Decremental Response to High-Frequency Trains of Acetylcholine Pulses but Unaltered Fractional Ca2+ Currents in a Panel of “Slow-Channel Syndrome” Nicotinic Receptor Mutants

The slow-channel congenital myasthenic syndrome (SCCMS) is a disorder of the neuromuscular junction caused by gain-of-function mutations to the muscle nicotinic acetylcholine (ACh) receptor (AChR). Although it is clear that the slower deactivation time course of the ACh-elicited currents plays a central role in the etiology of this disease, it has been suggested that other abnormal properties of these mutant receptors may also be critical in this respect. We characterized the kinetics of a panel of five SCCMS AChRs (αS269I, βV266M, ɛL221F, ɛT264P, and ɛL269F) at the ensemble level in rapidly perfused outside-out patches. We found that, for all of these mutants, the peak-current amplitude decreases along trains of nearly saturating ACh pulses delivered at physiologically relevant frequencies in a manner that is consistent with enhanced entry into desensitization during the prolonged deactivation phase. This suggests that the increasingly reduced availability of activatable AChRs upon repetitive stimulation may well contribute to the fatigability and weakness of skeletal muscle that characterize this disease. Also, these results emphasize the importance of explicitly accounting for entry into desensitization as one of the pathways for burst termination, if meaningful mechanistic insight is to be inferred from the study of the effect of these naturally occurring mutations on channel function. Applying a novel single-channel–based approach to estimate the contribution of Ca2+ to the total cation currents, we also found that none of these mutants affects the Ca2+-conduction properties of the AChR to an extent that seems to be of physiological importance. Our estimate of the Ca2+-carried component of the total (inward) conductance of wild-type and SCCMS AChRs in the presence of 150 mM Na+, 1.8 mM Ca2+, and 1.7 mM Mg2+ on the extracellular side of cell-attached patches turned out be in the 5.0–9.4 pS range, representing a fractional Ca2+ current of ∼14%, on average. Remarkably, these values are nearly identical to those we estimated for the NR1-NR2A N-methyl-d-aspartate receptor (NMDAR), which has generally been considered to be the main neurotransmitter-gated pathway of Ca2+ entry into the cell. Our estimate of the rat NMDAR Ca2+ conductance (using the same single-channel approach as for the AChR but in the nominal absence of extracellular Mg2+) was 7.9 pS, corresponding to a fractional Ca2+ current of 13%.

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Lipid‐protein Interactions Studies in the αC418W Mutant Suffering from Slow Channel Congenital Myasthenic Syndrome

The FASEB Journal ◽

10.1096/fasebj.24.1_supplement.lb26 ◽

2010 ◽

Vol 24 (S1) ◽

Author(s):

Jose Gabriel Grajales ◽

Gary Grajales ◽

Carlos Baez ◽

Haipeng Zhu ◽

Oreste Quesada ◽

...

Keyword(s):

Protein Interactions ◽

Congenital Myasthenic Syndrome ◽

Slow Channel ◽

Myasthenic Syndrome ◽

Lipid Protein Interactions

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Congenital myasthenic syndrome, slow channel syndrome

Neuromuscular Disease: A Case-Based Approach ◽

10.1017/cbo9780511735905.039 ◽

2013 ◽

pp. 111-114

Author(s):

John H. J. Wokke ◽

Pieter A. van Doorn ◽

Jessica E. Hoogendijk ◽

Marianne de Visser

Keyword(s):

Congenital Myasthenic Syndrome ◽

Slow Channel ◽

Myasthenic Syndrome

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Slow-Channel Congenital Myasthenic Syndrome

Genetic Neuromuscular Disorders ◽

10.1007/978-3-319-07500-6_44 ◽

2014 ◽

pp. 191-194

Author(s):

Corrado Angelini

Keyword(s):

Congenital Myasthenic Syndrome ◽

Slow Channel ◽

Myasthenic Syndrome

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The Extracellular Linker of Muscle Acetylcholine Receptor Channels Is a Gating Control Element

The Journal of General Physiology ◽

10.1085/jgp.116.3.327 ◽

2000 ◽

Vol 116 (3) ◽

pp. 327-340 ◽

Cited By ~ 84

Author(s):

