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Next Article 
Volume 17, Number 15,
Issue of August 1, 1997
pp. 5651-5665
Copyright ©1997 Society for Neuroscience
Slow-Channel Myasthenic Syndrome Caused By Enhanced Activation,
Desensitization, and Agonist Binding Affinity Attributable to
Mutation in the M2 Domain of the Acetylcholine Receptor  Subunit
Margherita Milone1,
Hai-Long Wang2,
Kinji Ohno1,
Takayasu Fukudome1,
J. Ned Pruitt1,
Nina Bren2,
Steven M. Sine2, and
Andrew G. Engel1
1 Muscle Research Laboratory, Department of Neurology,
and 2 Receptor Biology Laboratory, Department of Physiology
and Biophysics, Mayo Foundation, Rochester, Minnesota 55905
 ABSTRACT
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- DISCUSSION
- FOOTNOTES
- REFERENCES
ABSTRACT 
We describe a novel genetic and kinetic defect in a
slow-channel congenital myasthenic syndrome. The severely disabled
propositus has advanced endplate myopathy, prolonged and
biexponentially decaying endplate currents, and prolonged acetylcholine
receptor (AChR) channel openings. Genetic analysis reveals the
heterozygous mutation  V249F in the propositus and mosaicism for
V249F in the asymptomatic father. Unlike mutations described
previously in the M2 transmembrane domain, V249F is located
N-terminal to the conserved leucines and is not predicted to face the
channel lumen. Expression of the V249F AChR in HEK fibroblasts
demonstrates increased channel openings in the absence of ACh,
prolonged openings in its presence, enhanced steady-state
desensitization, and nanomolar rather than micromolar affinity of one
of the two binding sites in the resting activatable state. Thus,
neuromuscular transmission is compromised because cationic overloading
leads to degenerating junctional folds and loss of AChR, because an
increased fraction of AChR is desensitized in the resting state, and
because physiological rates of stimulation elicit additional
desensitization and depolarization block of transmission.
Key words:
slow-channel congenital myasthenic syndrome;
neuromuscular transmission;
endplate myopathy;
acetylcholine receptor
subunit gene;
mutation analysis;
single-channel recording;
desensitization;
agonist binding affinity
INTRODUCTION
Recent studies of congenital myasthenic syndromes
(CMS) revealed mutations in acetylcholine receptor (AChR) subunit genes that either reduce expression of AChR (Engel et al., 1996a ) or alter
its kinetic properties to decrease (Ohno et al., 1996) or increase
(Ohno et al., 1995; Sine et al., 1995; Engel et al., 1996b; Gomez et
al., 1996) the response to ACh. Mutations that increase response to ACh
prolong elementary activation episodes, and these disorders are
referred to as slow-channel CMS (SCCMS). Mutations underlying SCCMS
have been identified in different AChR subunits, and in different
domains of the subunits, and affect AChR function through different
mechanisms. The mutation G153S is in the major extracellular domain
and near residues that contribute to ligand binding; it increases
affinity of ACh for the resting closed state and prolongs activation
episodes by allowing multiple reopenings before ACh can dissociate
(Sine et al., 1995). The mutation N217K is in the M1 transmembrane
domain and both slows the rate of channel closing and allows multiple
reopenings per activation episode (Engel et al., 1996b). Mutations in
the M2 domain,  T264P, L269F, and  V266M (Ohno et al., 1995;
Engel et al., 1996b), increase spontaneous opening of the channel,
increase apparent affinity for ACh, and slow the rate of channel
closing.
Here we describe and functionally characterize the mutation V249F in
the M2 domain that causes severe SCCMS through a novel combination of
mechanisms. Our mechanistic studies reveal functional consequences
attributable to local perturbation of the M2 domain, including
increased opening in the absence and prolonged opening in the presence
of ACh, and enhanced steady-state desensitization. Moreover, the
perturbation in M2 spreads to the binding site to increase affinity of
the resting closed state from the micromolar to the nanomolar range.
High affinity of the resting state predicts occupancy of sufficient
duration to allow desensitization at physiological rates of
stimulation. By enhancing both activation and desensitization, V249F
severely compromises the safety margin of neuromuscular transmission.
MATERIALS AND METHODS
Muscle specimens
Intercostal muscle specimens were obtained intact from origin to
insertion from the patient 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.
Endplate studies
Morphology and counts of AChR per endplate. For
electron microscopy, endplates (EPs) were localized and analyzed by
established methods (Engel, 1994a). Peroxidase-labeled -bungarotoxin
(-bgt) was used for the ultrastructural localization of AChR (Engel
et al., 1977b). The number of AChRs per EP was measured with
125I-labeled -bgt, as described previously (Engel et
al., 1993).
Intracellular microelectrode studies. Miniature EP potential
(MEPP), miniature EP current (MEPC) and EP potential (EPP) recordings, estimates of the number of transmitter quanta released by nerve impulse, and analysis of the ACh-induced current noise were performed as described previously (Engel et al., 1993; Uchitel et al., 1993).
