Congenital myasthenic syndromes due to heteroallelic nonsense/missense mutations in the acetylcholine receptor [epsilon] subunit gene: identification and functional characterization of six new mutations
Congenital myasthenic syndromes due to heteroallelic nonsense/missense mutations in the acetylcholine receptor [epsilon] subunit gene: identification and functional characterization of six new mutationsKinji Ohno1, Polly A. Quiram2, Margherita Milone1, Hai-Long Wang2, Michel C. Harper1, J. Ned Pruitt II1, Joan M. Brengman1, Linda Pao1, Kenneth H. Fischbeck3, Thomas O. Crawford4, Steven M. Sine2 and Andrew G. Engel1,*
1Department of Neurology and Neuromuscular Research Laboratory and 2Department of Physiology and Biophysics and Receptor Biology Laboratory, Mayo Clinic and Foundation, Rochester, MN 55905, USA, 3Department of Neurology, University of Pennsylvania Medical Center, Philadelphia, PA 19104-6146, USA and 4Department of Pediatric Neurology, The Johns Hopkins Hospital, Baltimore, MD 21298-8811, USA
Received December 18, 1996;Revised and Accepted February 21, 1997
We describe and functionally characterize six mutations of the acetylcholine receptor (AChR) [epsilon] subunit gene in three congenital myasthenic syndrome patients. Endplate studies demonstrated severe endplate AChR deficiency, dispersed endplate regions and well preserved junctional folds in all three patients. Electrophysiologic studies were consistent with expression of the fetal [gamma]-AChR at the endplates in one patient, prolongation of some channel events in another and [gamma]-AChR expression as well as some shorter than normal channel events in still another. Genetic analysis revealed two recessive and heteroallelic [epsilon] subunit gene mutations in each patient. One mutation in each ([epsilon]C190T [[epsilon]R64X], [epsilon]127ins5 and [epsilon]553del7) generates a nonsense codon that predicts truncation of the [epsilon] subunit in its N-terminal, extracellular domain; and one mutation in each generates a missense codon ([epsilon]R147L, [epsilon]P245L and [epsilon]R311W). None of the mutations was detected in 100 controls. Expression studies in HEK cells indicate that the three nonsense mutations are null mutations and that surface expression of AChRs harboring the missense mutations is significantly reduced. Kinetic analysis of AChRs harboring the missense mutations show that [epsilon]R147L is kinetically benign, [epsilon]P245L prolongs burst open duration 2-fold by slowing the rate of channel closing and [epsilon]R311W shortens burst duration 2-fold by slowing the rate of channel opening and speeding the rate of ACh dissociation. The modest changes in activation kinetics are probably overshadowed by reduced expression of the missense mutations. The consequences of the endplate AChR deficiency are mitigated by persistent expression of [gamma]-AChR, changes in the release of transmitter quanta and appearance of multiple endplate regions on the muscle fiber.
Congenital myasthenic syndromes (CMS) are heterogeneous disorders in which the safety margin of neuromuscular transmission is compromised by one or more specific mechanisms (1 ). The CMS identified to date include endplate (EP) acetylcholinesterase (AChE) deficiency (2 ,3 ), presynaptic abnormalities that affect the release (4 ) or size (5 ) of transmitter quanta, or postsynaptic abnormalities associated with marked deficiency of the acetylcholine receptor (AChR), a kinetic abnormality of the AChR or both (1 ,6 -8 ).
The adult AChR is a pentamer of homologous subunits with the composition of [alpha]2[beta][delta][epsilon]. We hypothesized (1 ,9 ) and subsequently confirmed (10 -13 ) that a kinetic abnormality of AChR detected at the single channel level predicts a mutation involving one or more of its subunits. Kinetically abnormal mutations have been identified in the [alpha], [beta] and [epsilon] subunits, and do not reduce AChR expression appreciably (10 -13 ). We also posited that a severe deficiency of AChR could stem from nonsense mutations in one or more AChR subunit genes, although severe EP AChR deficiency also could result from primary defects in mechanisms that regulate the synthesis, aggregation, cytoskeletal attachment and metabolic stability of EP AChR (14 -18 ). We confirmed this notion by discovery of nonsense mutations in the [epsilon] subunit gene that cause severe EP AChR deficiency (19 -22 ).
Here we report and functionally characterize six mutations of the AChR [epsilon] subunit gene in three CMS patients who have severe EP AChR deficiency. Each patient carries a nonsense and a missense mutation on different alleles of the [epsilon] subunit gene. Each nonsense mutation predicts truncation of the [epsilon] subunit in its extracellular domain, and expression studies in HEK cells indicate that these are null mutations. Each missense mutation significantly reduces AChR expression, two of the missense mutations result in kinetic abnormalities of the AChR and one missense mutation is kinetically benign.
We also describe compensatory mechanisms that mitigate the effects of these mutations in the [epsilon] subunit. Normally, fetal AChR is present at human EPs until the 31st week of gestation, reappears following denervation, is suppressed by nerve contact or electrical activity and contains a [gamma] in place of an [epsilon] subunit. [gamma]-AChR is readily distinguished from [epsilon]-AChR by its smaller single channel current amplitude and increased mean open duration (23 ,24 ). In two of the three patients, we detected significant [gamma]-AChR that appeared to compensate for deficiency of [epsilon]-AChR.
