Pathogenic point mutations in a transmembrane domain of the ε subunit increase the Ca<sup>2+</sup> permeability of the human endplate ACh receptor

Castro, Maria Amalia Di; Martinello, Katiuscia; Grassi, Francesca; Eusebi, Fabrizio; Engel, Andrew G.

doi:10.1113/jphysiol.2007.127977

Cited by 24 publications

(24 citation statements)

References 19 publications

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“…Single channel recordings obtained under outside-out or cell-attached conditions were filtered at 5 kHz and sampled at 25 kHz. Data were analysed with pCLAMP 9, using 50% threshold criterion, omitting events shorter than 0.12 ms, as previously described (Di Castro et al 2007). In cell-attached recordings, slope conductance was calculated by linear fitting of the unitary amplitudes recorded at least at three different pipette potentials for each cell.…”

Section: Patch Clamp Recordings In Cultured Cellsmentioning

confidence: 99%

Riluzole blocks human muscle acetylcholine receptors

Deflorio

Palma

Conti

et al. 2012

The Journal of Physiology

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Key points• Riluzole is the only drug available against amyotrophic lateral sclerosis (ALS), a fatal disease characterized by death of motor neurones.• Recently it has been shown to block muscle ACh receptors (AChRs), raising concerns about possible side-effects on neuromuscular transmission in patients.• In this work we studied the effect of riluzole on the function of muscle AChRs in vitro and on neuromuscular transmission in ALS patients.• Data indicate that riluzole is apparently safe regarding neuromuscular transmission in patients.• However, riluzole may affect the function of AChRs expressed in denervated muscle fibres of ALS patients, with biological consequences that remain to be investigated.Abstract Riluzole, the only drug available against amyotrophic lateral sclerosis (ALS), has recently been shown to block muscle ACh receptors (AChRs), raising concerns about possible negative side-effects on neuromuscular transmission in treated patients. In this work we studied riluzole's impact on the function of muscle AChRs in vitro and on neuromuscular transmission in ALS patients, using electrophysiological techniques. Human recombinant AChRs composed of α 1 β 1 δ subunits plus the γ or ε subunit (γ-or ε-AChR) were expressed in HEK cells or Xenopus oocytes. In both preparations, riluzole at 0.5 μM, a clinically relevant concentration, reversibly reduced the amplitude and accelerated the decay of ACh-evoked current if applied before coapplication with ACh. The action on γ-AChRs was more potent and faster than on ε-AChRs. In HEK outside-out patches, riluzole-induced block of macroscopic ACh-evoked current gradually developed during the initial milliseconds of ACh presence. Single channel recordings in HEK cells and in human myotubes from ALS patients showed that riluzole prolongs channel closed time, but has no effect on channel conductance and open duration. Finally, compound muscle action potentials (CMAPs) evoked by nerve stimulation in ALS patients remained unaltered after a 1 week suspension of riluzole treatment. These data indicate that riluzole, while apparently safe with regard to synaptic transmission, may affect the function of AChRs expressed in denervated muscle fibres of ALS patients, with biological consequences that remain to be investigated.

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Section: Patch Clamp Recordings In Cultured Cellsmentioning

confidence: 99%

Riluzole blocks human muscle acetylcholine receptors

Deflorio

Palma

Conti

et al. 2012

The Journal of Physiology

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“…However, at least two slow-channel mutations in the ε M2 domain of AChR (εT264P and εV259F) increase the Ca 2+ permeability 1.5-fold and twofold, respectively (Di Castro et al 2007) and further enhance the deleterious effects of the prolonged synaptic currents and the intrinsically high Ca 2+ permeability of human AChR.…”

Section: Slow-channel Cmsmentioning

confidence: 99%

What Have We Learned from the Congenital Myasthenic Syndromes

et al. 2009

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The congenital myasthenic syndromes have now been traced to an array of molecular targets at the neuromuscular junction encoded by no fewer than 11 disease genes. The disease genes were identified by the candidate gene approach, using clues derived from clinical, electrophysiological, cytochemical, and ultrastructural features. For example, electrophysiologic studies in patients suffering from sudden episodes of apnea pointed to a defect in acetylcholine resynthesis and CHAT as the candidate gene (Ohno et al., Proc Natl Acad Sci USA 98:2017–2022–2001); refractoriness to anticholinesterase medications and partial or complete absence of acetylcholinesterase (AChE) from the endplates (EPs) has pointed to one of the two genes (COLQ and ACHET) encoding AChE, though mutations were observed only in COLQ. After a series of patients carrying mutations in a disease gene have been identified, the emerging genotype–phenotype correlations provided clues for targeted mutation analysis in other patients. Mutations in EP-specific proteins also prompted expression studies that proved pathogenicity, highlighted important functional domains of the abnormal proteins, and pointed to rational therapy.

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“…Rat pituitary GH4C1 cells were plated onto poly-L-lysinecoated glass coverslips and cultured in F10 medium supplemented by 10% FBS and 1% penicillin/streptomycin as previously described [7]. GH4C1 …”

Section: Cell Culture and Transfectionmentioning

confidence: 99%

Mutant human β4 subunit identified in amyotrophic lateral sclerosis patients impairs nicotinic receptor function

Moriconi

Angelantonio

Piccioni

et al. 2010

Pflugers Arch - Eur J Physiol

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Recently identified mutations in the genes encoding the neuronal nicotinic ACh receptor (nAChR) subunits in patients affected by sporadic amyotrophic lateral sclerosis (sALS) may represent a factor which enhances disease susceptibility, in particular in association with ambient causes such as cigarette smoking. In this work, we characterize the functional properties of nAChRs containing the β4R349C subunit, the mutation most frequently encountered in sALS patients. The mutation was coexpressed with wild-type α3 or α4 subunits or with mutant α4R487Q subunit, which has been detected in one patient together with β4R349C mutation. None of the functional parameters examined showed differences between α4β4 and α4R487Qβ4 nAChRs. By contrast, β4R349C mutation, independent of the companion α subunit, caused the reduction in potency of both ACh and nicotine, decreased the density of whole-cell current evoked by maximal transmitter concentrations, and altered the kinetics of ACh-evoked whole-cell currents. These data confirm that sALS-associated mutations in nicotinic subunits may markedly perturb cholinergic transmission in individuals bearing the mutations.

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Pathogenic point mutations in a transmembrane domain of the ε subunit increase the Ca²⁺ permeability of the human endplate ACh receptor

Cited by 24 publications

References 19 publications

Riluzole blocks human muscle acetylcholine receptors

Riluzole blocks human muscle acetylcholine receptors

What Have We Learned from the Congenital Myasthenic Syndromes

Mutant human β4 subunit identified in amyotrophic lateral sclerosis patients impairs nicotinic receptor function

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Pathogenic point mutations in a transmembrane domain of the ε subunit increase the Ca2+ permeability of the human endplate ACh receptor

Cited by 24 publications

References 19 publications

Riluzole blocks human muscle acetylcholine receptors

Riluzole blocks human muscle acetylcholine receptors

What Have We Learned from the Congenital Myasthenic Syndromes

Mutant human β4 subunit identified in amyotrophic lateral sclerosis patients impairs nicotinic receptor function

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Pathogenic point mutations in a transmembrane domain of the ε subunit increase the Ca²⁺ permeability of the human endplate ACh receptor