Decremental Response to High-Frequency Trains of Acetylcholine Pulses but Unaltered Fractional Ca2+ Currents in a Panel of "Slow-Channel Syndrome" Nicotinic Receptor Mutants (original) (raw)
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We describe a novel genetic and kinetic defect in a slow-channel congenital myasthenic syndrome. The severely disabled propositus has advanced endplate myopathy, prolonged and biexponentially decaying endplate currents, and prolonged acetylcholine receptor (AChR) channel openings. Genetic analysis reveals the heterozygous mutation ␣V249F in the propositus and mosaicism for ␣V249F in the asymptomatic father. Unlike mutations described previously in the M2 transmembrane domain, ␣V249F is located N-terminal to the conserved leucines and is not predicted to face the channel lumen. Expression of the ␣V249F AChR in HEK fibroblasts demonstrates increased channel openings in the absence of ACh, prolonged openings in its presence, enhanced steady-state desensitization, and nanomolar rather than micromolar affinity of one of the two binding sites in the resting activatable state. Thus, neuromuscular transmission is compromised because cationic overloading leads to degenerating junctional folds and loss of AChR, because an increased fraction of AChR is desensitized in the resting state, and because physiological rates of stimulation elicit additional desensitization and depolarization block of transmission.
The Journal of Physiology, 2003
The discovery of the genetic basis for many of the human congenital myasthenic syndromes has added to the inherent interest in investigation of the relationship between structure and function in the nicotinic acetylcholine receptor (AChR) of the muscle endplate. The slow channel congenital myasthenic syndromes result from single amino acid 'gain-of-function' mutations in the receptor protein that give rise to prolonged endplate currents. Muscle weakness is thought to result from endplate damage caused by excess calcium entry. In addition, at physiological rates of stimulation the prolonged endplate potentials summate, leading to persistent depolarisation at the endplate and the consequent inactivation of the voltage-gated sodium channels.
The Journal of Neuroscience, 1997
We describe a novel genetic and kinetic defect in a slow-channel congenital myasthenic syndrome. The severely disabled propositus has advanced endplate myopathy, prolonged and biexponentially decaying endplate currents, and prolonged acetylcholine receptor (AChR) channel openings. Genetic analysis reveals the heterozygous mutation ␣V249F in the propositus and mosaicism for ␣V249F in the asymptomatic father. Unlike mutations described previously in the M2 transmembrane domain, ␣V249F is located N-terminal to the conserved leucines and is not predicted to face the channel lumen. Expression of the ␣V249F AChR in HEK fibroblasts demonstrates increased channel openings in the absence of ACh, prolonged openings in its presence, enhanced steady-state desensitization, and nanomolar rather than micromolar affinity of one of the two binding sites in the resting activatable state. Thus, neuromuscular transmission is compromised because cationic overloading leads to degenerating junctional folds and loss of AChR, because an increased fraction of AChR is desensitized in the resting state, and because physiological rates of stimulation elicit additional desensitization and depolarization block of transmission.
Pfl�gers Archiv European Journal of Physiology, 1991
Skeletal muscles of adult mice and frogs were dissociated enzymatically and prepared for patchclamping within less than 6 h. Outside-out patches were superfused with repetitive pulses of acetylcholine (ACh) with switching times of about 0.2 ms. Peak responses were reached within 1 ms. In mouse muscle the average channel conductance was 65 pS and the average open time 1 ms (20~ Between 1 and 10 txM ACh, the peak responses increased proportional to the second to third power of the ACh concentration, and less steeply between 10 and 1000 laM ACh. The apparent Km of the doseresponse curve was about 100 gM. After the peak, channel opening probability declined with time constants decreasing from about 1 s with 1 ~tM ACh to 15-50 ms with 1 mM ACh. After 100 ms desensitization the channel opening had decreased to less than 1/300 peak value. The rate of desensitization increased with rising temperature, with Qlo values of 1.7-2.5 between 10 and 30~ The desensitization characteristics of channels from frog muscle were similar to that from mice. With pulses of 100 ixM ACh the channels opened with a probability of 0.55, the open probability declining with a time constant of about 60 ms and dropping to less than 0.001 after 300 ms. The results support the view that three binding steps of ACh are necessary for opening of the channel. Desensitization in the presence of high ACh concentrations is slower than the decay of synaptic currents.