All three CMS patients had myasthenic symptoms since the neonatal period. Patient 1, an 11-year-old male, had decreased movements in utero, a weak cry and a feeble suck after birth, ptosis of the eyelids since 5 months of age and ophthalmoparesis by the age of 2 years. He always fatigued easily, could never run well and had difficulty climbing steps. Patient 2, an 8-year-old female, had a weak cry at birth, ptosis since the age of 8 months and ophthalmoparesis since the age of 2 years. She learned to walk at the age of 18 months, always fatigued easily and could not run. Patient 3, a 31-year-old female, had a poor suck and cry after birth, ptosis in the neonatal period and breathing and swallowing difficulties at 4 months of age. Since then she had numerous episodes of impaired respiration and fatigued easily on exertion. She could not run, climbed steps with difficulty and could walk only a few hundred yards without having to rest. Patients 1 and 2 both have a similarly affected sibling. Patient 3 has no history of similarly affected relatives. All three have negative tests for anti-AChR antibodies, a decremental electromyographic response on stimulation of motor nerves and respond favorably but incompletely to AChE inhibitors.
EP morphology. This was similar in the three patients. The configuration of the EPs, evaluated from the cytochemical reaction product for AChE on teased single muscle fibers, was abnormal, with an increased number of small EP regions distributed over a 3- to 7-fold increased span of the muscle fiber surface (Fig. 1 A and B). Electron microscopy revealed that the structural integrity of most EPs was well preserved; a few junctional folds were degenerating in patient 2, the junctional sarcoplasm harbored a few myeloid structures in patients 2 and 3, and in all three patients some postsynaptic regions appeared simplified (Fig. 1 C). At individual EP regions, the mean nerve terminal size was reduced to 59, 65 and 42% and the mean area of junctional folds and clefts to 45, 44 and 29% of normal. However, owing to the increased number of regions per EP, the total nerve terminal and postsynaptic volumes per EP were probably greater rather than smaller than normal.
To determine whether the EP AChR deficiency of the CMS patients was caused by mutations in genes encoding AChR subunits, we used genomic DNA to sequence the exons and their flanking intronic regions of the [alpha], [beta], [delta] and [epsilon] subunit genes in patients 1 and 2 and of the [epsilon] subunit gene in patient 3.Patient 1. Direct sequencing revealed two [epsilon] subunit gene mutations and three polymorphisms in the [alpha], [beta] and [delta] subunit genes. The first mutation is a C -> T transition in [epsilon] exon 4 at nucleotide 190 ([epsilon]C190T) that converts an arginine codon to a TGA stop codon at position 64 ([epsilon]R64X) (Fig. 3 A) and predicts truncation of the [epsilon] subunit in its extracellular domain. The second mutation is a G -> T transversion in [epsilon] exon 5 at nucleotide 440 ([epsilon]G440T) that converts an arginine to a leucine codon at position 147 ([epsilon]R147L) in the extracellular domain of [epsilon] (Fig. 3 A). The mutated arginine is conserved across [epsilon] subunits of other species, but not in other subunits (Fig. 3 C). As [epsilon]G440T ([epsilon]R147L) alters a nucleotide at the end of exon 5, we searched for aberrant splicing due to this mutation using RT-PCR but detected none (data not shown). [epsilon]R64X causes gain of a ScaI and [epsilon]R147L loss of a BsaWI site. Restriction analysis of DNA samples demonstrated that patient 1 and his affected brother have both mutations, the asymptomatic mother has [epsilon]R64X and the asymptomatic father and brother have [epsilon]R147L (Fig. 3 B). Screening for the mutations by allele-specific PCR revealed neither mutation in 100 normal controls or 58 other unrelated CMS patients. Table 3 lists the three polymorphisms identified in patient 1.
Each patient carries a nonsense and a missense mutation on different alleles of the AChR [epsilon] subunit gene. Expression studies indicate that none of the three [epsilon] subunits with nonsense mutations associates with the [alpha] subunit and none incorporates into surface pentamers in the presence of complementary subunits. Thus, these are null mutations, and the clinical phenotype in each patient is defined by the consequences of the corresponding missense mutation. The [epsilon] subunits with missense mutations incorporate into surface pentamers but with reduced efficiency. AChRs with these subunits bind ACh with normal affinity and are opened by ACh. Channel kinetics, however, are normal only for [epsilon]R147L-AChR but not for [epsilon]P245L- or [epsilon]R311W-AChRs. The effects of the prolonged bursts caused by [epsilon]P245L are mitigated by the paucity of AChRs at the EP, but shortening of the burst open duration by [epsilon]R311W may further impair the safety margin of neuromuscular transmission.