Patch-clamp recordings from EP AChRs. These were performed
in the cell-attached mode by a slight modification of the methods described previously (Milone et al., 1994; Ohno et al., 1995). 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
using an Axopatch 200A amplifier (Axon Instruments, Foster City, CA)
and analyzed at a final bandwidth of 5.8 kHz using the program pClamp 6 (Axon Instruments). 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 (Colquhoun and Sakmann, 1985). Dwell time histograms
were plotted on logarithmic abscissa and fitted to the sum of
exponentials by maximum likelihood (Sigworth and Sine, 1987).
Mutation analysis
Single-strand conformation polymorphism (SSCP) and
sequencing. mRNA, first-strand cDNA, and genomic DNA were obtained
as described previously (Ohno et al., 1996). PCR primers were designed
to amplify exons with their flanking regions from each AChR subunit as
previously described (Ohno et al., 1995). Published cDNA sequences of
the human (Noda et al., 1983), (Beeson et al., 1989),  (Luther et al., 1989), and (Beeson et al., 1993) subunits were used to design cDNA primers. The "cold" SSCP procedure was used as described previously (Ohno et al., 1996). PCR-amplified fragments of
genomic DNA or cDNA were purified by Wizard PCR Preps (Promega, Madison, WI). Plasmids were purified by QIAwell 8 Plus Plasmid kit
(Qiagen, Santa Clarita, CA). DNA fragments and plasmids were sequenced
with an Applied Biosystems model 377 DNA sequencer using fluorescently
labeled dideoxy terminators.
Allele-specific PCR. We used allele-specific PCR to search
for the V249F mutation in the patient's relatives and normal
controls. The respective wild-type and mutant sense primers were
5 -AAGATGACTCTGAGCATCTgTG-3 and 5-AAGATGACTCTGAGCATCTgTT-3.
Mismatched nucleotide "g" was deliberately introduced two
nucleotides upstream of the 3 end of the primer to avoid misannealing
of the primer to the opposite allele. The antisense primer was
5-GTTGATGACGATGACAGTGA-3.
Mutagenic PCR plus restriction analysis. We also synthesized
a mutagenic PCR primer to check for mosaicism for the V249F mutation
in the patient's family. A 157 bp fragment of genomic DNA spanning the
V249F mutation was amplified with primers
5-AGGACTCAGGACTTCCACATA-3 in intron 6 and
5-AGAAGGAACACAGTCAAAGACAGTgAGA-3 in exon 7. The mismatched
nucleotide "g," which introduces a mutation four nucleotides
downstream from the G745T (V249F) mutant site, enables
BsmAI to differentiate among PCR products with or without the V249F: BsmAI yields 134 and 23 bp fragments from the
PCR product lacking V249F mutation, and a 157 bp fragment from the PCR product harboring V249F. The PCR product was digested with BsmAI (New England Biolabs, Beverly, MA).
Cloning of PCR products to detect mosaicism. A 157 bp
fragment spanning the V249F mutation was amplified from genomic DNA from the patient and his father and was ligated into a vector using the
TA cloning kit (Invitrogen, Carlsbad, CA). White colonies were picked
up from Luria-Bertani plates, boiled for 10 min in 50 µl of distilled
water, and immediately cooled on ice. Five microliters of boiled sample
was mixed with three PCR primers, 5-GTAAAACGACGGCCAGT-3 (forward
generic primer for M13), 5-GGAAACAGCTATGACCATG-3 (reverse generic
primer for M13), and 5-AAGATGACTCTGAGCATCTgTT-3 (mutant-allele-specific primer, as shown in the section for
allele-specific PCR) in 25 µl of reaction mixture. All clones with
inserts yield a 355 bp fragment, clones with no inserts produce a 198 bp fragment, and clones harboring V249F additionally produce a 166 or 137 bp fragment, depending on the direction of the insert. Thus, we could characterize the inserts by size fractionation of the PCR products. Presence of V249F in the 161 and 137 bp fragments was verified by BsmAI digestion, as described above.
Expression studies
Construction of human wild-type and mutant AChR cDNAs and
expression in 293 HEK cells. Human and subunit cDNAs were
generously provided by Dr. Jon Lindstrom (Schoepfer et al., 1988;
Luther et al., 1989). and subunit cDNAs were cloned from normal
human skeletal muscle as described previously (Ohno et al., 1996). All four cDNAs were subcloned into the CMV-based expression vector pRBG4
(Lee et al., 1991) for expression in 293 HEK cells.
The V249F mutation was constructed using QuickChange Site-Directed
Mutagenesis Kit (Stratagene, La Jolla, CA) according to the
manufacturer's recommendations. The presence of the desired mutation
and the absence of unwanted mutations were confirmed by sequencing the
entire insert.
HEK cells were transfected with mutant or wild-type AChR subunit cDNAs
using calcium phosphate precipitation as described (Bouzat et al.,
1994). For patch recordings, plasmid-encoding green fluorescent protein
(Life Technologies, Grand Island, NY) was included for identification
of transfected cells.
