New mutations in acetylcholine receptor subunit genes reveal heterogeneity in the slow-channel congenital myasthenic syndrome
New mutations in acetylcholine receptor subunit genes reveal heterogeneity in the slow-channel congenital myasthenic syndromeAndrew G. Engel*, Kinji Ohno, Margherita Milone, Hai-Long Wang1, Satoshi Nakano, Cecilia Bouzat1, J. Ned Pruitt II, David O. Hutchinson, Joan M. Brengman, Nina Bren1, Joern P. Sieb and Steven M. Sine1
Department of Neurology and Neuromuscular Research Laboratory and 1Department of Physiology and Biophysics and Receptor Biology Laboratory, Mayo Clinic and Foundation, Rochester, MN 55905, USA
Received May 9, 1996;Revised and Accepted June 24, 1996
Mutations in genes encoding the [epsilon], [delta], [beta] and [alpha] subunits of the end plate acetylcholine (ACh) receptor (AChR) are described and functionally characterized in three slow-channel congenital myasthenic syndrome patients. All three had prolonged end plate currents and AChR channel opening episodes and an end plate myopathy with loss of AChR from degenerating junctional folds. Genetic analysis revealed heterozygous mutations: [epsilon]L269F and [delta]Q267E in Patient 1, [beta]V266M in Patient 2, and [alpha]N217K in Patient 3 that were not detected in 100 normal controls. Patients 1 and 2 have no similarly affected relatives; in Patient 3, the mutation cosegregates with the disease in three generations. [epsilon]L269F, [delta]Q267E and [beta]V266M occur in the second and [alpha]N217K in the first transmembrane domain of AChR subunits; all have been postulated to contribute to the lining of the upper half of the channel lumen and all but [delta]Q267E are positioned toward the channel lumen, and introduce an enlarged side chain. Expression studies in HEK cells indicate that all of the mutations express normal amounts of AChR. [epsilon]L269F, [beta]V266M, and [alpha]N217K slow the rate of channel closure in the presence of ACh and increase apparent affinity for ACh; [epsilon]L269F and [alpha]N217K enhance desensitization, and [epsilon]L269F and [beta]V266M cause pathologic channel openings in the absence of ACh, rendering the channel leaky. [delta]Q267E has none of these effects and is therefore a rare polymorphism or a benign mutation. The end plate myopathy stems from cationic overloading of the postsynaptic region. The safety margin of neuromuscular transmission is compromised by AChR loss from the junctional folds and by a depolarization block owing to temporal summation of prolonged end plate potentials at physiologic rates of stimulation.
After the discovery of the autoimmune origin of myasthenia gravis in the 1970s (1 ), it was realized that defects of neuromuscular transmission occurring in a congenital setting and in the absence of antibodies against the acetylcholine receptor (AChR) have different etiologies. Until recently, the congenital myasthenic syndromes (CMS) were defined by clinical, morphological, and electrophysiological criteria. The syndromes identified in this manner included end plate (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 AChR deficiency, or a kinetic abnormality of the AChR, or both (6 -9 ). In each syndrome, the safety margin of neuromuscular transmission is compromised by one or more specific mechanisms (9 ).
It was also hypothesized that a kinetic abnormality of AChR detected at the single channel level predicts a mutation involving one or more AChR subunits (7 ,9 ). This notion was subsequently confirmed by the discovery of mutations in two of the four homologous subunits of AChR, and in different structural domains of the subunits, that either increase (10 ,11 ) or decrease (12 ) response to acetylcholine (ACh). In the slow-channel CMS (SCCMS), the increased response to ACh consists of prolonged opening episodes of the AChR channel. Here we report identification and functional characterization of mutations of AChR in three patients with SCCMS. In each patient, cationic overloading of the postsynaptic region caused by cholinergic overactivity results in an EP myopathy with loss of AChR due to destruction of the junctional folds and temporal summation of the prolonged end plate potentials results in a depolarization block at physiologic rates of stimulation. Despite this common final pathway in pathogenesis, the mutations resulting in SCCMS are found in different AChR subunits, and even in different structural domains of the subunits. The findings reported here have implications for treatment of CMS, genetic counseling, and structure-function relationships of AChR.