[epsilon]R147L. Our findings, together with previous mutagenesis studies, suggest that [epsilon]R147L leads to a novel predominance of [gamma]-AChR at the patient EP. [epsilon]R147L lies between Il45 and T150, two residues crucial for progression of assembly beyond [alpha][epsilon] dimers (36 ); our observation of reduced expression in HEK cells suggests that [epsilon]R147L adversely affects assembly. As indicated by the predominance of [gamma]-AChR in experiments in HEK cells co-expressing [gamma] and [epsilon]R147L subunits, [gamma] expressed in adult muscle may compete efficiently with [epsilon]R147L for assembly with complementary subunits. Evidence for this competition mechanism in adult muscle is the predominance of [gamma]-AChR in the MEPC, noise analysis and single channel recordings from patient 1. Persistent [gamma]-AChR is compatible with the recent observation that homozygous deletion of the [epsilon] subunit gene in mice results in persistent, though reduced [gamma] subunit gene expression in the adult mutants (37 ).[epsilon]R311W. This mutation borders the carboxy-terminus of the M3 transmembrane domain where the long cytoplasmic loop begins. In this location, [epsilon]R311 is an example of the `positive inside' rule for membrane protein topology in which positively charged residues contribute to orienting and anchoring the adjacent transmembrane domain (38 ). Our findings that [epsilon]R311W reduces channel open durations by slowing channel opening and speeding agonist dissociation indicate a perturbation of the gating apparatus and the ACh-binding site to which it is linked. The kinetic effect of [epsilon]R311W may be related to the contribution of the long cytoplasmic loop to the fetal-to-adult change in AChR kinetics (39 ). Owing to its location between transmembrane and cytoplasmic domains, [epsilon]R311W may reduce surface expression by impairing folding during AChR assembly. The expression studies also indicate that the long components of [tau]MEPC and [tau]noise observed at EPs of patient 3 are not due to mutant channels but rather to fetal [gamma]-AChR. As in the case of [epsilon]R147L, the fetal [gamma] subunit may compete efficiently with the [epsilon] subunit harboring [epsilon]R311W for [alpha], [beta] and [delta] subunits. [epsilon]P245L. The mutated P245 residue is part of the palindromic sequence thought to mark the carboxy-terminus of the M1 transmembrane domain. The structural contribution of P245 may be to align the M1 domain within the membrane and to produce a kink on the cytoplasmic face of the AChR, allowing the adjacent M2 channel domain to reverse direction and extend back across the membrane. Our expression studies indicate that [epsilon]P245L increases channel open duration by selectively slowing the rate of channel closing, perhaps owing to perturbation of the channel gating apparatus. The reduced efficiency of surface expression may result from impaired folding during assembly of the [epsilon]P245L-AChR. The presence of [epsilon]P245L in EP AChRs is evidenced by a population of prolonged channel opening episodes recorded from EPs of patient 2.
The sparse AChRs expressed at each patient's EPs probably comprise different species of the receptor macromolecule. These include AChR harboring the missense mutations [epsilon]R147L, [epsilon]P245L and [epsilon]R311W in patients 1, 2, and 3, respectively, [gamma]-AChR in patients 1 and 3, and possibly [epsilon]-omitted [alpha]2[beta][delta]2 AChR in any of the three patients.
We previously reported a low level of [gamma]-AChR expression at the EP in another patient with severe AChR deficiency due to heteroallelic null mutations in the long cytoplasmic loop of [epsilon] (19 ), and proposed that [gamma]-AChR expression may serve as the means of phenotypic rescue from potentially fatal mutations in the [epsilon] gene. As the [epsilon] subunit gene is located on chromosome 17 (40 ) and the [gamma] subunit gene on chromosome 2 (41 ), the persistent [gamma] expression in these diseases is not due to altered regulation of a single gene cluster by a common locus control region.
As regards the expression of [alpha]2[beta][delta]2 AChR, even if this species of AChR were expressed, it may not function efficiently due to its reduced affinity for ACh (13 ). The kinetic signature of the human [alpha]2[beta][delta]2 AChR is still unknown, and this precludes identifying it at the EP by single channel recordings.
In addition to persistent expression of the fetal [gamma]-AChR at the EPs (as in patients 1 and 3), other mechanisms may also help to improve neuromuscular transmission in our patients. The nerve sprouts, associated with the multiple focal EP regions (see Fig. 1 A), have an imprinting influence on the underlying nuclei to induce transcription of AChR subunit genes (14 ,42 ,43 ), augmenting the total amount of EP AChR. A trophic influence of muscle on nerve may be at work in increasing the number of ACh quanta released by nerve impulse (as in patients 2 and 3); alternatively, the increased quantal release stems from an increased number of active zones per EP due to the increased number of EP regions. Despite these compensatory mechanisms, the safety margin of neuromuscular transmission remains severely compromised by the AChR deficiency.
The postsynaptic CMS are associated with a kinetic abnormality of the AChR, a deficiency of the AChR or both (1 ). The kinetic abnormalities are associated with mutations in AChR subunits that enhance (the slow channel CMS) (10 -12 ,44 ) or reduce (the low-affinity fast channel syndrome) (13 ) the response to ACh.
The slow channel syndromes stem from dominant missense mutations that confer a pathological gain of function. The resulting prolonged opening episodes of the AChR channel overload the junctional sarcoplasm with cations, causing an EP myopathy with secondary loss of AChR from degenerating junctional folds, and cause a depolarization block due to staircase summation of prolonged EP potentials (10 -12 ). HEK cells expressing slow-channel mutations produce either normal (11 ,12 ) or only modestly reduced (10 ) amounts of AChR. Therefore, in the slow channel syndromes, the EP AChR deficiency is due predominantly to the EP myopathy and not to diminished expression of the mutated subunit.