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 22°C, and with bath
and pipette solutions containing (in mM): KCl 142, NaCl
5.4, CaCl2 1.8, MgCl2 1.7, HEPES 10, pH 7.4 (Bouzat et al., 1994; Ohno et al., 1996). 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, Great Neck, NY), and detected by the half-amplitude threshold criterion using the program MacTac (Instrutech) at a final
bandwidth of 10 kHz. For V249F receptors, infrequent subconductance states before or after an opening were excluded from the measured open
durations, whereas those interrupting an opening were included. Open-
and closed-duration histograms were constructed using a logarithmic
abscissa and square root ordinate (Sigworth and Sine, 1987) and fitted
to the sum of exponentials by maximum likelihood. The resulting time
constants, relative areas, and total numbers of events were used to
determine opening, closing, and agonist dissociation rate constants as
described (Sine and Steinbach, 1986). For openings interrupted by
closings approaching the instrumentation dead time (18 µsec), we
determined the true mean open duration from the total open duration in
the recording (apparent mean open duration multiplied by the total
number of openings) divided by the total number of brief closings
(determined from the area of the brief component of closings) as
described (Sine et al., 1990).
To confirm our analysis method, we simulated single-channel events
according to Scheme 1 (see text) using the parameters obtained from the
experimental data. We simulated open and closed intervals as described
by Clay and deFelice (1983) and constructed bursts using our
experimental closed time criterion (1 msec). After compiling the bursts
into histograms, we fitted the histograms by the sum of exponentials,
as described above for the experimental data. We computed the
probability of openings in the three types of burst events (P1,
P2, P3) by multiplying the relative area by the corresponding mean open
duration.
Scheme 1.
[View Larger Version of this Image (15K GIF file)]
-Bgt and ACh binding measurements. The total number of
125I--bgt sites and ACh competition against the initial
rate of 125I--bgt binding were determined as described
previously (Ohno et al., 1996). ACh competition measurements were
analyzed according to either the monophasic Hill equation (Eq. 1) or
the sum of two distinct binding sites (Eq.
2): 1 Y = 1/(1 + ([ACh]/Kov)n), (1)1 Y = fractA(1/(1 + [ACh]/KA)) + (1 fractA)(1/(1 + [ACh]/KB)), (2)
where Y is fractional occupancy by ACh,
n is the Hill coefficient, 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.
RESULTS
Clinical data
The patient, a 12-year-old male, had myasthenic symptoms involving
the ocular, bulbar, trunkal, and limb muscles since early infancy.
Between the ages of 3 and 8 years, he could walk only short distance
before having to rest and subsequently he became wheelchair-dependent.
He had negative tests for anti-AChR antibodies, a decremental
electromyographic response on stimulation of motor nerves, and a
repetitive action potential response to single nerve stimuli, as seen
in the SCCMS (Engel et al., 1982) or in EP acetylcholinesterase deficiency (Engel et al., 1977a).
Morphological observations and counts of AChR per EP
Acetylcholinesterase activity was preserved at all EPs.
Ultrastructural studies revealed an EP myopathy with honeycomb networks in junctional folds (Fig. 1A),
degeneration of junctional folds with widening of the synaptic space
(Fig. 1B) and loss of AChR (Fig.
1D), degenerating organelles in the junctional
sarcoplasm (Fig. 1C), and apoptosis of some junctional
nuclei (Fig. 1E). The number of
125I--bgt binding sites/EP was 5.02 × 106, or 39% of control.
Fig. 1.
EP fine structure. A, Many
junctional folds are honeycombed by membranous networks. This is a
common ultrastructural reaction of the EP in states of cholinergic
overactivity. Magnification, 18,100×. B, Degeneration
of the junctional folds leaves globular debris in the widened synaptic
space (asterisk). Magnification, 21,500×.
C, The junctional sarcoplasm at the left
is filled with degenerating organelles; the star
indicates remnants of degenerated junctional folds. Magnification,
17,500×. D, Localization of EP AChR with peroxidase
labeled -bungarotoxin. Note loss of AChR from degenerating
junctional folds (arrowhead). Magnification, 23,800×.
E, The junctional sarcoplasm harbors nuclei in early (x) and advanced (X) stages
of apoptosis. The star indicates remnants of degenerated
junctional folds. Magnification, 14,800×.
[View Larger Version of this Image (180K GIF file)]
Intracellular microelectrode studies
Quantal release by nerve impulse was normal. MEPPs and MEPCs had a
reduced amplitude, consistent with EP AChR deficiency. The MEPCs
decayed biexponentially, suggesting two populations of channel
openings, with one  MEPC close to normal and one markedly prolonged (Fig. 2, top panels, and Table
1). Spectral analysis of the ACh-induced current noise
indicated two channel open times, with one noise normal
and one markedly prolonged (Table 1). These findings pointed to a
kinetic abnormality of at least some of the EP AChRs.
Fig. 2.
MEPCs and channel events recorded from control and
patient EPs. Top traces, MEPCs; bottom
traces, channel events; left, control EP;
right, patient EP. Note markedly prolonged MEPC and some
highly prolonged channel events in the SCCMS. The MEPC decay is best fitted by two exponentials. The vertical arrows indicate
decay time constants. MEPCs filtered at 500 Hz and channel currents at
5.8 kHz; 80 mV; temperature, 22°C.
