Congenital myasthenic syndromes due to heteroallelic nonsense ...

 1997 Oxford University Press

Human Molecular Genetics, 1997, Vol. 6, No. 5

753–766

Congenital myasthenic syndromes due to heteroallelic nonsense/missense mutations in the acetylcholine receptor ε subunit gene: identification and functional characterization of six new mutations Kinji 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) ε 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 γ-AChR at the endplates in one patient, prolongation of some channel events in another and γ-AChR expression as well as some shorter than normal channel events in still another. Genetic analysis revealed two recessive and heteroallelic ε subunit gene mutations in each patient. One mutation in each (εC190T [εR64X], ε127ins5 and ε553del7) generates a nonsense codon that predicts truncation of the ε subunit in its N-terminal, extracellular domain; and one mutation in each generates a missense codon (εR147L, εP245L and εR311W). 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 εR147L is kinetically benign, εP245L prolongs burst open duration 2-fold by slowing the rate of channel closing and εR311W 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 γ-AChR, changes in the release of transmitter quanta and appearance of multiple endplate regions on the muscle fiber. INTRODUCTION 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 α2βδε. 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 α, β and ε 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 ε subunit gene that cause severe EP AChR deficiency (19–22). Here we report and functionally characterize six mutations of the AChR ε subunit gene in three CMS patients who have severe

*To whom correspondence should be addressed. Tel: +1 507 284 5102; Fax: +1 507 284 5831; Email: [email protected]

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EP AChR deficiency. Each patient carries a nonsense and a missense mutation on different alleles of the ε subunit gene. Each nonsense mutation predicts truncation of the ε 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 ε 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 γ in place of an ε subunit. γ-AChR is readily distinguished from ε-AChR by its smaller single channel current amplitude and increased mean open duration (23,24). In two of the three patients, we detected significant γ-AChR that appeared to compensate for deficiency of ε-AChR. RESULTS Patients 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. Endplate studies 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. 1A 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. 1C). 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. EP AChR deficiency. This was established by several measures. The reaction for AChR, detected by fluorescence microscopy

Figure 1. (A) and (B) Cholinesterase-reactive EP regions in patient 2 (A) and in a control subject (B). Note the dispersion of EP regions over an extended length of the muscle fiber in patient 2. (C) and (D) Ultrastructural localization of AChR with peroxidase-labeled α-bgt at an EP from patient 3 (C) and at a control EP (D). The control EP shows heavy reaction for AChR on the terminal expansions of the junctional folds. At the patient’s EP, the junctional folds are simplified, the reaction for AChR is attenuated (arrows) and the length of the postsynaptic membrane reacting for AChR is reduced. (A) and (B), ×310; (C), ×21 900; (D), ×6500.

with rhodamine-labeled α-bungarotoxin (α-bgt) and by electron microscopy with peroxidase-labeled α-bgt (Fig. 1C and D), was markedly attenuated. The AChR index [defined as the ratio of the postsynaptic membrane reacting for AChR to the length of the primary synaptic cleft (25)] was only 14–23% of the control mean (Table 1) and the reaction for AChR on the junctional folds was patchy as well as of reduced intensity (Fig. 1C). The number of [125I]α-bgt-binding sites per EP was also markedly decreased (Table 1). Finally, the amplitudes of the miniature EP potential (MEPP) and of the miniature EP current (MEPC) were decreased to 8–26% and to 20–42% of normal, consistent with EP AChR deficiency. These values, however, may represent overestimates as the smallest potentials and currents were probably lost in the baseline noise. The number of transmitter quanta released by nerve impulse was normal in patient 1 but was increased in patients 2 and 3, perhaps as an adaptive response to decreased postsynaptic sensitivity to acetylcholine (ACh) (26,27) (Table 1).