The low-affinity fast channel syndrome results from a missense mutation that causes loss of function and requires a heteroallelic null mutation in the same subunit gene in order to become clinically manifest. There is no EP AChR deficiency, the structural integrity of the postsynaptic region is maintained, but AChR opening episodes are fewer and shorter than normal owing to a decreased affinity of AChR for ACh (13 ).
The CMS described in this report stem from nonsense and missense mutations in different alleles of the [epsilon] subunit gene. Only [epsilon] subunits carrying the missense mutations are expressed, but with reduced efficiency, and this results in severe EP AChR deficiency. The integrity of the postsynaptic region is preserved but the EPs now consist of multiple small regions that probably increase the area of synaptic contact. The [gamma]-AChR expression in patients 1 and 3 results in prolonged channel openings but the postsynaptic region is protected from cationic overloading because the synaptic current is restricted by the severe AChR deficiency, the [gamma]-AChR channel opens to a reduced conductance and [gamma]-AChR passes 3-fold less calcium than the mature [epsilon]-AChR (45 ).
Specimens of intercostal muscle were obtained intact from origin to insertion from patients and from control subjects without muscle disease undergoing thoracic surgery. All human studies were in accord with the guidelines of the Institutional Review Board of the Mayo Clinic.
Morphology and counts of AChR per EP.For electron microscopy, EPs were localized and analyzed by established methods (25 ). Peroxidase-labeled [alpha]-bgt was used for the ultrastructural localization of AChR (46 ). The number of AChRs per EP was measured with 125I-labeled [alpha]-bgt, as previously described (47 ).Intracellular microelectrode studies. MEPC and EPP recordings, estimates of the number of transmitter quanta released by nerve impulse and analysis of the ACh-induced current noise were carried out as previously described (8 ,47 ). Patch-clamp recordings from EP AChRs. These were performed in the cell-attached mode by a slight modification of the previously described method (10 ,48 ). For all patches, the membrane potential was set to -80 mV; when possible, recordings were also obtained at -40, -120 and -160 mV. These membrane potentials were achieved by applying a corresponding potential to the patch pipet, and assuming a resting potential of 0 mV; a null resting potential was confirmed by the absence of detectable channel events with 0 mV applied to the pipet. Channel currents were recorded using an Axopatch 200A amplifier (Axon Instruments), and analyzed at a final bandwidth of 5.8 kHz using the program PCLAMP 6 (Axon Instruments) or MacTac (Instrutech). Burst durations were determined by grouping openings separated by a specified closed time that misclassifies equal proportions of long closed times within bursts and short intervals between bursts (31 ). Dwell time histograms were plotted on logarithmic abscissa and fitted to the sum of exponentials by maximum likelihood (49 ).
mRNA and DNA samples. Genomic DNA was isolated from proteinase/SDS digest of blood or muscle by phenol-chloroform extraction followed by ethanol precipitation (50 ). mRNA was obtained using the Micro-FastTrack mRNA isolation kit (Invitrogen). First-strand cDNA was prepared from mRNA by using random hexamer primers with the cDNA Cycle kit (Invitrogen) and following the instructions of the manufacturer. Polymerase chain reaction procedures. PCR primers were designed to amplify exons with their flanking regions from each AChR subunit as previously described (10 ). Published cDNA sequences of the human [epsilon] subunit (40 ) were used to design cDNA primers. Sequence analysis. PCR-amplified fragments of genomic DNA or cDNA were purified by Wizard PCR Preps (Promega). Plasmids were purified by QIAwell 8 Plus Plasmid kit (Qiagen). DNA fragments and plasmids were sequenced with an Applied Biosystems model 377 DNA sequencer using fluorescently labeled dideoxy terminators.Restriction enzyme analysis and allele-specific PCR. We screened for all six mutations by restriction analysis in the patients' relatives, and we screened for [epsilon]127ins5 and [epsilon]553del7 by restriction analysis and for [epsilon]R64X, [epsilon]R147L, [epsilon]P245L and R311W by allele-specific PCR in other CMS patients and controls. The primers for amplifying DNA fragments for restriction analysis and for allele-specific PCR and the PCR conditions are available upon request.
Construction and expression of human wild-type and mutant AChRs.Human [alpha] and [delta] subunit cDNAs were generously provided by Dr Jon Lindstrom (51 ,52 ). The [beta] and [epsilon] subunit cDNAs were cloned from normal human skeletal muscle as described previously (13 ). cDNA for the normal human [gamma] subunit was cloned from a nested RT-PCR product amplified from normal muscle with Pfu DNA polymerase (Stratagene). The first primers for amplification were 5'-CTGCCCCAGACCTTGGAG-3' (nucleotides -124 to -107) and 5'-GAGAACGTGGCTGATAAGACT-3' (nucleotides 1540-1560); the nested primers were 5'-cggccggaattcTGTTGTCCCACCCCTGTCAC-3' (nucleotides -105 to -86) and 5'-cggccggaattcCTCCCACATGCCCCACAGTG-3' (nucleotides 1499-1518), the artificial lower case sequences serving to introduce an EcoRI site into the PCR product. The nested PCR product spanned nucleotides -105 to 1518, whereas the coding region of the [gamma] subunit extends from -66 to 1485. The nested PCR product was digested with EcoRI, gel purified and ligated into pBluescript II SK(-) (Stratagene). Sequencing of our [gamma] clone revealed no mutation or polymorphism when compared with the reference sequence of human [gamma] subunit gene (GenBank accession nos, X01715-X01721 and X04759) (53 ).