Patient 1, a 16-year-old male, had myasthenic symptoms since early infancy involving ocular, trunkal and limb muscles. Since the age of 7, he also experienced intermittent episodes of respiratory insufficiency requiring ventilatory support. Patient 2, a 19-year-old female, had myasthenic symptoms since birth involving ocular, other cranial, and limb muscles. Patient 3, a 30-year-old female, had ocular muscle weakness since early childhood, limb muscle weakness since the age of 8, and scoliosis since the age of 10. She cannot walk more than 100 yards without having to rest and cannot climb stairs without assistance. Patients 1 and 2 have no history of similarly affected relatives; in Patient 3, the family history is consistent with autosomal dominant inheritance through three generations. All patients have negative tests for anti-AChR antibodies, a decremental electromyographic response on stimulation of motor nerves, and a repetitive compound muscle action potential response to single nerve stimuli, as seen in the SCCMS (6 ) or EP AChE deficiency (2 ).
Ultrastructural studies in all three patients showed an EP myopathy typical of the SCCMS (6 ) with destruction of many junctional folds, widened synaptic spaces, degeneration of membranous organelles in the junctional sarcoplasm, and focal myofibrillar degeneration and sometimes vacuolar change near EPs. Ultrastructural localization of AChR with peroxidase-[alpha]-bungarotoxin ([alpha]-BGT) revealed a normal density of AChR on the crests of those junctional folds that were intact and loss of AChR where the folds were destroyed. Some fiber nuclei near EPs displayed condensed chromatin and were convoluted or fragmented, suggesting apoptosis (13 ). Abundant AChE activity was detected at all EPs.
The number of transmitter quanta released by nerve impulse was normal in Patients 2 and 3 and fell in the low-normal range in Patient 1 (data not shown). In all three patients, miniature EP currents (MEPCs) decayed biexponentially, with one decay time that fell in the normal range and one that was 8- to 13-fold prolonged (Fig. 1 ). The mean MEPC amplitude was reduced to 43-69% of the normal mean. EP potentials were even more prolonged than EP currents, extending to over 50 ms in duration. Indeed, in each patient at physiologic rates of stimulation consecutive EP potentials arose during the decay of the preceding potential resulting in staircase summation of progressively smaller potentials (data not shown). The prolonged EP currents and potentials suggested a kinetic defect in one or more populations of EP AChRs.
To search for a kinetic defect of AChR, we recorded single channel currents from EPs of each SCCMS patient and compared the findings with those at 34 EPs from eight controls. Simple inspection of the recordings from each patient revealed a population of markedly prolonged channel events (Fig. 2 , left). On formal analysis, at control EPs both the open intervals and bursts had a very brief minor ([tau]1) and longer major ([tau]2) component, as previously described (14 ). At the SCCMS EPs, open intervals and bursts had two components, [tau]1 and [tau]2, similar to those observed at the control EPs, and a third component, [tau]3, that was 3- to 4-fold and 5- to 8-fold longer than the respective open interval and burst [tau]2 (Table 1 and Fig. 2 , right). With 1 [mu]M ACh, at control EPs there was no clear clustering of channel events but at SCCMS EPs long channel events frequently occurred in clusters separated by intervals ranging from 1.4-12 s (Patient 1), 0.8-10 s (Patient 2), and 3-40 s (Patient 3), suggesting increased desensitization (15 ). In Patient 3, we also recorded channel events with 0.1 [mu]M ACh and still detected clustering of long-duration channel events.