[View Larger Version of this Image (15K GIF file)]
Patch-clamp analysis of EP AChR
To gain additional insight into the kinetic defect of AChR,
we recorded single-channel currents from EPs of the patient in the
presence of 1 µM ACh and compared the findings with those at control EPs. Simple inspection of the recordings from the patient EPs revealed a mixture of apparently normal and markedly prolonged channel events (Fig. 2, bottom panels). On formal analysis,
at control EPs both the open intervals and the bursts had a minor, brief (1) and a major, longer component
(2), as described previously (Milone et al.,
1994). At the patient EPs, the channel open intervals and bursts had
two components, 1 and 2, similar
to those observed at the control EPs, and a third component,
3, that was five- to sevenfold longer than the
respective 2 (Table 2). The
above channel events had a conductance of 60-66 pS, similar or up to 10% greater than the conductance of channel events at control EPs.
Approximately 12% of all channel events at the patient's EPs had a
reduced conductance of 45 pS. The open interval and burst distributions
of these channels showed two components, a brief, minor component,
1, similar to that of the 60-66 pS channels, and
a second, prolonged component (2 = 5.88 ± 0.58 msec for open intervals, and 10.86 ± 1.93 msec for bursts). The
conductance and open durations of these channels were typical of AChRs
containing the  instead of the subunit (-AChR) (Mishina et
al., 1986). In addition, at two EPs the 45 pS channels had an
additional longer component accounting for 15-20% of the 45 pS
channel events (3 = 11.27 and 17.4 msec for open
intervals, and 16.74 and 41.68 msec for bursts). The patch-clamp data
were compatible with different species of EP AChRs: adult-type AChR
composed of wild-type subunits, adult-type AChR harboring one or more
mutant subunits, -AChR composed of wild-type subunits, and -AChR
harboring one or more mutant subunits. To identify the structural basis
of the abnormal channel kinetics, we searched for mutations in AChR
subunit genes.
Mutation analysis
SSCP analysis of PCR-amplified fragments of genomic DNA encoding
the , , , and subunits revealed seven aberrant conformers, all commonly observed in controls (Table 3), but no
mutations. Sequencing of all exons and flanking intronic regions of
genomic DNA for the , , , and subunit genes as well as
overlapping cDNA fragments of the entire coding regions for the four
AChR subunit genes revealed one additional polymorphism (Table 3) and a
heterozygous G T transversion in exon 7 at nucleotide 745 (G745T) that converted a valine to a phenylalanine codon at position
249 (V249F) (Fig. 3A). The
altered valine is located in the M2 transmembrane domain that lines the
channel pore and is conserved across the subunit of all species and
the human , , and subunits (Fig. 3C).
Fig. 3.
Identification and analysis of mutation in the
AChR subunit. A, Automated sequencing of exon 7 around codon 249 in a control and the propositus. In the propositus,
both G and T nucleotides are present at position 745 (arrow), indicating a heterozygous GT transversion.
This mutation changes codon 249 from a GTC for valine to a TTC for
phenylalanine. B, Allele-specific PCR and mutagenic PCR
plus BsmAI analysis of genomic DNA in the propositus' family. Both wild-type and mutant-allele-specific primers amplify an
expected 168 bp fragment in propositus and his father, but only the
wild-type primer amplifies the expected fragment in the other family
members. On BsmAI analysis after mutagenic PCR, the wild-type allele gives rise to a 134 bp fragment (open
arrowhead) and a 23 bp fragment (not shown); the mutant allele
yields an undigested 157 bp fragment (closed arrowhead).
Both wild-type and mutant fragments appear in the propositus, but only
the wild-type fragment appears in other family members. The incongruity
between allele-specific PCR and restriction analysis in the father is attributable to the father being a mosaic for V249F (see text). The
arrow indicates propositus. C,
Multiple alignment of AChR M2 membrane-spanning domains. The
boxes enclose the conserved valine residues in human
AChR subunits and in AChR subunits of other species. The mutant
phenylalanine is shown at the bottom.
[View Larger Version of this Image (35K GIF file)]
Using genomic DNA, we searched for V249F by allele-specific PCR in
100 normal controls, 55 other unrelated CMS patients, and in the
propositus' relatives, and detected it only in the patient's
asymptomatic father (Fig. 3B). Direct sequencing, however, did not show V249F in the father's genomic DNA. We then searched for V249F in the father's genomic DNA by mutagenic PCR and
restriction analysis using BsmAI that digested only the
wild-type sequence. This revealed V249F only in the patient but not
in his father or other relatives (Fig. 3B). Repeated
experiments using newly drawn blood from the father gave the same
results, precluding contamination of father's DNA by that of the
propositus. Detection of V249F in the father by allele-specific PCR,
but not by direct sequencing or restriction analysis, suggested
paternal mosaicism for V249F. To prove this, we cloned the
PCR-amplified segment of DNA spanning V249F from the propositus and
his father. Allele-specific PCR of 20 clones from the propositus
revealed that 10 harbored V249F and 10 did not, consistent with
heterozygosity. By contrast, analysis of 106 clones from the father
revealed 2 clones with V249F and 104 clones without it (Fig.
4), confirming mosaicism in the father. Chimerism in the
father was excluded by failure of allele-specific PCR to detect
V249F in the paternal grandparents (Fig. 3B).
Fig. 4.
Allele-specific PCR reveals mosaicism for V247F
in paternal DNA. Only 2 of 106 clones of paternal genomic DNA harbored
V247F. The figure demonstrates V247F, represented by the 166 bp
fragment, in clone number 90.