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Table 1. Endplate studies

EPP quantal

contenta

MEPP amplitude, mVb MEPC amplitude,

nAc

τMEPC, msc

Patient 1

Patient 2

Patient 3

Controls

33 ± 2.0 (14)

46 ± 4 (15)

45 ± 6 (15)

31 ± 1 (190)

0.26 ± 0.037 (14)

0.081 ± 0.003 (10)

0.20 ± 0.015 (15)

1.00 ± 0.025 (165)

1.66 ± 0.15 (7)

0.78 ± 0.09 (4)

1.38 ± 0.08 (13)

3.95 ± 0.10 (79)

(i) 2.27 ± 0.09 (7)

3.97 ± 0.23

(i) 0.61 ± 0.10 (9)

3.23 ± 0.06 (79)

(ii) 9.54 ± 0.23 (7) τnoise,

(ii) 6.63 ± 0.87 (9)

(i) 2.32 ± 0.06 (4)

msc

(i) 1.85 ± 0.48 (3)

ND

(ii) 9.75 ± 0.13 (4)

2.30 ± 0.043 (52)

(ii) 9.19 ± 1.15 (3)

[125I]α-bgt binding sites/EP

0.8 E6

1.0 E6

0.63 E6

12.8 ± 0.79 E6 (13)

AChR index

0.77 ± 0.10 (27)

0.46 ± 0.04 (56)

0.45 ± 0.04 (35)

3.3 ± 0.08 (155)

Values represent mean ± SE; numbers in parenthesis indicate number of EPs except for [125I]α-bgt binding sites/EP where they indicate number of controls. T = 29 ± 0.5C for EPP and MEPP recordings and 22 ± 0.5C for noise analysis and MEPC studies. ND, not determined. aQuantal content of EPP at 1 Hz stimulation corrected for resting membrane potential of –80 mV, non-linear summation, and non-Poisson release. bCorrected for resting membrane potential of –80 mV and normalized for a fiber diameter of 60 µm. c–80 mV. Table 2. Kinetic parameters of opening bursts of AChR channels at control and patient EPs Subjects

Controls

Patient 1

Patient 2

Bursts

No. of EPs

Conductance, pS

τ1, ms (a1)

τ2, ms (a2)

τ3, ms (a3)

60 ± 0.5

0.12 ± 0.012

3.04 ± 0.17

ND

41a

(0.16 ± 0.01)

(0.85 ± 0.01)

0.17 ± 0.03

9.54 ± 0.89

ND

7

(0.19 ± 0.03)

(0.81 ± 0.03)

0.048

2.09

6.51

4b

(0.37)

(0.52)

(0.11)

46 ± 1.0

62

Values indicate mean ±SE. τn and an indicate time constants and relative areas for burst components. ACh concentration = 1 µM; potential = –80 mV; T = 22C ± 0.5C. ND, not detected. aFirst component not detected at two EPs. bCombined data from four EPs.

Kinetic properties of the EP AChR channels. We estimated the duration of channel activation episodes from the decay time constant of the MEPC (τMEPC) (28) (Table 1, patients 1, 2 and 3) and from spectral analysis of the ACh-induced current noise (τnoise) (29) (Table 1, patients 1 and 3) and obtained more precise measurements of the open duration and conductance of channel events from single channel patch-clamp recordings (Table 2, patients 1 and 2). In patient 1, the MEPC decayed biexponentially, suggesting two populations of channel openings, with one τMEPC close to normal and one significantly prolonged; noise analysis yielded similar results. Patch-clamp recordings demonstrated that 99% of the channel events opened to a conductance of 46 pS compared with the normal of 60 pS and that the predominant component of both the channel open intervals and bursts was ∼3-fold prolonged (Fig. 2). Less than 1% of the recorded channel events resembled those found at normal EPs. These findings suggest that the EP AChRs contain mostly the fetal γ subunit (γ-AChR) in place of the adult ε subunit (ε-AChR) (23) but that mutant AChR channels are probably also expressed. In patient 2, the τMEPC was mildly prolonged. Patch-clamp recordings captured only 442 channel openings at four EPs. These