The above channel events at the SCCMS EPs, like more than 99% of channel events at normal EPs (14 ), had a conductance of ~60 pS. Approximately 30% of the channel events in Patient 1, 17% in Patient 3, and none in Patient 2 had a reduced conductance of ~46 pS and prolonged burst open durations (8 ms), typical of AChRs containing the [gamma] instead of the [epsilon] subunit ([gamma]AChR) (16 ). In addition, about 7% of the 46 pS channels in Patient 3 had a prolonged burst open duration of 20 ms. As in a previously reported case of SCCMS (10 ), we detected clear immunostaining for [gamma]AChR at some EPs of Patient 1; we found no staining for [gamma]AChR in Patient 2, and only trace staining in Patient 3 (17 ).
In each patient, the abnormally prolonged EP currents combined with a major class of prolonged ACh-evoked channel openings suggested a mutation in one or more AChR subunit genes.
. Kinetic parameters of open intervals and bursts of 60 pS channels at control and patient EPs
Subjects
Open intervals
Bursts
No. of EPs
[tau]1, ms (a1)
[tau]2, ms (a2)
[tau]3, ms (a3)
[tau]1, ms (a1)
[tau]2, ms (a2)
[tau]3, ms (a3)
Controls
0.20 +- 0.02
1.9 +- 0.09
ND
0.12 +- 0.012
3.04 +- 0.17
ND
34a
(0.16 +- 0.02)
(0.85 +- 0.02)
(0.16 +- 0.01)
(0.85 +- 0.01)
Patient 1
0.15 +- 0.05
1.86 +- 0.04
7.21 +- 1.26
0.19 +- 0.030
2.92 +- 0.13
16.67 +- 2.75
3
(0.09 +- 0.01)
(0.51 +- 0.13)
(0.4 +- 0.12)
(0.15 +- 0.04)
(0.53 +- 0.16)
(0.32 +- 0.13)
Patient 2
0.18 +- 0.06
2.09 +- 0.16
8.12 +- 0.53
0.15 +- 0.02
2.63 +- 0.23
18.41 +- 1.81
13b
(0.14 +- 0.01)
(0.40 +- 0.07)
(0.51 +- 0.06)
(0.14 +- 0.02)
(0.47 +- 0.05)
(0.46 +- 0.06)
Patient 3
0.21 +- 0.03
1.68 +- 0.13
5.69 +- 0.06
0.19 +- 0.025
2.65 +- 0.23
13.05 +- 1.74
6c
(0.24 +- 0.06)
(0.51 +- 0.04)
(0.30 +- 0.02)
(0.29 +- 0.07)
(0.57 +- 0.06)
(0.17 +- 0.04)
Time constants, [tau]n, and relative areas, an, for each component are presented as mean +- SE. Recordings were in the presence of 1 [mu]M ACh. Potential = -80 mV; T = 22oC +- 0.5oC. ND, not detected. aFirst component not detected at 2 EPs. bSecond or third component not detected at single EPs. cThird component not detected at 1 EP.
To evaluate pathogenicity of the four mutations, we engineered each mutation into the corresponding subunit cDNA of mouse AChR and coexpressed it with complementary wild-type subunit cDNAs in human embryonic kidney (HEK) fibroblasts. Measurements of [alpha]-BGT binding revealed robust expression of AChR containing each of the mutant subunits. Relative to wild type, amounts of [alpha]-BGT binding for the mutants were: 109 +- 22 % for [alpha]N217K, 109 +- 27 % for [beta]V266M, 102 +- 33 % for [epsilon]L269F, and 74 +- 11 % for [delta]Q267E (mean +- SD of three experiments).
We here report two new mutations, [beta]V266M and [alpha]N217K, in adult muscle AChR subunit genes that cause SCCMS. We functionally characterize these as well as a third mutation, [epsilon]L269F, previously reported by us (20 ) and also described by Gomez and Gamack (18 ). In the patient studied by us, [epsilon]L269F occurred together with [delta]Q267E. We now show that [delta]Q267E has no effect on AChR function. Therefore, it is a very infrequent polymorphism or, because its frequency in heterozygotes is less than 2%, a benign mutation (21 ). [alpha]N217K is also noteworthy in that it occurs in the M1 transmembrane domain whereas the other SCCMS mutations described here or reported previously are in the M2 domain of different subunits or in the extracellular domain of the [alpha] subunit (10 ,11 ).