[View Larger Version of this Image (33K GIF file)]
Expression studies
To evaluate pathogenicity of V249F, we transfected 293 HEK
cells with either wild-type or mutant human plus complementary ,
, and subunit cDNAs. Measurements of 125I--bgt
binding revealed 55 ± 10% (mean ± SD for 10 determinations) surface expression of V249F relative to wild-type
AChR.
Activation of the V249F receptor by ACh
To investigate the mechanistic consequences of V249F, we
recorded ACh-activated, single-channel currents from HEK cells
expressing the V249F AChR. We initially attempted to record currents
in the presence of desensitizing concentrations of ACh (10-100
µM), because this allows identification of clusters of
events attributable to a single channel; analysis of open and closed
intervals within clusters would have allowed determination of rate
constants for each step in a scheme for receptor activation, as
described previously (Sine et al., 1990; Ohno et al., 1996). However,
V249F receptors showed no channel activity at ACh concentrations >1
µM, presumably because of enhanced desensitization.
Decreasing the ACh concentration into the nanomolar range resulted in
vigorous activation of V249F receptors and the appearance of
markedly prolonged channel openings (Fig. 5, left
panels).
Fig. 5.
Activation of human wild-type and V249F
AChRs by nanomolar concentrations of ACh. The left
panels show single-channel currents elicited by the indicated
concentrations of ACh at a bandwidth of 10 kHz for wild-type and
V249F receptors. The center and right panels show corresponding closed and burst-duration histograms fitted to the sum of exponentials. Note three components of bursts for
both receptor types, the increased contribution of the long-duration component with increasing ACh concentration, and the increased duration
of the long component attributable to V249F.
[View Larger Version of this Image (27K GIF file)]
Current amplitudes of V249F receptor channels were noticeably
increased. The conductance of V249F channels, 94 ± 1.0 pS (± SEM, 3 patches), was significantly greater than that for wild type,
84 ± 0.9 pS (4 patches); p < 0.001. V249F
receptors also infrequently showed subconductance states of half the
full amplitude and lasting several milliseconds; these preceded,
followed, or interrupted bursts of prolonged channel openings.
Effect of V249F on burst durations
To investigate the molecular basis of the prolonged openings
caused by V249F, we recorded currents elicited by 3-100
nM ACh, constructed burst-duration histograms, and fitted
the histograms by the sum of exponentials. For this analysis, bursts
were defined as openings separated by closed intervals <200 µsec.
For both wild-type and V249F receptors, the distributions of burst
durations show three exponential components (Fig. 5, right
panels). The two brief components predominate at low ACh
concentrations, whereas the long component predominates at the highest
concentration. This dependence on ACh concentration suggests that long
bursts correspond to receptors with two bound agonists, whereas the two types of brief bursts correspond to receptors with one bound agonist. Because the receptor contains two ACh binding sites, we hypothesize that one type of brief burst corresponds to occupancy of the site, whereas the other type corresponds to occupancy of the site. Mean durations of singly occupied bursts are not significantly affected by V249F, but doubly occupied bursts are prolonged
~10-fold.
Kinetic analysis of the V249F receptor
Determination of each rate constant underlying receptor activation
relies on identifying clusters of open and closed intervals attributable to a single channel. However, because openings of V249F
receptors did not cluster, we used a novel analysis to define a subset
of the activation parameters. First, we determined the time constants
and relative areas of closed-duration components over the concentration
range of 3-100 nM ACh. Second, we identified two types of
burst events and determined the open- and closed-duration components
associated with each type. Third, we determined rate and equilibrium
constants for gating of the two types of bursts and, based on their
dependence on ACh concentration, assigned one type to singly and the
other type to doubly occupied receptors. Fourth, we determined the
probability of occurrence of singly and doubly occupied bursts as a
function of ACh concentration and combined the measured gating
equilibrium constants to estimate the dissociation constant for ACh
binding.
Analysis of closed durations
For wild-type receptors, closed-duration histograms are well
described as the sum of two exponentials (Fig. 5, middle
panels; 3-100 nM ACh). The long-duration component
corresponds to periods between independent bursts of openings and the
brief component to transient interruptions of single-channel bursts;
thus, the brief component corresponds to a closed state associated with activation. By contrast, for V249F receptors, closed-duration histograms are well described as the sum of five or six exponentials (Fig. 5, middle panels; 3-100 nM ACh). One or
more of the longer components are attributed to dwells in desensitized
states (Sakmann et al., 1980; Sine and Steinbach, 1987), because of
profound desensitization even at nanomolar ACh concentrations, whereas
one or more of the brief components are candidates for a closed state
associated with activation.
Temporal association of openings and closings
To determine activation rate constants from the open- and
closed-duration histograms, we first determined which component of
openings is associated with which component of closings. We searched
recordings for sequences of two or more closely spaced channel
openings, defined as openings separated by closed intervals <1 msec;
this duration was chosen to include closings belonging to the two
briefest components for the V249F receptor and could be increased to
10 msec with similar results. We then looked for bursts with different
properties by examining the distribution of the mean duration of
openings within bursts of closely spaced events. V249F receptors
exhibited two readily distinguishable distributions of bursts, with
mean open durations of 0.49 ± 0.2 msec and 6.0 ± 1.1 msec.
