Structural basis of the different gating kinetics of fetal and adult acetylcholine receptors

doi:10.1016/0896-6273(94)90424-3

Neuron

Volume 13, Issue 6, December 1994, Pages 1395-1402

https://doi.org/10.1016/0896-6273(94)90424-3 Get rights and content

Summary

Structure-function studies have identified key functional motifs in the acetylcholine receptor, including residues that contribute to the ion channel and to the ligand-binding sites. Little is known, however, about determinants of channel gating kinetics. To identify structural correlates of gating, we examined the structual basis of the fetal-to-adult decrease in channel open time conferred by the presence of the ε subunit in place of the γ subunit. By constructing chimeras composed of segments of the ε and γ subunits, we show that the main determinant of this kinetic change is a 30 residue segment of a predicted amphipathic helix located between transmembrane domains M3 and M4. Further subdividing the amphipathic helix revealed that either multiple residues or its overall conformation confers this regulation of channel kinetics. We also show that L440 and M442, conserved residues within M4 of the γ subunit, contribute to long duration openings characteristic of the fetal receptor.

References (23)

LeeY.-H. et al.
Mutations in the M4 channel domain dramatically alterion channel function
Biophys. J.
(1994)
MonyerH. et al.
Glutamateoperated channels: developmentally early and mature forms arise by alternative splicing
Neuron
(1991)
SigworthF. et al.
Data transformations for improved display and fitting of single-channel dwell time histograms
Biophys. J.
(1987)
SineS.M. et al.
Conserved tyrosines in the α subunit of the nicotinic acetylcholine receptor stabilize quaternary ammonium groups of agonists and curariform antagonists
J. Biol. Chem.
(1994)
CamachoP. et al.
The ε subunit confers fast channel gating on multiple classes of acetylcholine receptors
J. Neurosci.
(1993)
CockcroftV.B. et al.
Ligand-gated ion channels, homology and diversity
Mol. Neurobiol.
(1992)
EngelA.G. et al.
Myasthenic syndromes attributed to mutations affecting the s subunit of the acetylcholine receptor
Ann. NY Acad. Sci.
(1993)
Finer-MooreJ. et al.
Amphipathic analysis and possible formation of the ion channel in an acetylcholine receptor
GardnerP.
Nucleotide sequence of the ε subunit of the mouse muscle nicotinic receptor
Nucleic Acids Res.
(1990)
HamillO.P. et al.
Improved patch-clamp techniques for high resolution current recordings from cells and cell-free membrane patches
Pflügers Arch.
(1981)

HallZ.W. et al.

Synaptic structure and development: the neuromuscular junction

Cell

(1993)

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Abstract
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Citation Excerpt :
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Neuron

Structural basis of the different gating kinetics of fetal and adult acetylcholine receptors

Summary

Biophys. J.

Neuron

Biophys. J.

J. Biol. Chem.

The ε subunit confers fast channel gating on multiple classes of acetylcholine receptors

J. Neurosci.

Ligand-gated ion channels, homology and diversity

Mol. Neurobiol.

Myasthenic syndromes attributed to mutations affecting the s subunit of the acetylcholine receptor

Ann. NY Acad. Sci.

Amphipathic analysis and possible formation of the ion channel in an acetylcholine receptor

Nucleotide sequence of the ε subunit of the mouse muscle nicotinic receptor

Nucleic Acids Res.

Improved patch-clamp techniques for high resolution current recordings from cells and cell-free membrane patches

Pflügers Arch.

Synaptic structure and development: the neuromuscular junction

Cell