The present study reconfirms the notion that a kinetic abnormality of AChR is presumptive evidence for a mutation in one or more AChR subunit genes (9 -11 ). In the cases investigated, a kinetic abnormality is indicated by abnormally prolonged decay of the end plate currents and by prolonged opening episodes of AChR channels. All channel events in Patient 2 and most channel events in Patients 1 and 3 had a normal 60 pS conductance. A minor proportion of channel events in Patients 1 and 3 had a reduced conductance of 46 pS and prolonged open durations typical of [gamma]AChR. In Patient 3, however, about 7% of the 46 pS channels had an additional even longer component. This component can be readily explained by [gamma]AChR that harbor one or possibly two [alpha] subunits with [alpha]N217K. That the 46 pS channels represent immature [gamma]AChRs rather than a second pathogenic mutation is also evidenced by discovery of only one pathogenic mutation in each patient, by normal conductance of the expressed mutant channels, and by the immunocytochemical localization of [gamma]AChR at some EPs of Patients 1 and 3. Thus, all SCCMS mutations described in this as well as in previous reports (10 ,11 ,18 ,20 ) can be attributed to a single mutation that causes a pathologic gain of function. However, mutations occurring in different domains of AChR exert their effect by different molecular mechanisms.
The expression studies confirmed that three mutations, one detected in each of the three SCCMS patients, result in prolonged activation episodes of AChR and are therefore pathogenic. In addition, they allowed studies of the mutant AChRs in the absence of non-mutant or [gamma]-subunit-containing AChRs. All four mutations lead to normal amounts of functional AChR in HEK cells, indicating a high probability of being present in functional AChRs at the synapse. Pathogenicity results from prolonged activation episodes caused by mutations in the M2 domain, [epsilon]L269F and [beta]V266M, and by mutation in the M1 domain [alpha]N217K. Increased spontaneous opening by the M2 mutations may also contribute to pathogenicity. The prolonged activation episodes are due in part to a slower than normal rate of channel closing, indicating stabilization of the open state by these mutations. Effects on opening rate or rate of ACh dissociation are also possible, but cannot be defined from the present studies. The increase in burst duration for each mutation correlates well with the time constant of the slowly decaying component in the MEPCs observed in the corresponding patient.
The expression system also allowed measurements of equili- brium ACh binding, which can detect changes in the contributions of high affinity open channel or desensitized states. Each of the pathogenic mutations lead to increased apparent affinity for ACh. The apparent affinity of the [beta]V266M AChR increased two-fold relative to wild type, that for [epsilon]L269F increased nine-fold, and that for [alpha]N217K increased 34-fold. A two-fold increase in apparent affinity can be accounted for by about a 50-fold increase in the open channel equilibrium constant (22 ); although open channel equilibrium constants were not measured, the observed seven- to 41-fold increases in burst duration are sufficiently large to suggest increases in the open channel equilibrium constant of this magnitude. Increases in ACh affinity greater than about two-fold are therefore likely to be due to increased desensitization. Thus we hypothesize that the extent of desensitization is not altered by [beta]V266M, is enhanced by [epsilon]L269F, and is markedly enhanced by [alpha]N217K. Indeed, the extent of equilibrium desensitization by [alpha]N217K is expected to be near maximal, as its binding profile coincides with that of the fully desensitized AChR (11 ).
