Activation of acetylcholine receptors on clonal mammalian BC3H‐1 cells by low concentrations of agonist.

Sine, Steven M.; Steinbach, Joe Henry

doi:10.1113/jphysiol.1986.sp016039

Cited by 128 publications

(153 citation statements)

References 33 publications

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“…Open-and closed-duration histograms were constructed using a logarithmic abscissa and square root ordinate (Sigworth and Sine, 1987) and fitted to the sum of exponentials by maximum likelihood. The resulting time constants, relative areas, and total numbers of events were used to determine opening, closing, and agonist dissociation rate constants as described (Sine and Steinbach, 1986). For openings interrupted by closings approaching the instrumentation dead time (18 sec), we determined the true mean open duration from the total open duration in the recording (apparent mean open duration multiplied by the total number of openings) divided by the total number of brief closings (determined from the area of the brief component of closings) as described (Sine et al, 1990).…”

Section: Construction Of Human Wild-t Ype and Mutant Achr Cdnas And Ementioning

confidence: 99%

Slow-Channel Myasthenic Syndrome Caused By Enhanced Activation, Desensitization, and Agonist Binding Affinity Attributable to Mutation in the M2 Domain of the Acetylcholine Receptor α Subunit

Milone¹,

Wang

Ohno³

et al. 1997

J. Neurosci.

133

140

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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.Key words: slow-channel congenital myasthenic syndrome; neuromuscular transmission; endplate myopathy; acetylcholine receptor ␣ subunit gene; mutation analysis; single-channel recording; desensitization; agonist binding affinity Recent studies of congenital myasthenic syndromes (CMS) revealed mutations in acetylcholine receptor (AChR) subunit genes that either reduce expression of AChR (Engel et al., 1996a) or alter its kinetic properties to decrease (Ohno et al., 1996) or increase Sine et al., 1995; Engel et al., 1996b;Gomez et al., 1996) the response to ACh. Mutations that increase response to ACh prolong elementary activation episodes, and these disorders are referred to as slow-channel C MS (SCCMS). Mutations underlying SCC MS have been identified in different AChR subunits, and in different domains of the subunits, and affect AChR f unction through different mechanisms. The mutation ␣G153S is in the major extracellular domain and near residues that contribute to ligand binding; it increases affinity of ACh for the resting closed state and prolongs activation episodes by allowing multiple reopenings before ACh can dissociate . The mutation ␣N217K is in the M1 transmembrane domain and both slows the rate of channel closing and allows multiple reopenings per activation episode (Engel et al., 1996b). Mutations in the M2 domain, ⑀T264P, ⑀L269F, and ␤V266M Engel et al., 1996b), increase spontaneous opening of the channel, increase apparent affinity for ACh, and slow the rate of channel closing.Here we describe and functionally characterize the mutation ␣V249F in the M2 domain that causes severe SCCMS through a novel combination of mechanisms. Our mechanistic studies reveal functional consequences attributable to local perturbation of the M2 domain, including increased opening in the absence and prolonged opening in the p...

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Section: Construction Of Human Wild-t Ype and Mutant Achr Cdnas And Ementioning

confidence: 99%

Slow-Channel Myasthenic Syndrome Caused By Enhanced Activation, Desensitization, and Agonist Binding Affinity Attributable to Mutation in the M2 Domain of the Acetylcholine Receptor α Subunit

Milone¹,

Wang

Ohno³

et al. 1997

J. Neurosci.

133

140

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“…These pioneering studies of single channel currents from mouse myotubes led to the estimate that the unliganded gating equilibrium constant of γ-AChRs is ∼1.6 × 10 −6 . γ-AChRs were also the first channels for which the rate and equilibrium constants for closed channel binding and liganded gating were measured (13,(20)(21)(22).We have used improved methods of analysis to extend these studies of γ-AChRs with regard to both the rate constants and thermodynamics of activation. In adult AChRs, the two binding sites are functionally equivalent, but in the fetal subtype, the α−γ site has a higher affinity for ACh and provides more energy for gating than α−δ/e.…”

mentioning

confidence: 99%

mentioning

confidence: 99%

Asymmetric transmitter binding sites of fetal muscle acetylcholine receptors shape their synaptic response

Nayak

Auerbach

2013

Proc. Natl. Acad. Sci. U.S.A.

