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Modal gating of endplate acetylcholine receptors: A proposed mechanism.

Geng Y, Magleby KL - J. Gen. Physiol. (2015)

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Affiliation: Department of Physiology and Biophysics, University of Miami Miller School of Medicine, Miami, FL 33136.

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In retrospect, such stochastic variation of single ion channel gating was predictable from the observed exponential decays of macroscopic ionic currents recorded from cells when large numbers of open channels were closing ; the exponential decays arose from exponentially distributed effective open durations (burst durations)... With further analysis, the stochastic variation in ion channel gating was found to be far more complex... In mammals, neuromuscular transmission requires the presynaptic release of acetylcholine from the nerve terminals of motor neurons whose cell bodies are located in the spinal cord and brainstem onto AChRs located in the postsynaptic muscle membrane... In an extensive series of experiments like those in Fig. 2, found that: (a) WT channels gated in a single mode. (b) Point mutations at 10 of the 12 residues comprising loop C at the α–δ binding pocket produced stable modal gating... The number of stable modes ranged from two to four, depending on the site and identity of the substituted residue. (c) Point deletions in the C loop at the α–δ binding pocket could also produce modal gating. (d) C loop mutations at the α–ε binding pocket did not produce modal gating. (e) Partial agonists generated fewer modes than ACh. (f) Modal gating was not observed in the absence of agonist. (g) Removing a tryptophan sidechain in the δ subunit (δW57A) prevented modal gating. (h) ACh affinity at the α–δ binding pocket was increased in modes with increased Po and decreased in modes with decreased Po... These observations clearly focus the culprit for stable modal gating of AChRs on changes in the structure of the binding pocket for ACh at the α–δ interface... For all of these nebulous mechanisms, it seems highly unlikely that only mutations at the α–δ binding pocket, but not the α–ε binding pocket, would produce such modal gating... Given the conclusion that modal gating in AChRs arises from mutation-induced structural changes in the α–δ binding pocket, how is it possible that a single-point mutation or deletion can produce from two to four different stable modes? If such alternative stable conformations of the binding pocket cannot be found, then it is possible that the different conformational subtypes might arise indirectly from different multiple stable conformations in other parts of the channel feeding back to regulate the conformations and affinities of the binding pocket in a mutation-specific manner... The authors conclude that the different stable modes arise from different stable affinities at the α–δ binding pocket, thus providing a mechanism for modal gating in AChRs 30 years after the initial observation... Finally, their system could perhaps be tweaked by additional mutations to decrease the stability of the modes so that the process of mode switching could itself be routinely observed and studied... In future studies, it will be interesting to examine to what extent their conclusions apply to transient modal gating in WT adult endplate AChRs and in other ion channels where modal gating has been reported.

No MeSH data available.


Stable modal gating in adult endplate AChRs. (A) Stable normal-mode gating. (Left) Continuous single-channel recording displayed as seven traces obtained with on-cell patch-clamp recording from HEK cells with expressed WT mouse AChRs. Opening is downward. The clusters of activity arise from AChRs coming out of desensitization (D) to gate (C-O), and then returning to desensitization. (Middle) Histogram of the number of clusters versus their mean Po. Only a single normal mode of activity is seen with a Po of 0.7. (Right) Dwell-time distributions of shut- and open-interval durations are each described by a single exponential, indicating gating in two states (C-O). (B) Four stable modes of gating after the mutation P197A in the α subunits. (Left) Single-channel current record during continuous recording displayed as seven traces. (Middle) Gating in four different stable modes with mean durations of 0.01, 0.20, 0.67, and 0.96. (Right) The dwell-time distributions of the entire current record are described by multiple exponentials, indicating different kinetics for the different modes. See Fig. 2 of Vij et al. (2015) for further details.
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fig2: Stable modal gating in adult endplate AChRs. (A) Stable normal-mode gating. (Left) Continuous single-channel recording displayed as seven traces obtained with on-cell patch-clamp recording from HEK cells with expressed WT mouse AChRs. Opening is downward. The clusters of activity arise from AChRs coming out of desensitization (D) to gate (C-O), and then returning to desensitization. (Middle) Histogram of the number of clusters versus their mean Po. Only a single normal mode of activity is seen with a Po of 0.7. (Right) Dwell-time distributions of shut- and open-interval durations are each described by a single exponential, indicating gating in two states (C-O). (B) Four stable modes of gating after the mutation P197A in the α subunits. (Left) Single-channel current record during continuous recording displayed as seven traces. (Middle) Gating in four different stable modes with mean durations of 0.01, 0.20, 0.67, and 0.96. (Right) The dwell-time distributions of the entire current record are described by multiple exponentials, indicating different kinetics for the different modes. See Fig. 2 of Vij et al. (2015) for further details.