Both cryoelectron microscopy (19 ) and site-directed mutagenesis studies (23 ) indicate that the M2 transmembrane domain consists of two alpha helical regions joined in the center by an extended structure of approximately three residues. Within the extended structure is a highly conserved leucine residue (human [alpha]L251, [beta]L262, [delta]L265, [epsilon]L261) that may form a hydrophobic barrier to ion flow. All three of the M2 mutations reported here, as well as the previously reported [epsilon]T264P mutation (10 ) are within the outer helical region facing the synapse (Fig. 8 ). The site of the mutation in the [beta] subunit, V266, is the fourth residue in the C-terminal direction from the central leucine, is accessible to labeling reagents when converted to cysteine (23 ), and is predicted to face the lumen of the channel. The site of the mutation in the [epsilon] subunit, L269, is the eighth residue C-terminal to the central leucine, is not affected by labeling reagents when converted to cysteine, but is predicted to face the lumen of the channel. The site of the mutation in the [delta] subunit, Q267, is the second residue C-terminal to the central leucine, is not susceptible to labeling reagents when converted to cysteine, and is predicted to face away from the lumen of the channel. The non-lumenal position of [delta]Q267 or that the side-chain length of [delta]E267 is unchanged may account for the lack of effect on ion permeation or gating when mutated to the negatively charged glutamate. Common features of the three pathogenic M2 mutations are location in the synaptic half of M2, positioning toward the channel lumen, and introduction of a larger side-chain. Pathogenicity of the three M2 mutations suggests a role for these residues in stabilizing both the open and closed states of the channel.
SCCMS can have several adverse effects on neuromuscular transmission. Cationic overloading of the postsynaptic region caused by the cholinergic overactivity results in an EP myopathy with loss of AChR due to destruction of the junctional folds (6 ,26 ). Abnormal channel openings occurring even in the absence of ACh might also contribute to the abnormal postsynaptic ionic milieu and therefore to the EP myopathy. The temporal summation of EP potentials at physiological rates of stimulation predicts a depolarization block of transmission, as has also been demonstrated in experimental organophosphate poisoning (27 ). Finally, an increased proportion of the mutant AChRs is likely to become desensitized in the presence of AChE inhibitors (28 ). The duration of the synaptic response is likely to determine the severity of the EP myopathy and the degree of depolarization block, whereas the variable desensitization of the different mutant AChRs in the presence of ACh will govern the adverse response to AChE inhibitors. It follows that AChE inhibitors, which are beneficial in myasthenic disorders caused by a decreased number of kinetically normal AChRs, should be avoided in the SCCMS. On the other hand, long-lived channel blockers that curtail the quantal conductance change may be of benefit.
The predominant effect of the above slow-channel AChR mutations, together with the previously reported [epsilon]T264P mutation (10 ), is to slow the rate of channel closure; a previously described [alpha]G153S mutation (11 ) in the extracellular domain of the [alpha] subunit prolongs channel open episodes primarily by enhancing the affinity for ACh, allowing repeated opening during ACh occupancy. These findings indicate genetic heterogeneity in the slow-channel syndrome and that multiple molecular mechanisms influence the duration of opening episodes of the AChR channel.
Specimens of intercostal muscle were obtained intact from origin to insertion from the 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 cytochemistry. For electron microscopy, EPs were localized and analyzed by established methods (29 ). Peroxidase-labeled [alpha]-BGT was used for the ultrastructural localization of AChR (30 ). [gamma]-AChR was immunolocalized in cryosections with an affinity-purified [gamma]-subunit-specific antibody (17 ) (gift from Z. W. Hall) and a biotin-avidin-based detection system. This antibody recognizes AChR in fetal but not adult human muscle.Intracellular microelectrode studies. All studies were done at 22 +- 0.5oC. MEPC and EPP recordings and estimates of the number of transmitter quanta released by nerve impulse were obtained as previously described (31 ).Patch-clamp recordings from EP AChRs. These were performed at 22 +- 0.5oC in the cell-attached mode by a slight modification of the previously described method (10 ,14 ). 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 pipette, 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 pipette. Channel currents were recorded at 10 kHz using an Axopatch 200A amplifier (Axon Instruments), digitized at 50 kHz, stored on hard disk, and analyzed using the program MacTac (Instrutech) at a final bandwidth of 5.8 kHz. 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 (32 ). Dwell time histograms were plotted on logarithmic abscissa and fitted to the sum of exponentials by maximum likelihood (33 ).