The Journal of General Physiology, 2000
We describe the genetic and kinetic defects in a congenital myasthenic syndrome due to the mutation ⑀ A411P in the amphipathic helix of the acetylcholine receptor (AChR) ⑀ subunit. Myasthenic patients from three unrelated families are either homozygous for ⑀ A411P or are heterozygous and harbor a null mutation in the second ⑀ allele, indicating that ⑀ A411P is recessive. We expressed human AChRs containing wild-type or A411P ⑀ 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 ⑀ A411P. Prolines engineered into positions flanking residue 411 of the ⑀ subunit greatly increase the range of activation kinetics similar to ⑀ A411P, whereas prolines engineered into positions equivalent to ⑀ A411 in  and ␦ subunits are without effect. Thus, the amphipathic helix of the ⑀ 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. key words: congenital myasthenic syndrome • single channel kinetics • hidden Markov modeling • channel gating • energy landscape I N T R O D U C T I O N
The dissociation of acetylcholine from open nicotinic receptor channels
Proceedings of the National Academy of Sciences, 2001
Ligand-gated ion channels bind agonists with higher affinity in the open than in the closed state. The kinetic basis of this increased affinity has remained unknown, because even though the rate constants of agonist association to and dissociation from closed receptors can be estimated with reasonable certainty, the kinetics of the binding steps in open receptors have proven to be elusive. To be able to measure the agonist-dissociation rate constant from open muscle nicotinic receptors, we increased the probability of ligand unbinding from the open state by engineering a number of mutations that speed up opening and slow down closing but leave the ligand-binding properties unchanged. Single-channel patchclamp recordings from the wild-type and mutant constructs were performed at very low concentrations of acetylcholine (ACh). The durations of individual channel activations were analyzed assuming that ''bursts'' of fully liganded (diliganded) receptor openings can be terminated by ligand dissociation from the closed or open state (followed by fast closure) or by desensitization. This analysis revealed that ACh dissociates from diliganded open receptors at Ϸ24 s ؊1 , that is, Ϸ2,500 times more slowly than from diliganded closed receptors. This change alone without a concomitant change in the association rate constant to the open state quantitatively accounts for the increased equilibrium affinity of the open channel for ACh. Also, the results predict that both desensitization and ACh dissociation from the open state frequently terminate bursts of openings in naturally occurring gain-of-function mutants (which cause slow-channel congenital myasthenia) and therefore would contribute significantly to the time course of the endplate current decay in these disease conditions.
Human Molecular Genetics, 1997
Congenital myasthenic syndromes are a group of rare genetic disorders that compromise neuromuscular transmission. A subset of these disorders, the slowchannel congenital myasthenic syndrome (SCCMS), is dominantly inherited and has been shown to involve mutations within the muscle acetylcholine receptor (AChR). We have identified three new SCCMS mutations and a further familial case of the αG153S mutation. Single channel recordings from wild-type and mutant human AChR expressed in Xenopus oocytes demonstrate that each mutation prolongs channel activation episodes. The novel mutations αV156M, αT254I and αS269I are in different functional domains of the AChR α subunit. Whereas αT254I is in the pore-lining region, like five of six previously reported SCCMS mutations, αS269I and αV156M are in extracellular domains. αS269I lies within the short extracellular sequence between M2 and M3, and identifies a new region of muscle AChR involved in ACh binding/channel gating. αV156M, although located close to αG153S which has been shown to increase ACh binding affinity, appears to alter channel function through a different molecular mechanism. Our results demonstrate heterogeneity in the SCCMS, indicate new regions of the AChR involved in ACh binding/channel gating and highlight the potential role of mutations outside the pore-lining regions in altering channel function in other ion channel disorders.
A ?-subunit mutation in the acetylcholine receptor channel gate causes severe slow-channel syndrome
Annals of Neurology, 1996
Point mutations in the genes encoding the acetylcholine receptor (AChR) subunits have been recognized in some patients with slow-channel congenital myasthenic syndromes (CMS). Clinical, electrophysiological, and pathological differences between these patients may be due to the distinct effects of individual mutations. We report that a spontaneous mutation of the p subunit that interrupts the leucine ring of the AChR channel gate causes an eightfold increase in channel open time and a severe CMS characterized by severe endplate myopathy and extensive remodeling of the postsynaptic membrane. The pronounced abnormalities in neuromuscular synaptic architecture and function, muscle fiber damage and weakness, resulting from a single point mutation are a dramatic example of a mutation having a dominant gain of function and of hereditary excitotoxicity.
Human Molecular Genetics, 1996
Mutations in genes encoding the ε, δ, β and α subunits of the end plate acetylcholine (ACh) receptor (AChR) are described and functionally characterized in three slow-channel congenital myasthenic syndrome patients. All three had prolonged end plate currents and AChR channel opening episodes and an end plate myopathy with loss of AChR from degenerating junctional folds. Genetic analysis revealed heterozygous mutations: εL269F and δQ267E in Patient 1, βV266M in Patient 2, and αN217K in Patient 3 that were not detected in 100 normal controls. Patients 1 and 2 have no similarly affected relatives; in Patient 3, the mutation cosegregates with the disease in three generations. εL269F, δQ267E and βV266M occur in the second and αN217K in the first transmembrane domain of AChR subunits; all have been postulated to contribute to the lining of the upper half of the channel lumen and all but δQ267E are positioned toward the channel lumen, and introduce an enlarged side chain. Expression studies in HEK cells indicate that all of the mutations express normal amounts of AChR. εL269F, βV266M, and αN217K slow the rate of channel closure in the presence of ACh and increase apparent affinity for ACh; εL269F and αN217K enhance desensitization, and εL269F and βV266M cause pathologic channel openings in the absence of ACh, rendering the channel leaky. δQ267E has none of these effects and is therefore a rare polymorphism or a benign mutation. The end plate myopathy stems from cationic overloading of the postsynaptic region. The safety margin of neuromuscular transmission is compromised by AChR loss from the junctional folds and by a depolarization block owing to temporal summation of prolonged end plate potentials at physiologic rates of stimulation.