Mentions: An example of stable modal gating from the paper by Vij et al. (2015) for adult mouse endplate AChRs expressed in HEK cells is shown in Fig. 2, which presents excerpts from their Fig. 2. The horizontal panel in Fig. 2 A characterizes normal mode gating. On the left is a continuous single-channel record displayed as seven traces (opening in this instance is downward) showing clusters of activity (C-O) separated by longer closed intervals indicating desensitized channels (D) resulting from the continuous presence of 30 µM ACh. A histogram of the number of clusters versus their Po shows a single, normal mode of activity with a mean Po of 0.7 (middle). The open and closed dwell-time distributions of the entire record are each described by a single-exponential component (right). With a single WT channel, only normal mode activity (plus a few isolated openings) was observed in the clusters for up to 30 min of recorded activity, indicating stability of the normal mode in the absence of mutations. In a previous study of AChRs expressed naturally in Xenopus laevis myocytes, a single putative shift to a higher Po mode was observed (Auerbach and Lingle, 1986), indicating that mode shifts in WT endplate AChRs are possible but rare.


Modal gating of endplate acetylcholine receptors: A proposed mechanism.

Geng Y, Magleby KL - J. Gen. Physiol. (2015)

Stable modal gating in adult endplate AChRs. (A) Stable normal-mode gating. (Left) Continuous single-channel recording displayed as seven traces obtained with on-cell patch-clamp recording from HEK cells with expressed WT mouse AChRs. Opening is downward. The clusters of activity arise from AChRs coming out of desensitization (D) to gate (C-O), and then returning to desensitization. (Middle) Histogram of the number of clusters versus their mean Po. Only a single normal mode of activity is seen with a Po of 0.7. (Right) Dwell-time distributions of shut- and open-interval durations are each described by a single exponential, indicating gating in two states (C-O). (B) Four stable modes of gating after the mutation P197A in the α subunits. (Left) Single-channel current record during continuous recording displayed as seven traces. (Middle) Gating in four different stable modes with mean durations of 0.01, 0.20, 0.67, and 0.96. (Right) The dwell-time distributions of the entire current record are described by multiple exponentials, indicating different kinetics for the different modes. See Fig. 2 of Vij et al. (2015) for further details.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4664830&req=5