Wild-type receptors, by contrast, exhibited a single distribution of
bursts with a mean open duration of 1.2 ± 0.3 msec. To illustrate
the kinetic properties of each type of burst for V249F, we
eliminated all solitary openings from the table of detected events,
eliminated one or the other type of burst based on open duration,
joined the flanking long closed intervals, and constructed histograms
of the resulting open and closed intervals. We used a similar procedure
to select bursts for wild-type receptors, except that only solitary
events were eliminated from the event table.
The histograms of selected bursts confirm that wild-type receptors give
rise to only one type of coupled event, long openings interrupted by
brief closings; histograms of these events reveal only one brief closed
and one long burst component (Fig. 6, top panel). V249F receptors, by contrast, give rise to two
types of coupled events. The first type consists of long openings
interrupted by brief closings, corresponding to a predominant brief
closed and one long burst component (Fig. 6, middle
panel). The second type consists of intermediate openings
interrupted by intermediate closings, corresponding to one intermediate
closed and one intermediate burst component (Fig. 6, bottom
panel). Thus, for wild-type receptors, we assign the single
type of coupled event, long openings separated by brief closings, to
doubly occupied bursts because their relative frequency increases with
increasing ACh concentrations. Similarly, for V249F receptors, we
assign one type of coupled event, long openings separated by brief
closings, to doubly occupied bursts because their relative frequency
increases with increasing ACh concentrations. We assign the other type
of coupled event for V249F, intermediate openings separated by
intermediate closings, to singly occupied bursts because their relative
frequency decreases with increasing ACh concentrations.
Fig. 6.
Temporal association of openings and closings
elicited by wild-type and V249F receptors. For each recording, the
table of detected events was searched for consecutive openings
separated by closed intervals <1 msec, and these coupled events were
preserved. The remaining solitary openings were eliminated, and the
flanking long closed intervals joined and retained in the table.
Top panels show analysis of the only type of coupled
event observed for wild-type receptors, long openings interrupted by
brief closings. The center and bottom panels show
analysis of the two types of coupled events observed for V249F
receptors; bursts corresponding to the first type were recognized as
those with mean open durations >1 msec, whereas those of the second
type had mean open durations <1 msec. To analyze each type of burst
alone, the other type of burst was eliminated from the event table
based on mean open duration, and the flanking closed intervals joined
and retained in the table. The left panels show traces
of each type of coupled event filtered at 10 kHz. The
center and right panels show
corresponding histograms of the coupled events fitted by the sum of
exponentials. Note that each type of coupled event shows one component
of bursts and a major component of brief closings.
[View Larger Version of this Image (22K GIF file)]
Estimation of rate and equilibrium constants for the
V249F receptor
We used the following kinetic scheme to describe
activation of wild-type and V249F receptors: where two
agonists A bind to the receptor R with association rates
k+1 through k+4 and
dissociate from the receptor with rates k 1
through k4. The two types of singly occupied
receptors, AR and AR, open with rates 1 and
1, whereas open receptors AR* and AR* close
with rates 1 and 1. Fully occupied
receptors A2R open with rate 2, and
open receptors A2R* close with rate 2. Scheme 1 allows ACh to bind independently to the two sites and receptors with one or two bound agonists to open; it is a general form
of the standard sequential scheme that has successfully described the
kinetics of activation of Torpedo, fetal mouse, and adult human AChRs
(Sine et al., 1990; Zhang et al., 1995; Ohno et al., 1996). We consider
Scheme 1 to be the simplest description of the data because of its
consistency with previous work and its ability to account for three
kinetically distinct types of openings.
Because brief closings and long openings are temporally associated
(Fig. 6, top and middle panels) and the
corresponding long bursts increase with increasing ACh concentration
(Fig. 5, right panels), for wild-type and V249F AChRs, we
assign the brief closings to dwells in A2R and the long
openings to dwells in A2R*. Given these assignments, the
mean duration of brief closings equals (2 + kdiss)1, the number of
brief closings per burst of long openings equals 2/kdiss, and the
mean burst duration equals (1 + 2/kdiss)/2 (Colquhoun and Hawkes, 1981). The parameter
kdiss is the sum of the dissociation rate
constants k1 and k4,
representing the two pathways for loosing bound ACh; if one
dissociation rate constant greatly exceeds the other,
kdiss equals the faster of the two rate
constants. These relationships lead to estimates of
2, 2, and
kdiss presented in Table 4,
showing that V249F speeds the rate of channel opening and slows the
rates of channel closing and agonist dissociation. Changes in all three
of these parameters contribute to the increase in burst duration
observed with V249F.
Table 4.