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Neuromuscular acetylcholine receptors (AChRs) have two transmitter binding sites: at α−δ and either α−γ (fetal) or α-e (adult) subunit interfaces. The γ-subunit of fetal AChRs is indispensable for the proper development of neuromuscular synapses. We estimated parameters for acetylcholine (ACh) binding and gating from single channel currents of fetal mouse AChRs expressed in tissuecultured cells. The unliganded gating equilibrium constant is smaller and less voltage-dependent than in adult AChRs. However, the α−γ binding site has a higher affinity for ACh and provides more binding energy for gating compared with α−e; therefore, the diliganded gating equilibrium constant at −100 mV is comparable for both receptor subtypes. The −2.2 kcal/mol extra binding energy from α−γ compared with α−δ and α−e is accompanied by a higher resting affinity for ACh, mainly because of slower transmitter dissociation. End plate current simulations suggest that the higher affinity and increased energy from α−γ are essential for generating synaptic responses at low pulse [ACh].are ligand-gated ion channels comprised of five subunits: two α(1) and one each of β, δ, and either γ or e. In most species, the γ-subunit is replaced by e during postnatal development. Electrical activity in muscle cells induced by the neurotransmitter acetylcholine (ACh) is important for the molecular maturation of γ-to e-AChRs (1, 2).Without γ-subunit, neuromuscular synapses do not develop properly (3) and are abnormal in innervation patterns (4) and muscle fiber-type composition (5). In mice, the γ-subunit KO is lethal (6), and in humans, mutations in the cholinergic receptor, nicotinic, gamma (CHRNG) gene that encodes for γ-subunit are associated with lethal forms of multiple pterygium and Escobar syndromes (7-9).AChRs have two transmitter binding sites located in the extracellular domain at α-δ and α-γ/e-subunit interfaces. In adult AChRs, the α−δ and α−e sites are equal and independent insofar as each has the same affinity for ACh and supplies the same amount of binding energy for gating (10). In fetal AChRs, the two binding sites are asymmetric with regard to agonist affinity, with estimates for the ratio of resting equilibrium dissociation constants for ACh ranging from ∼5-(11, 12) to >100-fold (13, 14). γ-AChRs also have a longer open channel lifetime, a smaller unitary conductance, and a lower Ca 2+ permeability than e-AChRs (15-17).Fetal AChRs hold a special place in the history of ion channel biophysics. The first single channel recordings from cells were of γ-AChRs (18). They were also the first ion channels to be analyzed according to a thermodynamic cycle, which required quantifying constitutive activity in the absence of agonists (19). These pioneering studies of single channel currents from mouse myotubes led to the estimate that the unliganded gating equilibrium constant of γ-AChRs is ∼1.6 × 10 −6 . γ-AChRs were also the first channels for which the rate and equilibrium constants for closed channel binding and liganded gating were measured (13,(...

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“…Further- (for review, see Brehm and Henderson, 1988;Witzeman et al, 1989;Brenner et al, 1990). Only the early phenotype had previously been described, both by molecular and physiological methods, in cultured rodent muscle cells (Siegelbaum et al, 1984;Buonanno and Merlie, 1986;Changeux et al, 1986;Sine and Steinbach, 1986;Yu et al, 1986;Evans et al, 1987;Mulle et al, 1988). However, recently, expression of the c-subunit gene was detected in cultured mouse muscle cells (Martinou an Merlie, 1991 the muscle from which they were derived.…”

Section: Introductionmentioning

confidence: 99%

“…On the other hand, aneural Xenopus myotomal muscle maintained in culture expresses the same percentage of high conductance channels as in vivo, suggesting that conversion from embryonic to adult type of AChR is at best only weakly dependent on innervation, at least in this species (Brehm et al, 1984;Owens and Kullberg, 1989a,b). Yet, aneural cultures of rodent muscle cells [rat primary cultures (Siegelbaum et al, 1984) and mouse muscle cell line C2 (Changeux et al, 1986), Sol 8 (Mulle et al, 1988) and BC3H1 (Sine and Steinbach, 1986) (Figure 4). Since cells differentiated on plastic dishes frequently detached from dishes after eight days, this process was difficult to analyse at later stages in culture.…”

Section: Introductionmentioning

confidence: 99%

Functional adult acetylcholine receptor develops independently of motor innervation in Sol 8 mouse muscle cell line.

et al. 1991

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Activation of acetylcholine receptors on clonal mammalian BC3H‐1 cells by low concentrations of agonist.

Cited by 128 publications

References 33 publications

Slow-Channel Myasthenic Syndrome Caused By Enhanced Activation, Desensitization, and Agonist Binding Affinity Attributable to Mutation in the M2 Domain of the Acetylcholine Receptor α Subunit

Slow-Channel Myasthenic Syndrome Caused By Enhanced Activation, Desensitization, and Agonist Binding Affinity Attributable to Mutation in the M2 Domain of the Acetylcholine Receptor α Subunit

Asymmetric transmitter binding sites of fetal muscle acetylcholine receptors shape their synaptic response

Functional adult acetylcholine receptor develops independently of motor innervation in Sol 8 mouse muscle cell line.

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