channels opened to a normal conductance. The open intervals and bursts of openings had a very brief minor and a longer major component, similar to those at the control EPs, and a third component that was 2-fold longer than normal (Fig. 2). Even these limited observations indicate the presence of a kinetically abnormal AChR at the EPs. In patient 3, the MEPC decayed biexponentially, with one τMEPC shorter and one 2-fold longer than normal; similarly, noise analysis revealed two time constants, one shorter and one 3-fold longer than normal. These findings suggest expression of γ-AChR as well as a mutant AChR with reduced open duration, or expression of two mutant AChRs, one with a prolonged and one with a reduced open duration. To summarize, the EP studies reveal severe EP AChR deficiency and a kinetic abnormality of at least a fraction of the AChR channels in each case. In patient 1, patch-clamp recordings are consistent with expression of γ-AChR but τnoise and τMEPC suggest an additional population of normal duration openings. In patient 2, τMEPC suggests slightly prolonged channel opening events and patch-clamp recordings resolve two populations of events, one normal and one prolonged. In patient 3, both τnoise and τMEPC indicate two

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Figure 2. AChR channel currents (shown as upward deflections) elicited by 1 µM ACh at a control EP and at EPs of patient 1 and 2. Left: in patient 1, the amplitude of the channel currents is lower than normal. In both patients, some of the opening bursts are prolonged. Right: open duration histograms fitted by the sum of exponentials. Note the prolonged second time constant in patient 1 and the prolonged third time constant in patient 2. Control:τ1 = 0.11 ms, a1 = 0.14, τ2 = 3.42 ms, a2 = 0.86, total bursts, 2530. Patient 1: τ1 = 0.26 ms, a1 = 0.21, τ2 = 8.34 ms, a2 = 0.79, total bursts, 2192. Patient 2: τ1 = 0.05 ms, a1 = 0.37, τ2 = 2.09 ms, a2 = 0.52, τ3 = 6.51 ms, a3 = 0.11, total bursts, 241.

populations of channel events, one shorter than normal and one prolonged, with the latter again representing γ-AChR. Mutation analysis 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 α, β, δ and ε subunit genes in patients 1 and 2 and of the ε subunit gene in patient 3. Patient 1. Direct sequencing revealed two ε subunit gene mutations and three polymorphisms in the α, β and δ subunit genes. The first mutation is a C→T transition in ε exon 4 at nucleotide 190 (εC190T) that converts an arginine codon to a TGA stop codon at position 64 (εR64X) (Fig. 3A) and predicts truncation of the ε subunit in its extracellular domain. The second mutation is a G→T transversion in ε exon 5 at nucleotide 440 (εG440T) that converts an arginine to a leucine codon at position 147 (εR147L) in the extracellular domain of ε (Fig. 3A). The mutated arginine is conserved across ε subunits of other species, but not in other subunits (Fig. 3C). As εG440T (εR147L) 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). εR64X causes gain of a ScaI and εR147L 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 εR64X and the asymptomatic father and brother have εR147L (Fig. 3B). 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.

Table 3. Identified polymorphisms in AChR subunit genes and their allelic frequencies in humans Patient 1

Patient 2

αG-18+59T* (11/28)

αG-18+59T (11/28)

βA26G [βE9G] (40/124)

α130-13insT (29/70)

δA-52G (32/64)

βA26G [βE9G] (40/124) βT541+6C (13/58) βT1296+17C (13/64) δG57A* (32/64)

Nucleotides and codons are numbered from the beginning of the mature peptide. Negative nucleotide numbers are in the signal peptide region. + or – symbols after nucleotide numbers indicate the position in an intron relative to the nearest last or first base of an exon. Allelic frequencies are shown in parentheses. Codon changes, if any, are shown in square brackets. *Homozygous polymorphism. Nomenclature for designating polymorphisms is according to Beaudet and Tsui (55).