fig2: Stable modal gating in adult endplate AChRs. (A) Stable normal-mode gating. (Left) Continuous single-channel recording displayed as seven traces obtained with on-cell patch-clamp recording from HEK cells with expressed WT mouse AChRs. Opening is downward. The clusters of activity arise from AChRs coming out of desensitization (D) to gate (C-O), and then returning to desensitization. (Middle) Histogram of the number of clusters versus their mean Po. Only a single normal mode of activity is seen with a Po of 0.7. (Right) Dwell-time distributions of shut- and open-interval durations are each described by a single exponential, indicating gating in two states (C-O). (B) Four stable modes of gating after the mutation P197A in the α subunits. (Left) Single-channel current record during continuous recording displayed as seven traces. (Middle) Gating in four different stable modes with mean durations of 0.01, 0.20, 0.67, and 0.96. (Right) The dwell-time distributions of the entire current record are described by multiple exponentials, indicating different kinetics for the different modes. See Fig. 2 of Vij et al. (2015) for further details.
Mentions: An example of stable modal gating from the paper by Vij et al. (2015) for adult mouse endplate AChRs expressed in HEK cells is shown in Fig. 2, which presents excerpts from their Fig. 2. The horizontal panel in Fig. 2 A characterizes normal mode gating. On the left is a continuous single-channel record displayed as seven traces (opening in this instance is downward) showing clusters of activity (C-O) separated by longer closed intervals indicating desensitized channels (D) resulting from the continuous presence of 30 µM ACh. A histogram of the number of clusters versus their Po shows a single, normal mode of activity with a mean Po of 0.7 (middle). The open and closed dwell-time distributions of the entire record are each described by a single-exponential component (right). With a single WT channel, only normal mode activity (plus a few isolated openings) was observed in the clusters for up to 30 min of recorded activity, indicating stability of the normal mode in the absence of mutations. In a previous study of AChRs expressed naturally in Xenopus laevis myocytes, a single putative shift to a higher Po mode was observed (Auerbach and Lingle, 1986), indicating that mode shifts in WT endplate AChRs are possible but rare.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology and Biophysics, University of Miami Miller School of Medicine, Miami, FL 33136.

AUTOMATICALLY GENERATED EXCERPT
Please rate it.

In retrospect, such stochastic variation of single ion channel gating was predictable from the observed exponential decays of macroscopic ionic currents recorded from cells when large numbers of open channels were closing ; the exponential decays arose from exponentially distributed effective open durations (burst durations)... With further analysis, the stochastic variation in ion channel gating was found to be far more complex... In mammals, neuromuscular transmission requires the presynaptic release of acetylcholine from the nerve terminals of motor neurons whose cell bodies are located in the spinal cord and brainstem onto AChRs located in the postsynaptic muscle membrane... In an extensive series of experiments like those in Fig. 2, found that: (a) WT channels gated in a single mode. (b) Point mutations at 10 of the 12 residues comprising loop C at the α–δ binding pocket produced stable modal gating... The number of stable modes ranged from two to four, depending on the site and identity of the substituted residue. (c) Point deletions in the C loop at the α–δ binding pocket could also produce modal gating. (d) C loop mutations at the α–ε binding pocket did not produce modal gating. (e) Partial agonists generated fewer modes than ACh. (f) Modal gating was not observed in the absence of agonist. (g) Removing a tryptophan sidechain in the δ subunit (δW57A) prevented modal gating. (h) ACh affinity at the α–δ binding pocket was increased in modes with increased Po and decreased in modes with decreased Po... These observations clearly focus the culprit for stable modal gating of AChRs on changes in the structure of the binding pocket for ACh at the α–δ interface... For all of these nebulous mechanisms, it seems highly unlikely that only mutations at the α–δ binding pocket, but not the α–ε binding pocket, would produce such modal gating... Given the conclusion that modal gating in AChRs arises from mutation-induced structural changes in the α–δ binding pocket, how is it possible that a single-point mutation or deletion can produce from two to four different stable modes? If such alternative stable conformations of the binding pocket cannot be found, then it is possible that the different conformational subtypes might arise indirectly from different multiple stable conformations in other parts of the channel feeding back to regulate the conformations and affinities of the binding pocket in a mutation-specific manner... The authors conclude that the different stable modes arise from different stable affinities at the α–δ binding pocket, thus providing a mechanism for modal gating in AChRs 30 years after the initial observation... Finally, their system could perhaps be tweaked by additional mutations to decrease the stability of the modes so that the process of mode switching could itself be routinely observed and studied... In future studies, it will be interesting to examine to what extent their conclusions apply to transient modal gating in WT adult endplate AChRs and in other ion channels where modal gating has been reported.

No MeSH data available.