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Fundamental gating mechanism of nicotinic receptor channel revealed by mutation causing a congenital myasthenic syndrome.

Wang HL, Ohno K, Milone M, Brengman JM, Evoli A, Batocchi AP, Middleton LT, Christodoulou K, Engel AG, Sine SM - J. Gen. Physiol. (2000)

Bottom Line: Prolines engineered into positions flanking residue 411 of the epsilon subunit greatly increase the range of activation kinetics similar to epsilonA411P, whereas prolines engineered into positions equivalent to epsilonA411 in beta and delta subunits are without effect.The findings suggest that analogous stabilizing structures are present in other ion channels, and possibly allosteric proteins in general, and that they evolved to maintain uniformity of activation episodes.The findings further suggest that the fundamental gating mechanism of the AChR channel can be explained by a corrugated energy landscape superimposed on a steeply sloped energy well.

View Article: PubMed Central - PubMed

Affiliation: Receptor Biology Laboratory, Department of Physiology and Biophysics, Mayo Foundation, Rochester, Minnesota 55905, USA.

ABSTRACT
We describe the genetic and kinetic defects in a congenital myasthenic syndrome due to the mutation epsilonA411P in the amphipathic helix of the acetylcholine receptor (AChR) epsilon subunit. Myasthenic patients from three unrelated families are either homozygous for epsilonA411P or are heterozygous and harbor a mutation in the second epsilon allele, indicating that epsilonA411P is recessive. We expressed human AChRs containing wild-type or A411P epsilon subunits in 293HEK cells, recorded single channel currents at high bandwidth, and determined microscopic rate constants for individual channels using hidden Markov modeling. For individual wild-type and mutant channels, each rate constant distributes as a Gaussian function, but the spread in the distributions for channel opening and closing rate constants is greatly expanded by epsilonA411P. Prolines engineered into positions flanking residue 411 of the epsilon subunit greatly increase the range of activation kinetics similar to epsilonA411P, whereas prolines engineered into positions equivalent to epsilonA411 in beta and delta subunits are without effect. Thus, the amphipathic helix of the epsilon subunit stabilizes the channel, minimizing the number and range of kinetic modes accessible to individual AChRs. The findings suggest that analogous stabilizing structures are present in other ion channels, and possibly allosteric proteins in general, and that they evolved to maintain uniformity of activation episodes. The findings further suggest that the fundamental gating mechanism of the AChR channel can be explained by a corrugated energy landscape superimposed on a steeply sloped energy well.

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Distribution of open probabilities from individual activation episodes for AChRs containing the indicated mutations. (A) The absence of consequences when mutations are introduced into β and δ subunits at positions equivalent to εA411P. (B) Broadening of the open probability distributions for mutations introduced along a five-residue stretch of the amphipathic helix of the ε subunit.
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Figure 8: Distribution of open probabilities from individual activation episodes for AChRs containing the indicated mutations. (A) The absence of consequences when mutations are introduced into β and δ subunits at positions equivalent to εA411P. (B) Broadening of the open probability distributions for mutations introduced along a five-residue stretch of the amphipathic helix of the ε subunit.

Mentions: We next asked whether the increased kinetic variability is unique to mutations in the ε subunit, and further whether it is unique to mutation of alanine 411. We engineered proline into positions equivalent to εA411 in the homologous α, β, and δ subunits, as well as into positions 409–413 of the ε subunit, and recorded single channel currents through individual mutant AChRs. Corresponding mutations in either β or δ subunits do not affect the distribution of open probabilities (Fig. 8 A), while the mutated α subunit does not support expression of AChR, as indicated by lack of ACh-induced single-channel currents. On the other hand, proline mutations scanning this region of the ε subunit, εF409P, εV410P, εE412P, and εS413P, all broaden the distribution of open probabilities similar to the original εA411P mutation (Fig. 8 B). Thus, increased kinetic variability of the AChR is specific to mutations of the ε subunit, where the local region flanking alanine 411 maintains uniformity of AChR activation kinetics.