Patient 2. Direct sequencing revealed two mutations in the ε subunit gene and six polymorphisms in the α, β and δ subunit genes. The first mutation is duplication in ε exon 2 of ‘CTCAC’ at nucleotide 123–127 (ε127ins5). ε127ins5 converts Leu–Asn– Glu codons to Pro–His–Stop at position 43–45 (Fig. 4A) and predicts truncation of the ε subunit in its extracellular domain. Although the ε127ins5 is positioned close to the end of exon 2, no aberrant splicing was detected by RT-PCR spanning the mutation (data not shown). The second mutation was a C→T transition in ε exon 7 at nucleotide 734 (εC734T) that converts a proline to a leucine codon at position 245 (εP245L) (Fig. 4A). The mutated proline is part of the palindromic sequence marking the C-terminal end of the M1 domain and is conserved across all species and subunits (Fig. 2C). ε127ins5 causes loss of a BslI and εP245L loss of a MspI site. Restriction analysis of DNA samples


Figure 3. (A) Left: automated sequencing of AChR ε exon 4 around codon 64 in patient 1. Both C and T nucleotides are present at position 190 (arrow), indicating a heterozygous C→T transition. This mutation changes codon 64 from a CGA for arginine to a TGA stop codon (εR64X). Right: automated sequencing of AChR ε exon 5 around codon 147 in patient 1. Both G and T nucleotides are present at position 440 (arrow), indicating a heterozygous G→T transversion. This mutation changes codon 147 from a CGC for arginine to a CTC for leucine (εR147L). (B) Restriction enzyme analysis using genomic DNA from blood of patient 1 and his relatives. For the εR64X mutation (upper panel), the wild-type allele yields an undigested fragment of 134 bp; the mutant allele gives rise to two fragments of 115 and 19 bp. The 19 bp fragment is not shown in the figure. Both wild-type and mutant fragments are present in the mother, the patient and an affected brother. The father and an asymptomatic brother show only the wild-type fragment. For the εR147L mutation (lower panel), the wild-type allele yields two fragments of 185 and 26 bp; the mutant allele gives rise to an undigested fragment of 211 bp. The 26 bp fragment is not shown in the figure. The mutant fragment is present in four family members but not in the mother. The arrow indicates patient 1. Closed symbols show affected individuals. (C) Multiple alignment of a part of the extracellular domain of muscle nicotinic AChR. Boxes enclose the conserved arginine in AChR ε subunits of other species.

demonstrated that patient 2 and her affected brother have both mutations, the asymptomatic maternal grandmother has no mutation and the asymptomatic mother and father have ε127ins5

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Figure 4. (A) Left: automated sequencing of AChR ε exon 2 around the duplication in patient 2. Nucleotides ‘CTCAC’ (underlined) at 123–127 are duplicated in one allele (ε127ins5). The respective wild-type and mutant sequences are shown in the upper and lower rows. Right: automated sequencing of AChR ε exon 7 around codon 245 in patient 2. Both C and T nucleotides are present at position 734 (arrow), indicating a heterozygous C→T transition. This mutation changes codon 245 from a CCG for proline to a CTG for leucine (εP245L). (B) Restriction enzyme analysis using genomic DNA from blood of patient 2 and her relatives. For the ε127ins5 mutation (upper panel), the wild-type allele yields three fragments of 119, 45 and 24 bp; the mutant allele gives rise to two fragments of 119 and 74 bp. The 24 bp fragment is not shown in the figure. Both wild-type and mutant fragments are present in the mother, the patient and an affected brother. The father and the maternal grandmother show only a wild-type fragment. For the εP245L mutation (lower panel), the wild-type allele yields four fragments of 82, 80, 67 and 42 bp; the mutant allele gives rise to three fragments of 124, 80 and 67 bp. The 42 bp fragment is not shown in the figure. The mutant fragment is present in the patient, the father and the affected brother, but not in the mother or maternal grandmother. The arrow indicates patient 2. Closed symbols show affected individuals. (C) Multiple alignment of M1 domain of muscle nicotinic AChR. Boxes enclose the conserved proline in other human AChR subunits and in AChR ε subunits of other species.

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Figure 6. Schematic representation of the AChR ε subunit and the missense (open circles) and nonsense (closed circles) mutations identified in the three CMS patients.

and εP245L, respectively (Fig. 4B). Screening for ε127ins5 by restriction analysis and for εP245L by allele-specific PCR revealed neither mutation in 100 normal controls or 58 other unrelated CMS patients. Table 3 lists the six polymorphisms identified in patient 2.

