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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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Single-channel currents and dwell time histograms from AChRs containing mutations of αVal 46. Currents elicited by 300 μM ACh are shown at a bandwidth of 10 kHz, with channel openings upward deflections. Histograms of dwell times within identified clusters of events are shown on logarithmic time axes with probability density functions generated from global kinetic fitting overlaid (see Materials and methods; fitted rate constants are given in Tables I and II).
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fig5: Single-channel currents and dwell time histograms from AChRs containing mutations of αVal 46. Currents elicited by 300 μM ACh are shown at a bandwidth of 10 kHz, with channel openings upward deflections. Histograms of dwell times within identified clusters of events are shown on logarithmic time axes with probability density functions generated from global kinetic fitting overlaid (see Materials and methods; fitted rate constants are given in Tables I and II).

Mentions: Substitutions of Val 46 also reduce the efficiency of channel gating, again by prolonging closed times and shortening open times (Fig. 5). Kinetic analyses of single-channel dwell times reveals that the small Gly and Ala substitutions profoundly decrease the gating equilibrium constant, up to 1,800-fold, while the larger Leu and Ile substitutions produce modest decreases (Table II). Thus similar to αVal 132, Val at position α46 is optimal.


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)

Single-channel currents and dwell time histograms from AChRs containing mutations of αVal 46. Currents elicited by 300 μM ACh are shown at a bandwidth of 10 kHz, with channel openings upward deflections. Histograms of dwell times within identified clusters of events are shown on logarithmic time axes with probability density functions generated from global kinetic fitting overlaid (see Materials and methods; fitted rate constants are given in Tables I and II).
© Copyright Policy
Related In: Results  -  Collection

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

fig5: Single-channel currents and dwell time histograms from AChRs containing mutations of αVal 46. Currents elicited by 300 μM ACh are shown at a bandwidth of 10 kHz, with channel openings upward deflections. Histograms of dwell times within identified clusters of events are shown on logarithmic time axes with probability density functions generated from global kinetic fitting overlaid (see Materials and methods; fitted rate constants are given in Tables I and II).
Mentions: Substitutions of Val 46 also reduce the efficiency of channel gating, again by prolonging closed times and shortening open times (Fig. 5). Kinetic analyses of single-channel dwell times reveals that the small Gly and Ala substitutions profoundly decrease the gating equilibrium constant, up to 1,800-fold, while the larger Leu and Ile substitutions produce modest decreases (Table II). Thus similar to αVal 132, Val at position α46 is optimal.

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