Fundamental gating mechanism of nicotinic receptor channel revealed by mutation causing a congenital myasthenic syndrome.

Wang HL, Ohno K, Milone M, Brengman JM, Evoli A, Batocchi AP, Middleton LT, Christodoulou K, Engel AG, Sine SM - J. Gen. Physiol. (2000)

Distribution of open probabilities from individual activation episodes for AChRs containing the indicated mutations. (A) The absence of consequences when mutations are introduced into β and δ subunits at positions equivalent to εA411P. (B) Broadening of the open probability distributions for mutations introduced along a five-residue stretch of the amphipathic helix of the ε subunit.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2233692&req=5

Figure 8: Distribution of open probabilities from individual activation episodes for AChRs containing the indicated mutations. (A) The absence of consequences when mutations are introduced into β and δ subunits at positions equivalent to εA411P. (B) Broadening of the open probability distributions for mutations introduced along a five-residue stretch of the amphipathic helix of the ε subunit.
Mentions: We next asked whether the increased kinetic variability is unique to mutations in the ε subunit, and further whether it is unique to mutation of alanine 411. We engineered proline into positions equivalent to εA411 in the homologous α, β, and δ subunits, as well as into positions 409–413 of the ε subunit, and recorded single channel currents through individual mutant AChRs. Corresponding mutations in either β or δ subunits do not affect the distribution of open probabilities (Fig. 8 A), while the mutated α subunit does not support expression of AChR, as indicated by lack of ACh-induced single-channel currents. On the other hand, proline mutations scanning this region of the ε subunit, εF409P, εV410P, εE412P, and εS413P, all broaden the distribution of open probabilities similar to the original εA411P mutation (Fig. 8 B). Thus, increased kinetic variability of the AChR is specific to mutations of the ε subunit, where the local region flanking alanine 411 maintains uniformity of AChR activation kinetics.

Bottom Line: Prolines engineered into positions flanking residue 411 of the epsilon subunit greatly increase the range of activation kinetics similar to epsilonA411P, whereas prolines engineered into positions equivalent to epsilonA411 in beta and delta subunits are without effect.The findings suggest that analogous stabilizing structures are present in other ion channels, and possibly allosteric proteins in general, and that they evolved to maintain uniformity of activation episodes.The findings further suggest that the fundamental gating mechanism of the AChR channel can be explained by a corrugated energy landscape superimposed on a steeply sloped energy well.

View Article: PubMed Central - PubMed

Affiliation: Receptor Biology Laboratory, Department of Physiology and Biophysics, Mayo Foundation, Rochester, Minnesota 55905, USA.

ABSTRACT
We describe the genetic and kinetic defects in a congenital myasthenic syndrome due to the mutation epsilonA411P in the amphipathic helix of the acetylcholine receptor (AChR) epsilon subunit. Myasthenic patients from three unrelated families are either homozygous for epsilonA411P or are heterozygous and harbor a mutation in the second epsilon allele, indicating that epsilonA411P is recessive. We expressed human AChRs containing wild-type or A411P epsilon subunits in 293HEK cells, recorded single channel currents at high bandwidth, and determined microscopic rate constants for individual channels using hidden Markov modeling. For individual wild-type and mutant channels, each rate constant distributes as a Gaussian function, but the spread in the distributions for channel opening and closing rate constants is greatly expanded by epsilonA411P. Prolines engineered into positions flanking residue 411 of the epsilon subunit greatly increase the range of activation kinetics similar to epsilonA411P, whereas prolines engineered into positions equivalent to epsilonA411 in beta and delta subunits are without effect. Thus, the amphipathic helix of the epsilon subunit stabilizes the channel, minimizing the number and range of kinetic modes accessible to individual AChRs. The findings suggest that analogous stabilizing structures are present in other ion channels, and possibly allosteric proteins in general, and that they evolved to maintain uniformity of activation episodes. The findings further suggest that the fundamental gating mechanism of the AChR channel can be explained by a corrugated energy landscape superimposed on a steeply sloped energy well.

Show MeSH
Related in: MedlinePlus