Figure 5. (A) Left: automated sequencing of AChR ε exon 7 around the deletion in patient 3. A train of double peaks indicates a heterozygous 7 bp deletion at nucleotides 553–559 (ε553del7). The deleted seven nucleotides are ‘TGGGCCA’. The respective wild-type and mutant sequences are shown in the upper and lower rows. Right: automated sequencing of AChR ε exon 9 around codon 311 in patient 3. Both C and T nucleotides are present at position 931 (arrow), indicating a heterozygous C→T transition. This mutation changes codon 311 from a CGG for arginine to a TGG for tryptophan (εR311W). (B) Restriction analysis using genomic DNA from blood of patient 3 and her relatives. For the ε553del7 mutation (upper panel), the wild-type allele yields an undigested fragment of 271 bp; the mutant allele gives rise to two fragments of 218 and 46 bp. The 46 bp fragment is not shown in the figure. Both wild-type and mutant fragments are present in the mother, the patient and her sister. Her father and two children show only the wild-type fragment. For the εR311W mutation (lower panel), the wild-type allele yields six fragments of 114, 51, 28, 19, 9 and 2 bp; the mutant allele gives rise to five fragments of 142, 51, 19, 9 and 2 bp. Fragments smaller than 114 bp are not shown in the figure. Both wild-type and mutant fragments are present in the patient, her father and her two children. The patient’s mother and sister show only the wild-type fragment. The arrow indicates patient 3. None of the relatives are affected. (C) Multiple alignment of part of the M3 membrane-spanning domains (underlined) and long cytoplasmic loop of muscle nicotinic AChR. Boxes enclose the conserved arginine in other human AChR subunits and in AChR ε subunits of other species.

Patient 3. Direct sequencing of the ε subunit gene revealed two mutations and no polymorphisms. The first mutation is a frameshifting 7 bp deletion at nucleotides 553–559 (ε553del7) predicting premature termination of translation at codon 191, 18 bp downstream from the site of deletion (Fig. 5A), and truncation of the ε subunit in its extracellular domain. We previously have observed ε553del7 as a heterozygous and homozygous mutation in two other CMS patients, and have reported this in abstracts (20,21). The second mutation is a C→T transition in ε exon 9 at nucleotide 931 (εC931T) that converts an arginine to a tryptophan codon at position 311 (εR311W) (Fig. 5A). The mutated arginine residue is close to the N-terminal end of the long cytoplasmic loop of the ε subunit and is conserved across all species and all subunits (Fig. 5C). ε553del7 results in gain of a HinfI site and εR311W in loss of an AciI site. Restriction analysis of DNA samples demonstrated both mutations in patient 3, ε553del7 in her asymptomatic mother and sister and εR311W in her asymptomatic father and two children (Fig. 5B). Screening for ε553del7 by restriction analysis did not reveal it in 100 normal controls and 56 other CMS patients; and screening for ε311W by allele-specific PCR did not reveal it in 100 normal controls and 58 other CMS patients. To summarize, each of three CMS patients carries two recessive and heteroallelic ε subunit gene mutations. One mutation in each patient (εR64X, ε127ins5 and ε553del7) generates a nonsense codon, and one in each (εR147L, εP245L and εR311W) a missense codon. Figure 6 shows a schematic representation of the mutations in the ε subunit. Expression studies of mutant ε subunits AChR expression and affinity for ACh. To gain further insight into the molecular defects produced by the mutant ε subunits, we engineered each mutation into the human ε subunit and co-expressed


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Figure 8. Acetylcholine binding to intact cells transfected with the indicated missense (A) or nonsense (B) AChR cDNAs determined by competition against the initial rate of [125I]α-bgt binding. In (A), the competition profiles are fitted by the Hill equation (Equation 1, see Materials and Methods). In (B), the competition profiles are fitted by Equation 2, which describes binding to two sites with different affinity for ACh. For α2βδ2, KA = 9.6×10–8, KB = 1.5×10–2 and fractA = 0.48.

