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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.


Examples of transient modal gating (mode shifting) in BK channels taken from the study of McManus and Magleby (1988). (A) A continuous single-channel current record (divided into three traces) recorded from a single BK channel. The inside-out patch-clamp configuration (Hamill et al., 1981) was used to record the data. The patch of membrane was pulled from a skeletal muscle cell in a primary culture of rat skeletal muscle. Channel opening is upward. The downward and upward arrows indicate switching from normal mode gating into brief open mode gating and then back, respectively. (B) Stability plot of the moving average of open-interval durations taken 50 at a time. Shifting from normal mode gating, with a mean open interval duration of 2.8 ms, to brief open-mode gating, with a mean open interval duration of 0.5 ms, and then back to normal mode gating is readily apparent. (C and D) Current record (C) and stability plot (D) for mode shifting into and out of a buzz mode with a mean open interval of 0.05 ms. Symmetrical 144 mM KCl; 30 mV; 7.5 µM Ca2+i; pH 7.2.
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fig1: Examples of transient modal gating (mode shifting) in BK channels taken from the study of McManus and Magleby (1988). (A) A continuous single-channel current record (divided into three traces) recorded from a single BK channel. The inside-out patch-clamp configuration (Hamill et al., 1981) was used to record the data. The patch of membrane was pulled from a skeletal muscle cell in a primary culture of rat skeletal muscle. Channel opening is upward. The downward and upward arrows indicate switching from normal mode gating into brief open mode gating and then back, respectively. (B) Stability plot of the moving average of open-interval durations taken 50 at a time. Shifting from normal mode gating, with a mean open interval duration of 2.8 ms, to brief open-mode gating, with a mean open interval duration of 0.5 ms, and then back to normal mode gating is readily apparent. (C and D) Current record (C) and stability plot (D) for mode shifting into and out of a buzz mode with a mean open interval of 0.05 ms. Symmetrical 144 mM KCl; 30 mV; 7.5 µM Ca2+i; pH 7.2.

Mentions: Fig. 1 shows two examples of transient modal gating (McManus and Magleby, 1988). Fig. 1 A presents a short excerpt of a continuous recording of current through a single BK channel, divided into three consecutive traces. Channel opening and closing is indicated by upwards and downward steps in the current, respectively. The down arrow indicates a shift from normal mode gating to brief open mode gating. The upward arrow 590 ms later signals a return to normal mode gating. The modal shifts were readily identified by the stability plot in Fig. 1 B, which presents a moving average of the mean duration of open intervals taken 50 at a time. The abrupt decrease in the mean open-interval duration from 2.8 to 0.5 ms indicates a shift from normal mode gating to brief open mode gating, and the abrupt return indicates a shift back to normal mode gating. Another example in Fig. 1 of mode shifting from the same BK channel presented in A and B is depicted in C and D. The BK channel shifts from normal mode gating to (flicker) buzz mode gating with a mean open-interval duration of 0.05 ms, and then back to normal mode gating. The stability plot for this channel also indicated mode shifts to and from an intermediate mode with a 2.0-ms mean open duration (not depicted). 96% of the time was spent in normal mode gating, with infrequent entries into the other three modes.


Modal gating of endplate acetylcholine receptors: A proposed mechanism.

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

Examples of transient modal gating (mode shifting) in BK channels taken from the study of McManus and Magleby (1988). (A) A continuous single-channel current record (divided into three traces) recorded from a single BK channel. The inside-out patch-clamp configuration (Hamill et al., 1981) was used to record the data. The patch of membrane was pulled from a skeletal muscle cell in a primary culture of rat skeletal muscle. Channel opening is upward. The downward and upward arrows indicate switching from normal mode gating into brief open mode gating and then back, respectively. (B) Stability plot of the moving average of open-interval durations taken 50 at a time. Shifting from normal mode gating, with a mean open interval duration of 2.8 ms, to brief open-mode gating, with a mean open interval duration of 0.5 ms, and then back to normal mode gating is readily apparent. (C and D) Current record (C) and stability plot (D) for mode shifting into and out of a buzz mode with a mean open interval of 0.05 ms. Symmetrical 144 mM KCl; 30 mV; 7.5 µM Ca2+i; pH 7.2.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4664830&req=5

fig1: Examples of transient modal gating (mode shifting) in BK channels taken from the study of McManus and Magleby (1988). (A) A continuous single-channel current record (divided into three traces) recorded from a single BK channel. The inside-out patch-clamp configuration (Hamill et al., 1981) was used to record the data. The patch of membrane was pulled from a skeletal muscle cell in a primary culture of rat skeletal muscle. Channel opening is upward. The downward and upward arrows indicate switching from normal mode gating into brief open mode gating and then back, respectively. (B) Stability plot of the moving average of open-interval durations taken 50 at a time. Shifting from normal mode gating, with a mean open interval duration of 2.8 ms, to brief open-mode gating, with a mean open interval duration of 0.5 ms, and then back to normal mode gating is readily apparent. (C and D) Current record (C) and stability plot (D) for mode shifting into and out of a buzz mode with a mean open interval of 0.05 ms. Symmetrical 144 mM KCl; 30 mV; 7.5 µM Ca2+i; pH 7.2.
Mentions: Fig. 1 shows two examples of transient modal gating (McManus and Magleby, 1988). Fig. 1 A presents a short excerpt of a continuous recording of current through a single BK channel, divided into three consecutive traces. Channel opening and closing is indicated by upwards and downward steps in the current, respectively. The down arrow indicates a shift from normal mode gating to brief open mode gating. The upward arrow 590 ms later signals a return to normal mode gating. The modal shifts were readily identified by the stability plot in Fig. 1 B, which presents a moving average of the mean duration of open intervals taken 50 at a time. The abrupt decrease in the mean open-interval duration from 2.8 to 0.5 ms indicates a shift from normal mode gating to brief open mode gating, and the abrupt return indicates a shift back to normal mode gating. Another example in Fig. 1 of mode shifting from the same BK channel presented in A and B is depicted in C and D. The BK channel shifts from normal mode gating to (flicker) buzz mode gating with a mean open-interval duration of 0.05 ms, and then back to normal mode gating. The stability plot for this channel also indicated mode shifts to and from an intermediate mode with a 2.0-ms mean open duration (not depicted). 96% of the time was spent in normal mode gating, with infrequent entries into the other three modes.

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.