All five cDNAs were subcloned into the CMV-based expression vector pRBG4 (54 ) for expression in 293 HEK cells.
The identified mutations were introduced into the pRBG4 with the [epsilon] subunit cDNA insert using QuickChange Site-Directed Mutagenesis Kit (Stratagene). We synthesized two complementary oligonucleotides for each mutation. The sense sequences of the synthesized oligonucleotides were 5'-GATTGGCAGGATTACtGACTCAACTACAGC-3' for [epsilon]R64X, 5'-CGCTTATTTTCCtCTCTCAGACGTACAATG-3' for [epsilon]R147L, 5'-TCACCCTGAC- GAATCTCATCTCACctcacTGAATGAAAAAGAGGAGAC-3' for [epsilon]127ins5, 5'-CCTACTTCCTGCtGGCGCAGGCCGGC-3' for [epsilon]P245L and 5'-CAACGTGTCCCAGtGGACGCCCACCAC-3' for [epsilon]R311W. Lower case letters represent artificial nucleotides used to introduce the respective mutations. The site-directed mutagenesis reaction mixture contained 50 ng of pRBG4 vector harboring the [epsilon] subunit cDNA insert, 125 ng of oligonucleotide, 50 nM each dNTP, 10% dimethyl sulfoxide, 2.5 U of Pfu DNA polymerase and the supplied buffer in 50 [mu]l. To synthesize nicked circular double-stranded DNA with the mutation, temperature cycling was carried out in Thermal Cycler 480 (Perkin Elmer) with the following conditions: denaturation at 95oC for 30 s; 12 cycles of 95oC for 30 s, 55oC for 1 min, and 68oC for 12 min. The reaction mixture was then incubated with 10 U of DpnI at 37oC for 1 h to digest the methylated parental plasmids without the mutation. The nicked circular double-stranded DNA was introduced in XL1-Blue competent cells. The resultant pRBG4 vector containing the mutation was proliferated in LB broth and purified with Plasmid Maxi Kit (Qiagen) for transfection into 293 HEK cells. The presence of the desired mutation and the absence of unwanted mutations was confirmed by sequencing the entire insert.
A pRBG4 vector with the [epsilon]553del7 mutation was obtained by subcloning an RT-PCR product amplified from muscle mRNA of another patient who had the same mutation (20 ). The entire coding region of the [epsilon] subunit was amplified from mRNA using the nested PCR method with the same primers that we used to clone the wild-type [epsilon] subunit gene (13 ). The artificially introduced EcoRI sites at both ends of the nested RT-PCR product were digested with EcoRI and the mutant cDNA was then cloned into a pRBG4 vector.
Human embryonic kidney fibroblasts (293 HEK) were transfected with mutant or wild-type AChR subunit cDNAs using calcium phosphate precipitation as described (39 ).[alpha]-Bgt and ACh binding measurements. The total number of [125I][alpha]-bgt sites and ACh competition against the initial rate of [125I][alpha]-bgt binding were determined as previously described (13 ). ACh competition measurements were analyzed according to either the monophasic Hill equation (Equation 1) or the sum of two distinct binding sites (Equation 2):1 - Y= 1/(1 + ([ACh]/Kov)n)11 - Y=fractA{1/(1 + [ACh]/KA)} + (1 - fractA){1/(1 + [ACh]/KB)} 2
where Y is the fractional occupancy by ACh, Kov is an overall dissociation constant for a monophasic binding profile, KA and KB are intrinsic dissociation constants for two binding sites and fractA is the fraction of sites with dissociation constant KA.Patch-clamp recordings from AChRs expressed in HEK cells. Recordings were obtained in the cell-attached configuration, with a membrane potential of -70 mV, a temperature of 22oC and with bath and pipet solutions containing (mM): KCl 142, NaCl 5.4, CaCl2 1.8, MgCl2 1.7, HEPES 10, pH 7.4 (13 ,39 ). Single channel currents were recorded using an Axopatch 200A at a bandwidth of 50 kHz, digitized at 100 kHz, transferred to a Macintosh computer using the program Acquire (Instrutech Corp.) and detected by the half-amplitude threshold criterion using the program MacTac (Instrutech Corp.) at a final bandwidth of 10 kHz. Open and closed duration histograms were constructed using a logarithmic abscissa and square root ordinate (49 ), and fitted to the sum of exponentials by maximum likelihood. For kinetic analysis according to Scheme 1, clusters of openings corresponding to a single channel were identified and then examined for homogeneity in terms of open probability and mean open duration as previously described (13 ). The resulting durations of open and closed intervals were transferred to an IBM RS6000 computer and analyzed according to Scheme 1 using an interval-based maximum likelihood method that incorporated corrections for missed events (35 ). Single channel dwell times, obtained from single patches at several concentrations of ACh, were fitted simultaneously. After fitting, error estimates for each parameter were determined as described (35 ). Open and closed duration histograms were calculated from the fitted rate constants and superimposed on the experimental dwell time histograms.