Figure 7. Expression of mutant ε subunits. (A) Total α-bgt binding to intact HEK cells transfected with the indicated missense ε plus complementary α, β and δ subunit cDNAs. The amount of bound [125I]α-bgt is normalized to that measured for the wild-type human AChR (α2βεδ). Non-specific binding is that measured in the presence of 300 µM d-tubocurarine (α-bgt + curare, filled bars). (B) Total α-bgt binding to intact HEK cells transfected with the indicated nonsense ε plus complementary α, β and δ subunit cDNAs. (C) Total α-bgt binding to saponin-permeabilized cells transfected with the indicated pairs of α and ε subunit cDNAs or with the α subunit alone. The amount of bound [125I]α-bgt is normalized to that measured for wild-type αε dimers. Nonspecific binding is that measured in the presence of 300 µM d-tubocurarine (α-bgt + curare, filled bars).

it with complementary α, β and δ subunits in 293 HEK cells. As controls, we co-expressed α, β and δ subunits with or without the wild-type ε subunit. Measurements of [125I]α-bgt binding to cell surface receptors revealed robust expression of wild-type receptors,

but reduced expression in the presence of each of the six mutant ε subunits. For each mutation, the number of α-bgt sites was similar to that observed for α2βδ2 pentamers (ε-omitted AChR) (Fig. 7A and B), indicating either no incorporation or reduced expression of surface pentamers containing the mutant ε subunits. To distinguish between lack of incorporation and reduced expression of the mutant subunits, we measured binding of ACh to AChRs harboring the mutant ε subunits by competition against the initial rate of [125I]α-bgt binding. AChRs harboring the three missense mutations, εR147L, εP245L and εR311W, bind ACh in a monophasic manner with dissociation constants coinciding with that of wild-type α2βεδ pentamers (Fig. 8A). Thus, ε subunits harboring the missense mutations incorporate into surface pentamers, but the level of expression is significantly reduced. By contrast, the three nonsense mutations, ε64X, ε127ins5 and ε553del7, give rise to biphasic ACh-binding profiles identical to that obtained with ε-omitted α2βδ2 AChR (Fig. 8B). The biphasic ACh-binding profile of the α2βδ2 AChR is attributed to loss of cooperative interactions between the two binding sites in the native pentamer, and is therefore a signature for pentamers lacking the ε subunit (13,30). Thus, ε subunits harboring the three nonsense mutations do not incorporate into surface pentamers. To determine whether the three nonsense ε mutations can assemble with the α subunit, one of the earliest steps in AChR assembly, we determined the number of αε complexes from the number of curare-displaceable α-bgt sites in cells permeabilized

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Figure 9. Single channel currents elicited by low concentrations of ACh for receptors containing the indicated ε subunits. Currents elicited by 30 nM ACh are shown filtered at 10 kHz with openings upward deflections (left column). The corresponding closed and burst duration histograms are shown fitted by the sum of exponentials (center and right columns). Bursts are defined as a series of closely spaced openings preceded and followed by closed intervals greater than a specified duration; this duration is taken as the point of intersection of the brief and long closed time components, or 200 µs in these recordings. The smooth curves are fits to the sum of two exponentials for closed and three exponentials for burst duration histograms. Table 1 lists the means of the brief component of closings and long component of bursts, and derived rate constants.

cDNAs. The current traces show that receptors harboring wild-type ε, εR147L, εP245L and εR311W open several times per activation episode, but that εP245L prolongs and εR311W shortens burst duration (Fig. 9). To examine the changes in burst duration quantitatively, we constructed burst duration histograms and fitted them with the sums of exponentials (Fig. 9). Burst distributions are best described as the sum of three exponential components for wild-type and the three mutant receptors. The two brief components are attributed to receptors with one bound agonist, because they vanish at high ACh concentrations (see Fig. 10), whereas the long component corresponds to receptors with two bound agonists. Durations of the brief bursts are not affected by any of the mutations, whereas durations of the long bursts are prolonged by εP245L, shortened by εR311W and not affected by εR147L (Table 4). Because long bursts predominate during synaptic activity, εR311 is expected to speed up and εP245L to slow down the decay of the EP current.