mRNA and DNA samples. 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. Genomic DNA was isolated from proteinase/SDS digest of blood or muscle by phenol-chloroform extraction followed by ethanol precipitation (34 ). Polymerase chain reaction procedures. Published cDNA sequences of the human [alpha] (35 ), [beta] (36 ), [delta] (37 ) and [epsilon] (38 ) subunit were used to design cDNA primers. PCR primers were designed to amplify each exon with its flanking regions of each subunit as previously described (10 ). PCR conditions for each primer were optimized by varying Mg2+ concentration and pH and by addition of 10% dimethyl sulfoxide for amplification of G + C-rich fragments. The typical PCR reaction mixture included 60 mM Tris-HCl (pH 8.5), 15 mM (NH4)2SO4, 1.5-2 mM MgCl2, 0.10-0.25 mM each dNTP, 0.8 [mu]M each primer, with 100 ng of DNA and 1.25 units of Taq DNA polymerase (Perkin-Elmer/Cetus) in 50 [mu]l. cDNA fragments were amplified by the nested PCR procedure using 1/50th of the first amplification product. The typical cycling protocol comprised (i) denaturation at 94oC for 2 min; (ii) 35 cycles of 94oC for 30 s, 55oC or 60oC for 30 s, 72oC for 3 min; and (iii) final extension at 72oC for 7 min. Positions of primers for amplifying DNA segments harboring mutations and polymorphisms are available on request. Exon scanning by SSCP analysis. The `cold' SSCP procedure was employed (39 ) with ~500 ng of PCR-amplified DNA. Denatured SSCP samples were loaded on 4-20% gradient polyacrylamide gel, electrophoresed at 12oC, stained with ethidium bromide, and examined under ultraviolet light.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 373A DNA sequencer using fluorescently labeled dideoxy terminators. Allele-specific PCR. The [beta]V266M mutation was identified by allele-specific PCR. The respective wild-type and mutant sense primers were 5'-CCCTGCTGACCCTTAaTG-3' and 5'-CCCTGCTGACCCTTAaTA-3' in [beta] exon 8, `a' representing a deliberately mismatched nucleotide to avoid misannealing of the primer to the opposite allele. The antisense primer was 5'-GTTGGAGGAGGAGATCTTTC-3' in [beta] intron 8. The length of the expected amplicon was 221 bp. The PCR reaction mixture contained 2.0 mM MgCl2, 0.25 mM each dNTP, 10% dimethyl sulfoxide and the annealing temperature was 55oC. Other PCR conditions were as described above.Restriction enzyme analysis. The [delta]Q267E mutation resulted in gain of HinfI site, the [epsilon]L269F mutation in loss of an MnlI site, and the [alpha]N217K mutation in loss of a HincII site. Using genomic DNA, a 163 bp fragment spanning [delta]Q267E was amplified with primers 5'-CCAAGGTCACAGCTAAGTCTG-3' and 5'-CGGGCTTGAGCGTCACTC-3'; a 188 bp fragment spanning [epsilon]L269F was amplified with primers 5'-CCGCTCTCACTGGCTCTC-3' and 5'-CCCCCACCCTTCACACTG-3'; and a 306 bp fragment spanning [alpha]N217K was amplified with primers 5'-CCAAACCTCACTTCCTTTCT-3' and 5'-AGACCCATCAGCGTCAGC-3'. After purification of the PCR products by ethanol precipitation, restriction enzyme digestion was carried out at 37oC for 2 h by adding 5 units of each enzyme in 20 [mu]l of reaction mixture. The digested fragments were size-fractionated either on 4% agarose gel containing ethidium bromide ([alpha]N217K) or on 20% acrylamide gel ([delta]Q267E and [epsilon]L269F) and then stained with ethidium bromide.