1 Engel, A.G. (1994) Myasthenic syndromes. In Engel, A.G. and Franzini-Armstrong, C. (eds), Myology: Basic and Clinical. 2nd Edn. McGraw-Hill, New York, pp. 1798-1835.
2 Engel, A.G., Lambert, E.H. and Gomez, M.R. (1977) A new myasthenic syndrome with end-plate acetylcholinesterase deficiency, small nerve terminals, and reduced acetylcholine release. Ann. Neurol., 1, 315-330. MEDLINE Abstract
4 Walls, T.J., Engel, A.G., Nagel, A.S., Harper, C.M. and Trastek, V.F. (1993) Congenital myasthenic syndrome associated with paucity of synaptic vesicles and reduced quantal release. Ann. N.Y. Acad. Sci., 681, 461-468. MEDLINE Abstract
5 Mora, M., Lambert, E.H. and Engel, A.G. (1987) Synaptic vesicle abnormality in familial infantile myasthenia. Neurology, 37, 206-214. MEDLINE Abstract
6 Engel, A.G., Lambert, E.H., Mulder, D.M., Torres, C.F., Sahashi, K., Bertorini, T.E. and Whitaker, J.N. (1982) A newly recognized congenital myasthenic syndrome attributed to a prolonged open time of the acetylcholine-induced ion channel. Ann. Neurol., 11, 553-569. MEDLINE Abstract
7 Engel, A.G., Hutchinson, D.O., Nakano, S., Murphy, L., Griggs, R.C., Gu, Y., Hall, Z.W. and Lindstrom, J. (1993) Myasthenic syndromes attributed to mutations affecting the epsilon subunit of the acetylcholine receptor. Ann. N.Y. Acad. Sci., 681, 496-508. MEDLINE Abstract
8 Uchitel, O., Engel, A.G., Walls, T.J., Nagel, A., Atassi, Z.M. and Bril, V. (1993) Congenital myasthenic syndromes. II. A syndrome attributed to abnormal interaction of acetylcholine with its receptor. Muscle Nerve, 16, 1293-1301. MEDLINE Abstract
9 Engel, A.G. (1993) The investigation of congenital myasthenic syndromes. Ann. N.Y. Acad. Sci., 681, 425-434. MEDLINE Abstract
10 Ohno, K., Hutchinson, D.O., Milone, M., Brengman, J.M., Bouzat, C., Sine, S.M. and Engel, A.G. (1995) Congenital myasthenic syndrome caused by prolonged acetylcholine receptor channel openings due to a mutation in the M2 domain of the [epsilon] subunit. Proc. Natl Acad. Sci. USA, 92, 758-762. MEDLINE Abstract
11 Sine, S.M., Ohno, K., Bouzat, C., Auerbach, A., Milone, M., Pruitt, J.N. and Engel, A.G. (1995) Mutation of the acetylcholine receptor a subunit causes a slow-channel myasthenic syndrome by enhancing agonist binding affinity. Neuron, 15, 229-239. MEDLINE Abstract
12 Engel, A.G., Ohno, K., Milone, M., Wang, H.-L., Nakano, S., Bouzat, C., Pruitt, J.N., Hutchinson, D.O., Brengman, J.M., Bren, N., Sieb, J.P. and Sine, S.M. (1996) New mutations in acetylcholine receptor subunit genes reveal heterogeneity in the slow-channel congenital myasthenic syndrome. Hum. Mol. Genet., 5, 1217-1227. MEDLINE Abstract
13 Ohno, K., Wang, H.-L., Milone, M., Bren, N., Brengman, J.M., Nakano, S., Quiram, P., Pruitt, J.N., Sine, S.M. and Engel, A.G. (1996) Congenital myasthenic syndrome caused by decreased agonist binding affinity due to a mutation in the acetylcholine receptor [epsilon] subunit. Neuron, 17, 157-170. MEDLINE Abstract
14 Hall, Z.W. and Sanes, J.R. (1993) Synaptic structure and development: the neuromuscular junction. Cell, 72 (Suppl.), 99-121. MEDLINE Abstract
15 Phillips, W.D. (1995) Acetylcholine receptors and the cytoskeletal connection. Clin. Exp. Pharmacol. Physiol., 22, 961-965. MEDLINE Abstract
16 Gautam, M., Noakes, P.G., Mudd, J., Nichol, M., Chu, G.C., Sanes, J.R. and Merlie, J.P. (1995) Failure of postsynaptic specialization to develop at neuromuscular junctions of rapsyn-deficient mice. Nature, 377, 232-236. MEDLINE Abstract
17 Gillespie, S.K.H., Balasubramanian, S., Fung, E.T. and Huganir, R.L. (1996) Rapsyn clusters and activates the synapse-specific receptor tyrosine kinase MuSK. Neuron, 16, 953-962.