Claudio Grosman ◽

Frank N. Salamone ◽

Steven M. Sine ◽

Anthony Auerbach

Keyword(s):

Acetylcholine Receptors ◽

Single Channel ◽

Channel Gating ◽

Congenital Myasthenic Syndrome ◽

Control Element ◽

Dependent Manner ◽

Α Subunit ◽

Transmembrane Segments ◽

Channel Opening ◽

Myasthenic Syndrome

We describe the functional consequences of mutations in the linker between the second and third transmembrane segments (M2–M3L) of muscle acetylcholine receptors at the single-channel level. Hydrophobic mutations (Ile, Cys, and Phe) placed near the middle of the linker of the α subunit (αS269) prolong apparent openings elicited by low concentrations of acetylcholine (ACh), whereas hydrophilic mutations (Asp, Lys, and Gln) are without effect. Because the gating kinetics of the αS269I receptor (a congenital myasthenic syndrome mutant) in the presence of ACh are too fast, choline was used as the agonist. This revealed an ∼92-fold increased gating equilibrium constant, which is consistent with an ∼10-fold decreased EC50 in the presence of ACh. With choline, this mutation accelerates channel opening ∼28-fold, slows channel closing ∼3-fold, but does not affect agonist binding to the closed state. These ratios suggest that, with ACh, αS269I acetylcholine receptors open at a rate of ∼1.4 × 106 s−1 and close at a rate of ∼760 s−1. These gating rate constants, together with the measured duration of apparent openings at low ACh concentrations, further suggest that ACh dissociates from the diliganded open receptor at a rate of ∼140 s−1. Ile mutations at positions flanking αS269 impair, rather than enhance, channel gating. Inserting or deleting one residue from this linker in the α subunit increased and decreased, respectively, the apparent open time approximately twofold. Contrary to the αS269I mutation, Ile mutations at equivalent positions of the β, ε, and δ subunits do not affect apparent open-channel lifetimes. However, in β and ε, shifting the mutation one residue to the NH2-terminal end enhances channel gating. The overall results indicate that this linker is a control element whose hydrophobicity determines channel gating in a position- and subunit-dependent manner. Characterization of the transition state of the gating reaction suggests that during channel opening the M2–M3L of the α subunit moves before the corresponding linkers of the β and ε subunits.

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Fluoxetine is neuroprotective in slow-channel congenital myasthenic syndrome

Experimental Neurology ◽

10.1016/j.expneurol.2014.10.008 ◽

2015 ◽

Vol 270 ◽

pp. 88-94 ◽

Cited By ~ 5

Author(s):

Haipeng Zhu ◽

Gary E. Grajales-Reyes ◽

Vivianette Alicea-Vázquez ◽

Jose G. Grajales-Reyes ◽

KaReisha Robinson ◽

...

Keyword(s):

Congenital Myasthenic Syndrome ◽

Slow Channel ◽

Myasthenic Syndrome

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Shaker potassium channel gating. I: Transitions near the open state.

The Journal of General Physiology ◽

10.1085/jgp.103.2.249 ◽

1994 ◽

Vol 103 (2) ◽

pp. 249-278 ◽

Cited By ~ 144

Author(s):

T Hoshi ◽

W N Zagotta ◽

R W Aldrich

Keyword(s):

Time Course ◽

Single Channel ◽

Activation Process ◽

Time Data ◽

Shaker Potassium Channel ◽

Voltage Dependent ◽

Average Current ◽

Shaker Potassium Channels ◽

Kinetics Of ◽

Deactivation Process

Kinetics of single voltage-dependent Shaker potassium channels expressed in Xenopus oocytes were studied in the absence of fast N-type inactivation. Comparison of the single-channel first latency distribution and the time course of the ensemble average current showed that the activation time course and its voltage dependence are largely determined by the transitions before first opening. The open dwell time data are consistent with a single kinetically distinguishable open state. Once the channel opens, it can enter at least two closed states which are not traversed frequently during the activation process. The rate constants for the transitions among these closed states and the open state are nearly voltage-independent at depolarized voltages (> -30 mV). During the deactivation process at more negative voltages, the channel can close directly to a closed state in the activation pathway in a voltage-dependent fashion.

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