with saponin. The three nonsense mutations fail to assemble with the α subunit, as the number of specific α-bgt-binding sites is similar to that observed in the presence of the α subunit alone (Fig. 7C). Thus, proteins produced by the three nonsense mutations are either unstable or not capable of associating with the α subunit. To summarize, the expression studies show that the three nonsense mutations are indeed null mutations and that the surface expression of AChRs harboring the three missense mutations is significantly reduced. The presence of a heteroallelic nonsense and a missense mutation in the ε subunit gene in each patient predicts markedly reduced expression of mutant AChRs at the EP. Kinetics of activation of AChRs with ε missense mutations. We studied functional consequences of the missense mutations by recording single channel currents activated by low concentrations (30–100 nM) of ACh from 293 HEK cells transfected with either wild-type or mutant ε plus complementary α, β and δ subunit Table 4. Kinetic parameters and derived rate constants at low ACh concentrations No. of patches

τgaps, µs

τburst, ms

Gaps/burst

β, s–1

k–2, s–1

α, s–1

Wild-type

5

17.6

3.54

4.55

47 000 ± 7300

10 700 ± 2300

1600 ± 310

εR147L

2

18.8

3.10

3.70

42 000 ± 1500

11 400 ± 350

1530 ± 230

εP245L

3

21.8

6.45

4.77

38 000 ± 6800

8300 ± 1500

960 ± 240

εR311W

2

22.6

1.63

2.32

30 800 ± 1500

13 800 ± 2900

2160 ± 730


761

Figure 10. Kinetics of activation of human AChRs containing the indicated wild-type or mutant ε subunits (A, B and C) analyzed over a range of high ACh concentrations. Left column shows individual clusters of single channel currents recorded in the presence of the indicated ACh concentrations at a bandwidth of 10 kHz. Center and right columns show histograms of closed and open durations for each ACh concentration with the results of the fit to Scheme 1 superimposed. The fits were derived from data obtained at 3, 10, 30 and 100 µM ACh. The resulting global rate constants are given in Table 5.

Closed duration histograms are well described as the sum of two exponentials for wild-type and the three mutant receptors. The long duration component corresponds to periods between independent bursts of openings, and the brief component to transient interruptions of single channel bursts. The mean duration of brief closings is similar for wild-type and the three mutant receptors, indicating similar latencies to reopening for each receptor type (Table 4). To identify kinetic steps underlying the changes in burst duration, we describe activation of mutant and wild-type receptors according to the following scheme: k b k 1 k 2 AR AR A A 2R A 2R * B A 2R * B k –1 k –2 k –b

Scheme 1

where two agonists A bind to the receptor R with association rates k+1 and k+2 and dissociate from the receptor with rates k–1 and k–2.

Fully occupied receptors A2R open with rate β, and open receptors A2R* close with rate α. ACh blocks the open channel with the forward rate k+b, and unblocks with the rate k–b. As described for other species of AChR, the human wild-type and mutant receptors show temporal association of long openings and brief closings (31,32). Thus, we assign brief closings to dwells in A2R and long openings to dwells in A2R* in Scheme 1. Given these assignments, the mean duration of brief closings equals (β + k–2)–1, the number of brief closings per burst of long openings equals β/k–2, and the mean burst duration equals (1 + β/k–2)/α (33). These relationships lead to estimates of α, β and k–2 presented in Table 4. No change in these parameters is detected for εR147L, confirming that it is a kinetically benign mutation. By contrast, εP245L primarily slows the rate of channel closing to prolong burst duration; εP245L is thus a moderate slow channel mutation. Conversely, εR311W slows the rate of channel opening and speeds the rate of agonist dissociation to shorten burst duration; εR311W is thus a moderate fast channel mutation.