18 Gautam, M., Noakes, P.G., Moscoso, L., Rupp, F., Scheller, R.H., Merlie, M.P. and Sanes, J.R. (1996) Defective neuromuscular synaptogenesis in agrin-deficient mutant mice. Cell, 85, 525-535. MEDLINE Abstract
19 Engel, A.G., Ohno, K., Bouzat, C., Sine, S.M. and Griggs, R.G. (1996) End-plate acetylcholine receptor deficiency due to nonsense mutations in the [epsilon] subunit. Ann. Neurol., 40, 810-817. MEDLINE Abstract
20 Ohno, K., Engel, A.G., Milone, M., Brengman, J.M., Sieb, J.P. and Iannaccone, S. (1995) A congenital myasthenic syndrome with severe acetylcholine receptor deficiency caused by heteroallelic frameshifting mutations in the epsilon subunit. Neurology, 45 (Suppl. 4), A283 (Abstract)
21 Milone, M., Ohno, K., Pruitt, J.N., Brengman, J.M., Sine, S.M. and Engel, A.G. (1996) Congenital myasthenic syndrome due to frameshifting acetylcholine receptor epsilon subunit mutation. Soc. Neurosci. Abstr., 22, 1942.
22 Ohno, K., Fukudome, T., Nakano, S., Milone, M., Feasby, T.E., Tyce, G.M. and Engel, A.G. (1996) Mutational analysis in a congenital myasthenic syndrome reveals a novel acetylcholine receptor epsilon subunit mutation. Soc. Neurosci. Abstr., 22, 234.
23 Mishina, M., Takai, T., Imoto, K., Noda, M., Takahashi, T., Numa, S., Methfessel, C. and Sakmann, B. (1986) Molecular distinction between fetal and adult forms of muscle acetylcholine receptor. Nature, 321, 406-411. MEDLINE Abstract
24 Schuetze, S.M. and Role, L.W. (1987) Developmental regulation of nicotinic acetylcholine receptors. Annu. Rev. Neurosci., 10, 403-457. MEDLINE Abstract
25 Engel, A.G. (1994) Quantitative morphological studies of muscle. In Engel, A.G. and Franzini-Armstrong, C. (eds), Myology: Basic and Clinical. 2nd Edn. McGraw-Hill, New York, pp. 1018-1045.
26 Plomp, J.J., van Kempen, G.Th.H. and Molenaar, P.C. (1992) Adaptation of quantal content to decreased postsynaptic sensitivity at single endplates in a-bungarotoxin treated rats. J. Physiol. (Lond.), 458, 487-499. MEDLINE Abstract
27 Plomp, J.J., van Kempen, G.Th.H., De Baets, M.Bsch., Graus, Y.M.F., Kuks, J.B.M. and Molenaar, P.C. (1995) Acetylcholine release in myasthenia gravis: regulation at single end-plate level. Ann. Neurol., 37, 627-636. MEDLINE Abstract
28 Magleby, K.L. and Stevens, C.F. (1972) A quantitative description of end-plate currents. J. Physiol. (Lond.), 223, 173-197. MEDLINE Abstract
29 Anderson, C.R. and Stevens, C.F. (1973) Voltage clamp analysis of acetylcholine produced end-plate current fluctuations at frog neuromuscular junction. J. Physiol. (Lond.), 235, 655-691. MEDLINE Abstract
30 Sine, S.M. and Claudio, T. (1991) Gamma and delta subunits regulate the affinity and the cooperativity of ligand binding to the acetylcholine receptor. J. Biol. Chem., 266, 19369-19377. MEDLINE Abstract
31 Colquhoun, D. and Sakmann, B. (1985) Fast events in single channel currents activated by acetylcholine and its analogues at the frog muscle end-plate. J. Physiol. (Lond.), 369, 501-557. MEDLINE Abstract
32 Sine, S.M., Claudio, T. and Sigworth, F.J. (1990) Activation of Torpedo acetylcholine receptors expressed in mouse fibroblasts: single-channel current kinetics reveal distinct agonist binding affinities. J. Gen. Physiol., 96, 395-437. MEDLINE Abstract
33 Colquhoun, D. and Hawkes, A.G. (1981) On the stochastic properties of single ion channels. Proc. R. Soc. Lond. Ser. B., 211, 205-235.
34 Sakmann, B., Patlak, J. and Neher, E. (1980) Single acetylcholine-activated channels show burst kinetics in the presence of desensitizing concentrations of agonist. Nature, 286, 71-73. MEDLINE Abstract
35 Qin, F., Auerbach, A. and Sachs, F. (1996) Estimating single-channel kinetic parameters from idealized patch-clamp data containing missed events. Biophys. J., 70, 264-280. MEDLINE Abstract
36 Kreienkamp, H.-J., Maeda, R.K., Sine, S.M. and Taylor, P. (1995) Intersubunit contacts governing assembly of the mammalian nicotinic acetylcholine receptor. Neuron, 14, 635-644.
