis. We find that in contributing to channel gating, alphaVal 46 and alphaVal 132 of the signature Cys loop couple energetically to alphaPro 272. Furthermore, these residues are optimized in both their size and hydrophobicity to mediate rapid and efficient channel gating, suggesting naturally occurring substitutions at these positions enable a diverse range of gating rate constants among the Cys-loop receptor superfamily. The overall results indicate that alphaPro 272 functionally couples to flanking Val residues extending from the beta1-beta2 and Cys loops within the ACh binding to channel opening transduction pathway.

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REFER analysis of mutations of αPro 272, αVal 132, and αVal 46. Channel opening rate constant is plotted against the channel gating equilibrium constant for the indicated substitutions of each residue. The values plotted are given in Tables I and II. Linear regression yielded the lines shown with the indicated slopes Φ and standard deviations.
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fig9: REFER analysis of mutations of αPro 272, αVal 132, and αVal 46. Channel opening rate constant is plotted against the channel gating equilibrium constant for the indicated substitutions of each residue. The values plotted are given in Tables I and II. Linear regression yielded the lines shown with the indicated slopes Φ and standard deviations.

Mentions: The timing of structural changes in the binding-gating transduction process has been inferred from rate-equilibrium free energy relationships (REFER) in which the channel opening rate constant is plotted against the channel gating equilibrium constant for a series of substitutions of a given residue. In many instances a log–log plot of these quantities is linear, and the slope (Φ) is interpreted as an index of the open- versus closed-state conformation of the specified residues in the gating transition state (Auerbach, 2007). Values of Φ approaching 1 suggest the conformation of the transition state is open-like, whereas values approaching 0 suggest the conformation of the transition state is closed-like. Our data for human muscle AChRs activated by ACh show Φ values for substitutions of αPro 272, αVal 46, and αVal 132, ranging from 0.87 to 0.93 (Fig. 9). If Φ reflects the relative time at which the transition state is achieved, the similar Φ values suggest the three residues move nearly simultaneously as they approach an open-like transition state. Thus, if the Torpedo AChR structural model accurately depicts the interloop triad in the resting closed state, our findings indicate that the three residues maintain physical continuity up to the transition state. This conclusion is tentative because although our REFER plots for αP272 and αVal 46 are linear, the plot for αVal 132 is curved. On the other hand, REFER analyses of mouse muscle AChRs activated by either ACh or choline appeared linear and yielded statistically different values (αPro 272 [Φ = 0.62], αVal 46 [Φ = 0.81], and αVal 132 [Φ = 0.75]), suggesting somewhat asynchronous movement of the three residues (Chakrapani, et al., 2004; Jha, et al., 2007).


Nicotinic receptor interloop proline anchors beta1-beta2 and Cys loops in coupling agonist binding to channel gating.

Lee WY, Free CR, Sine SM - J. Gen. Physiol. (2008)

REFER analysis of mutations of αPro 272, αVal 132, and αVal 46. Channel opening rate constant is plotted against the channel gating equilibrium constant for the indicated substitutions of each residue. The values plotted are given in Tables I and II. Linear regression yielded the lines shown with the indicated slopes Φ and standard deviations.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2483337&req=5

fig9: REFER analysis of mutations of αPro 272, αVal 132, and αVal 46. Channel opening rate constant is plotted against the channel gating equilibrium constant for the indicated substitutions of each residue. The values plotted are given in Tables I and II. Linear regression yielded the lines shown with the indicated slopes Φ and standard deviations.
Mentions: The timing of structural changes in the binding-gating transduction process has been inferred from rate-equilibrium free energy relationships (REFER) in which the channel opening rate constant is plotted against the channel gating equilibrium constant for a series of substitutions of a given residue. In many instances a log–log plot of these quantities is linear, and the slope (Φ) is interpreted as an index of the open- versus closed-state conformation of the specified residues in the gating transition state (Auerbach, 2007). Values of Φ approaching 1 suggest the conformation of the transition state is open-like, whereas values approaching 0 suggest the conformation of the transition state is closed-like. Our data for human muscle AChRs activated by ACh show Φ values for substitutions of αPro 272, αVal 46, and αVal 132, ranging from 0.87 to 0.93 (Fig. 9). If Φ reflects the relative time at which the transition state is achieved, the similar Φ values suggest the three residues move nearly simultaneously as they approach an open-like transition state. Thus, if the Torpedo AChR structural model accurately depicts the interloop triad in the resting closed state, our findings indicate that the three residues maintain physical continuity up to the transition state. This conclusion is tentative because although our REFER plots for αP272 and αVal 46 are linear, the plot for αVal 132 is curved. On the other hand, REFER analyses of mouse muscle AChRs activated by either ACh or choline appeared linear and yielded statistically different values (αPro 272 [Φ = 0.62], αVal 46 [Φ = 0.81], and αVal 132 [Φ = 0.75]), suggesting somewhat asynchronous movement of the three residues (Chakrapani, et al., 2004; Jha, et al., 2007).

Bottom Line: We find that in contributing to channel gating, alphaVal 46 and alphaVal 132 of the signature Cys loop couple energetically to alphaPro 272.Furthermore, these residues are optimized in both their size and hydrophobicity to mediate rapid and efficient channel gating, suggesting naturally occurring substitutions at these positions enable a diverse range of gating rate constants among the Cys-loop receptor superfamily.The overall results indicate that alphaPro 272 functionally couples to flanking Val residues extending from the beta1-beta2 and Cys loops within the ACh binding to channel opening transduction pathway.

View Article: PubMed Central - PubMed

Affiliation: Receptor Biology Laboratory, Department of Physiology and Biomedical Engineering, Mayo Clinic College of Medicine, Rochester, MN 55905, USA.

ABSTRACT
Nicotinic acetylcholine receptors (AChRs) mediate rapid excitatory synaptic transmission throughout the peripheral and central nervous systems. They transduce binding of nerve-released ACh into opening of an intrinsic channel, yet the structural basis underlying transduction is not fully understood. Previous studies revealed a principal transduction pathway in which alphaArg 209 of the pre-M1 domain and alphaGlu 45 of the beta1-beta2 loop functionally link the two regions, positioning alphaVal 46 of the beta1-beta2 loop in a cavity formed by alphaPro 272 through alphaSer 269 of the M2-M3 loop. Here we investigate contributions of residues within and proximal to this pathway using single-channel kinetic analysis, site-directed mutagenesis, and thermodynamic mutant cycle analysis. We find that in contributing to channel gating, alphaVal 46 and alphaVal 132 of the signature Cys loop couple energetically to alphaPro 272. Furthermore, these residues are optimized in both their size and hydrophobicity to mediate rapid and efficient channel gating, suggesting naturally occurring substitutions at these positions enable a diverse range of gating rate constants among the Cys-loop receptor superfamily. The overall results indicate that alphaPro 272 functionally couples to flanking Val residues extending from the beta1-beta2 and Cys loops within the ACh binding to channel opening transduction pathway.

Show MeSH
Related in: MedlinePlus