Kinetic parameters for activation of wild-type and V249F
receptors
|
1 |
1 |
 1 |
2 |
2 |
2 |
K1,
M |
kdiss |
k2
|
|
| Wild type |
6990 ± 1320 |
59.9
± 3.8 |
8.6E 3 |
1520 ± 306 |
46,000
± 6210 |
30.3 |
2.3E 5 |
10,060 ± 2370 |
12,700
± 597 |
| V249F |
2260 ± 465 |
9350
± 2850 |
4.1 |
690 ± 92 |
80,810
± 7530 |
117 |
8.4E 9 |
3300 ± 1060 |
8650
± 2610 |
|
|
Rate constants are as defined in Scheme 1 (see text), in units of
sec1. Channel opening equilibrium constants are
ratios of corresponding opening to closing rate constants, /. For
wild-type receptors, 1, k2, and
K1 are values determined previously from
analyses of clusters (Ohno et al., 1996). For V249F receptors,
1, 1, and k2
were determined from analyses of intermediate bursts of openings
elicited by 3-10 nM ACh, as described in the text (mean
values, ± SD from 4 patches). For both wild-type and V249F receptors, 2, 2 and
kdiss values were determined from analyses of
bursts of long openings as described in the text; values are means from
seven patches for each receptor type obtained from 3 to 100 nM ACh.
|
|
V249F receptors give rise to a second type of burst, intermediate
openings separated by intermediate closings (Fig. 6, bottom panel); because intermediate bursts are present at low ACh
concentrations and diminish with increasing concentrations (Fig. 5,
right panels), we assign these events to dwells in AR* and
AR, respectively. At this stage of analysis, these events could be
assigned equally to AR* and AR, but the subsequent analysis shows
that the binding step to form AR is one of low affinity and the step to
form A2R is one of high affinity. According to Scheme 1,
the mean duration of intermediate closings equals (1 + k2)1, the number of intermediate
closings per burst of intermediate openings equals
1/k2, and the mean burst
duration equals (1 + 1/k2)/1.
These relationships assume a negligible rate for the third pathway
leading away from the AR state, binding a second molecule of ACh to
form A2R; this is likely because a diffusion-limited
association rate constant of 108
M1 sec1 and a
concentration of 10 nM predict a rate of ~1
sec1 for ACh binding, whereas the apparent
(1 + k2) is 18,000 sec1 (Table 4).
The resulting estimates of 1,
1, and k2 for V249F
receptors are presented in Table 4. A comparable analysis was not
possible for wild-type receptors because brief openings occur in
isolation rather than in bursts. However, comparison with parameters for wild-type receptors, obtained by analysis of clusters at
desensitizing ACh concentrations (Ohno et al., 1996), shows that
V249F has little effect on k2, decreases
1, and increases 1 (Table 4). Thus
V249F receptors elicit bursts of singly liganded openings and
closings because 1 and k2
are of similar magnitude.
We estimated the dissociation constant for ACh binding to V249F
receptors from the dependence of the probabilities of long and
intermediate bursts on ACh concentration. According to Scheme 1, the
ratio of probabilities of long to intermediate openings, P3/P2, equals
[A]2/1/K1,
where [A] is ACh concentration, 2 equals
2/2, 1
equals 1/1, and
K1 is the dissociation constant for ACh
occupancy of the second binding site (Sine and Steinbach, 1984). Thus,
P3/P2 should increase linearly with increasing ACh concentration with a
slope of
2/1/K1.
Measurements of P3/P2 for V249F show the predicted linear dependence
on ACh concentration (Fig. 7A), and combined
with our kinetic estimates of 2 and
1, yield a value of K1 of
8.4 nM. This estimate of K1 for
V249F is considerably smaller than either K1
or K2 determined for wild-type receptors [23
and 116 µM, respectively (Ohno et al., 1996)] and can be
understood from the observation that the probabilities of long and
intermediate bursts change with nanomolar changes in ACh concentration.
These findings imply that although V249F is located in the M2
domain, the perturbation spreads to the binding site, some 30 Å away,
to increase affinity for ACh.
Fig. 7.
Ratios of probabilities of long (P3) to
intermediate (P2) or brief (P1) openings versus ACh concentration. For
each class of opening, probability is the product of the relative area
and the time constant determined by fitting dwell time histograms to
the sum of exponentials. A plots P3/P2 for the V249F
receptor. The line is the least-squares fit to
y = mx, with slope
m = 3.38 ± 0.14 × 109 M1. Combining the slope
with 1 and 2 yields the dissociation
constant K2 = 8.4 nM (see
text). B plots P3/P1 for the V249F receptor, with the
fitted m = 2.1 ± 0.26 × 1010 M1. C plots
P3/P1 for wild-type receptor, with the fitted m = 3.8 ± 0.24 × 109
M1. D plots P3/P2 for wild-type
receptor; the line is the best fit to the points from 1 to 50 nM, with m = 5.5 ± 0.83 × 108 M1.
[View Larger Version of this Image (17K GIF file)]
We checked additional predictions of Scheme 1 by examining the
dependence of the remaining types of openings on ACh concentration. For
V249F, the ratio of probabilities of long to brief openings, P3/P1,
also increases linearly with increasing ACh concentration, as expected
for AR* in Scheme 1 (Fig. 7B). However, because brief openings occur as single isolated events, the gating equilibrium constant 1 cannot be determined, thus preventing
estimation of the dissociation constant for the second binding site.
For wild-type receptors, the ratios P3/P2 and P3/P1 also increase linearly with ACh concentration (Fig.
7C,D), but again both intermediate and
brief openings occur as isolated events.