37 Witzemann, V., Schwartz, H., Koenen, M., Berberich, C., Villarroel, A., Wernig, A., Brenner, H.R. and Sakmann, B. (1996) Acetylcholine receptor [epsilon]-subunit gene deletion causes muscle weakness and atrophy in juvenile and adult mice. Proc. Natl Acad. Sci. USA, 93, 13286-13291. MEDLINE Abstract
38 von Heijne, G. and Gavel, Y. (1988) Topogenic signals in integral membrane proteins. Eur. J. Biochem., 174, 671-678. MEDLINE Abstract
39 Bouzat, C., Bren, N. and Sine, S.M. (1994) Structural basis of different gating kinetics of fetal and adult acetylcholine receptors. Neuron, 13, 1395-1402. MEDLINE Abstract
40 Beeson, D., Brydson, M., Betty, M., Jeremiah, S., Povey, S., Vincent, A. and Newsom-Davis, J. (1993) Primary structure of the human muscle acetylcholine receptor: cDNA cloning of the gamma and epsilon subunits. Eur. J. Biochem., 215, 229-238. MEDLINE Abstract
41 Cohen-Haguenauer, O., Barton, P.J., Buonanno, A., Cong, N.V., Masset, M., De Tand, M.F., Merlie, J. and Frezal, J. (1989) Localization of the acetylcholine receptor gamma subunit gene to human chromosome 2q32-qter. Cytogenet. Cell. Genet., 52, 124-127. MEDLINE Abstract
42 Sakmann, B., Witzemann, V. and Brenner, H. (1992) Developmental changes in acetylcholine receptor channel structure and function as a model for synaptic plasticity. Fidia Found. Neurosci. Award Lectures, 6, 51-103.
43 Moscoso, L.M., Chu, G.C., Gautam, M., Noakes, P.G., Merlie, J.P. and Sanes, J.R. (1995) Synapse-associated expression of an acetylcholine receptor-inducing protein, ARIA/heregulin, and its putative receptors, Erb2 and Erb3, in developing mammalian muscle. Dev. Biol., 172, 158-169. MEDLINE Abstract
44 Gomez, C.M., Maselli, R., Gammack, J., Lasalde, J., Tamamizu, S., Cornblath, D.R., Lehar, M., McNamee, M. and Kuncl, R. (1996) A beta-subunit mutation in the acetylcholine receptor gate causes severe slow-channel syndrome. Ann. Neurol., 39, 712-723. MEDLINE Abstract
45 Villarroel, A. and Sakmann, B. (1996) Calcium permeability increase of endplate channels in rat muscle during postnatal development. J. Physiol. (Lond.), 496, 331-338. MEDLINE Abstract
46 Engel, A.G., Lindstrom, J.M., Lambert, E.H. and Lennon, V.A. (1977) Ultrastructural localization of the acetylcholine receptor in myasthenia gravis and in its experimental autoimmune model. Neurology, 27, 307-315. MEDLINE Abstract
47 Engel, A.G., Nagel, A., Walls, T.J., Harper, C.M. and Waisburg, H.A. (1993) Congenital myasthenic syndromes. I. Deficiency and short open-time of the acetylcholine receptor. Muscle Nerve, 16, 1284-1292. MEDLINE Abstract
48 Milone, M., Hutchinson, D.O. and Engel, A.G. (1994) Patch-clamp analysis of the properties of acetylcholine receptor channels at the normal human endplate. Muscle Nerve, 17, 1364-1369. MEDLINE Abstract
49 Sigworth, F.J. and Sine, S.M. (1987) Data transformation for improved display and fitting of single-channel dwell time histograms. Biophys. J., 52, 1047-1054. MEDLINE Abstract
50 Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. 2nd Edn. Cold Spring Harbor Laboratory Press, Plainview, NY.
51 Schoepfer, R., Luther, M. and Lindstrom, J. (1988) The human medulloblastoma cell line TE671 expresses a muscle-like acetylcholine receptor. Cloning of the alpha-subunit cDNA. FEBS Lett., 226, 235-240. MEDLINE Abstract
52 Luther, M.A., Schoepfer, R., Whiting, P., Casey, B., Blatt, Y., Montal, M.S., Montal, M. and Lindstrom, J. (1989) A muscle acetylcholine receptor is expressed in the human cerebellar medulloblastoma cell line TE671. J. Neurosci., 9, 1082-1096. MEDLINE Abstract
53 Shibahara, S., Kubo, T., Perski, H.J., Takahashi, H., Noda, M. and Numa, S. (1985) Cloning and sequence analysis of human genomic DNA encoding gamma subunit precursor of muscle acetylcholine receptor. Eur. J. Biochem., 146, 15-22. MEDLINE Abstract
54 Lee, B.S., Gunn, R.B. and Kopito, R.R. (1991) Functional differences among nonerythroid anion exchangers expressed in a transfected human cell line. J. Biol. Chem., 266, 11448-11454. MEDLINE Abstract
55 Beaudet, A.L. and Tsui, L.-C. (1993) A suggested nomenclature for designating mutations. Hum. Mutat., 2, 245-248. MEDLINE Abstract
*To whom correspondence should be addressed. Tel: +1 507 284 5102; Fax: +1 507 284 5831; Email: age@mayo.edu
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