Construction and expression of wild type and mutant AChR. Mouse AChR subunit cDNAs were generously provided by Drs John Merlie, Norman Davidson [[alpha], [beta], and [delta] subunits; referenced in (40 )] and Paul Gardner [[epsilon] subunit; referenced in (41 )] and were subcloned into the CMV-based expression vector pRBG4 as described (40 ). The [epsilon]L269F and [alpha]N217K mutations were constructed by bridging restriction sites with synthetic double stranded oligonucleotides harboring the mutations. For [epsilon]L269F, a 65 bp oligonucleotide bridged from a NheI to a BspMI site, whereas for [alpha]N217K, a 31 bp oligonucleotide bridged from a HincII to a SapI site. The [beta]V266M and [delta]Q267E mutations were constructed by overlap PCR mutagenesis. For [beta]V266M, the final amplified fragment of 270 bp was ligated between NsiI and BglII sites, whereas for [delta]Q267E the final amplified fragment of 580 bp was ligated between KpnI and NdeI sites. The presence of each mutation and the absence of unwanted mutations was confirmed by dideoxy sequencing. Human embryonic kidney fibroblasts (293 HEK) were transfected with mutant or wild type AChR subunit cDNAs using calcium phosphate precipitation as described (41 ).Patch-clamp recordings from AChRs expressed in HEK cells. The methods were similar to those employed at the EP except that the bath and pipette solutions contained (in mM): KCl 142, NaCl 5.4, CaCl2 1.8, MgCl2 1.7, HEPES buffer 10, pH 7.4, with the pipette solution containing specified concentrations of ACh. Recordings were made with a membrane potential of -70 mV. Single channel currents were recorded at a bandwidth of 50 kHz, digitized with a pulse code modulation adapter at 94 kHz (VR-10B, Instrutech Corp.), transferred to a Macintosh computer using the program Acquire (Instrutech Corp.), and analyzed with the program MacTac at a final bandwidth of 8 kHz. Burst durations were determined by grouping openings separated by an interval greater than a specified closed time (100-150 [mu]s), which was taken as the intersection of the briefest closed time component with the next longer component.ACh binding measurements. Three days after transfection, intact HEK cells were harvested by gentle agitation in phosphate buffered saline (PBS) plus 5 mM EDTA. The esterase inhibitor diisopropylphosphofluoridate (1 [mu]M) was added to the PBS/EDTA solution, and cells were incubated for 15 min. Cells were briefly centrifuged, resuspended in high potassium Ringer's solution, and divided into aliquots for ACh binding measurements. Specified concentrations of ACh were added 30 min prior to addition of 125I-labeled-[alpha]-BGT (5 nM), which was allowed to bind for 30 min to occupy approximately half of the surface receptors. Binding of 125I-[alpha]-BGT was stopped by adding potassium Ringer's solution containing 300 [mu]M d-tubocurarine, followed by filtration using a cell harvester (Brandel, Inc.). Radioactivity retained by the glass fiber filters (Whatman GF-B, 1 [mu]m cutoff) was measured with a gamma counter. The total number of binding sites was determined by incubating cells for 1 h in the presence of 5 nM 125I-labeled-[alpha]-BGT. Nonspecific binding was determined in the presence of 300 [mu]M d-tubocurarine. The initial rate of 125I-[alpha]-BGT binding was determined to yield fractional occupancy of sites by ACh (42 ). Competition measurements were analyzed according to the Hill equation:
1-Y = 1/(1 + ([ACh]/Kapp)n), where Y is fractional occupancy by ACh, Kapp is the apparent dissociation constant, and n is the Hill coefficient.
This work was supported by NIH grant to A.G.E (NS6277) and S.M.S. (NS31744), an MDA grant to A.G.E., an MDA postdoctoral fellowship to K.O., an Italian Telethon Grant to M.M., and a Deutsche Forschungsgemeinschaft postdoctoral fellowship to J.P.S.
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