762


Table 5. Kinetic parameters for human wild-type, εP245L and εR311W mutant AChRs k+1

k–1

K1/µM

k+2

k–2

K2/µM

β

α

θ

k+block

k–block

KB/mM 3.2

Wild-type

151 ± 8

2880 ± 224

19

106 ± 3

15 200 ± 244

143

50 900 ± 1340

2160 ± 64

23

48 ± 14

155 000 ± 10 700

εP245L

97 ± 5

2380 ± 189

25

114 ± 4

15 000 ± 215

132

42 900 ± 1030

1100 ± 124

39

29 ± 9

154 000 ± 14 600

5.3

εR311W

176 ± 9

3810 ± 247

22

152 ± 4

23 600 ± 1060

155

36 200 ± 1060

2300 ± 44

16

19 ± 6

111 000 ± 16 000

5.8

Rate constants are as defined in Scheme 1 (see text) in units of µM–1s–1 for association rate constants and s–1 for all others. Values are results of a global fit of Scheme 1 to data obtained over the range 3–100 µM ACh, with standard errors (see Materials and Methods). The channel open equilibrium constant, θ, is ratio of the opening to the closing rate constant, β/α.

Kinetic analysis of currents elicited by high concentrations of ACh from AChRs harboring εP245L and εR311W. To determine whether additional steps in Scheme 1 are affected by the mutations, we examined the kinetics of εP245L and εR311W receptors by recording single channel currents over a range of desensitizing ACh concentrations (3–100 µM). Use of desensitizing ACh concentrations allows identification of clusters of events due to a single channel (34), while use of a range of concentrations allows all the states in Scheme 1 to contribute to the observed dwell times. For wild-type and mutant receptors, openings appear in readily recognizable clusters at concentrations from 3 to 100 µM ACh, with closed intervals within clusters becoming briefer with increasing ACh concentrations (Fig. 10). We then fitted Scheme 1 to the data by computing the likelihood of each open and closed dwell time, given a set of trial rate constants, and changing the rate constants to maximize the sum of the likelihoods (13,35). The results of the fit, shown as smooth curves superimposed on the open and closed duration histograms, reasonably describe the kinetics of wild-type, εP245L and εR311W receptors (Fig. 10). Comparison of the kinetics of wild-type and εP245L receptors reveals similar association, dissociation and channel opening rate constants for the two receptor types (Table 5). However, the rate constant for channel closing is slowed ∼2-fold, as observed in recordings obtained at limiting low ACh concentrations (Table 4). The estimates of α and β for mutant and wild-type were similar in the global and low concentration analyses, but the absolute values of k–2 were consistently greater in the global than in the low concentration analysis (Tables 4 and 5). We therefore reanalyzed the global set of data with k–2 fixed to the value obtained at low ACh concentrations, and allowed the other rate constants to vary. Comparison of likelihood values for the two analyses showed clearly superior descriptions of the data with the larger values of k–2. The global analysis is likely to be most reliable as it comprises considerably more data and describes the kinetics over a wide range of ACh concentrations. Thus, εP245L prolongs bursts solely by decreasing the rate of channel closing. Comparison of the kinetics of wild-type and εR311W receptors reveals similar rate constants for ACh association and channel closing, but increased rate constants for ACh dissociation and a decreased rate of channel opening. The global and low ACh concentration analyses give essentially the same estimates for β and α, while k–2 is somewhat greater in the global analysis. The opposite changes in β and k–2 lead to a decrease in the ratio β/k–2, indicating fewer openings per burst. Because α is unchanged, burst duration decreases in proportion to the decrease in β/k–2, as observed at low ACh concentrations (Table 4). Thus, εR311W reduces burst duration at the level of single channels, predicting faster decay of the EP current.

Patch-clamp recordings from HEK cells co-transfected with εR147L and wild-type γ subunit cDNAs. Although εR147L has no kinetic consequences, its expression may be curtailed in adult muscle by competition with the fetal γ subunit. To test this hypothesis, we co-expressed wild-type α, β, γ, δ and εR147L subunits in HEK cells and recorded ACh-induced single channel currents. This revealed predominantly channels with low conductance and long open duration characteristic of γ-AChR, with