Simulation of channel kinetics for aV249F and
wild-type receptor
To confirm our analysis method, we simulated single-channel events
according to Scheme 1 using the parameters obtained from the
experimental data (Table 4). Because the microscopic rate constants
underlying K1 were not determined
experimentally, we chose a diffusion-limited association rate constant
(108
M1sec1), and a
dissociation rate constant (1 sec1) corresponding
to K1 = 10 nM. Also, we used the
experimentally determined closing rate 1 for the second
of the two classes of monoliganded openings, but assigned a value of
1 sufficient to give enough of these events. We
simulated open and closed intervals assuming Markov kinetics,
constructed bursts using our experimental closed time criterion,
compiled the bursts into histograms, fitted the histograms by sums of
exponentials, and determined P3/P2 at different concentrations of ACh
(see Materials and Methods). For the V249F receptor, the simulated
burst-duration histograms closely resemble the experimental
burst-duration histograms, showing three components of bursts, with the
relative area of each depending on ACh concentration (Fig.
8A, left panels). Moreover,
for V249F, the ratio P3/P2 increases linearly with increasing ACh
concentration with a slope identical to the expected ratio
2/1/K1
(Fig. 8B). The slight departure observed at 100 nM ACh is likely attributable to difficulty in accurately
fitting the small component corresponding to P2 (see histogram at 100 nM ACh, Fig. 8A, left
panels).
Fig. 8.
Simulated activation kinetics at nanomolar ACh
concentrations for wild-type and V249F AChRs. A shows
simulated burst-duration histograms fitted by the sum of exponentials
at the indicated ACh concentrations. Channel events were simulated as
described in Materials and Methods using the following Scheme 1
parameters: V249F; k+1 = k+2 = k+3 = k+4 = 108
M1sec1,
k1 = 1 sec1,
k2 = 8700 sec1,
k3 = 2.6 sec1,
k4 = 3300 sec1,
1 = 30,000 sec1, 1 = 1 sec1, 1 = 2300 sec1, 1 = 9400 sec1, 2 = 690 sec1, 2 = 80,800 sec1: wild type: (parameters from Table 4 and Ohno
et al., 1996) k+1 = k+3 = 1.0 × 108
M1 sec1,
k+2 = k+4 = 1.1 × 108 M1
sec1, k1 = k3 = 2400 sec1,
k2 = k4 = 12,700 sec1, 1 = 30,000 sec1, 1 = 1 sec1, 1 = 6990 sec1, 1 = 58 sec1, 2 = 1520 sec1, 2 = 46,000 sec1. B plots the ratio of
probabilities of long to intermediate openings, P3/P2, against ACh
concentration for each receptor type.The dashed lines
indicate the theoretical slope, defined by
2/1/K1.
[View Larger Version of this Image (23K GIF file)]
We simulated activation kinetics for wild-type receptors using
parameters in Scheme 1 taken from our previous analysis of human
wild-type receptors (Ohno et al., 1996). Because only one class of
singly occupied openings was observed at the micromolar ACh
concentrations used in those experiments, for the second class of
singly occupied openings, we used the closing rate 1
determined in this study, but assigned a value of 1
sufficient to give enough of these events. The simulated burst-duration
histograms again exhibit three components, with the area of each
depending on ACh concentration (Fig. 8, right panel).
Again, the ratio P3/P2 increases linearly with increasing ACh
concentration with a slope identical to the expected ratio
2/1/K1.
In summary, the results from simulation of channel kinetics confirm the
analysis method applied to experimental data obtained at nanomolar ACh
concentrations.
Simulation of wild-type activation kinetics at nanomolar ACh
concentrations allows comparison with parameters determined previously from kinetic analysis of currents activated by micromolar
concentrations (Ohno et al., 1996). The ratio
2/1/K1
obtained at nanomolar ACh concentrations (Fig. 7C) is
approximately twofold greater than obtained at micromolar
concentrations. However, we consider this to be good agreement because
measurements at nanomolar concentrations yield the ratio of three
parameters, and measurements at micromolar concentrations yield each
parameter separately. The two methods yield similar estimates of
2 (30 at nanomolar and 24 at micromolar concentrations).
The parameter 1 was probably overestimated at micromolar
ACh concentrations because the corresponding monoliganded openings were
present in only a minor proportion of the data, whereas the remaining
parameter K1 was consistent with data obtained over a wide range of ACh concentrations.
Activation of V249F receptors by the competitive antagonist
dimethyl-D-tubocurarine
We further examined functional consequences of V249F by
recording single-channel currents activated by the competitive
antagonist dimethyl-d-tubocurarine (DMT). DMT only rarely
activates wild-type receptors, whereas it frequently activates V249F
receptors (Fig. 9). The increased frequency of
activation can be seen from the decrease in the mean of the major
closed-duration component, from 20 sec for wild type to 200 msec for
V249F (Fig. 9). The enhancement of activation is probably >100-fold
because the recording for wild type was especially active, whereas that
for V249F was typical. The recordings also show that DMT does not
significantly desensitize the V249F receptor; a major long component
of closings is observed in the presence of 10 µM DMT,
attributable to periods between independent activation episodes,
whereas multiple components with similar areas are observed with ACh
because of desensitization (compare Figs. 5 and 9, center
panels). Thus, V249F increases the ability of a competitive
antagonist to activate the receptor while retaining little or no
capacity to desensitize.
Fig. 9.
Activation of human wild-type and V249F AChRs
by DMT. The left panels show single-channel currents
elicited by the 10 µM DMT at a bandwidth of 10 kHz. The
center and right panels show corresponding closed and burst-duration histograms fitted to the sum of
exponentials. Note the major long-duration component of closings for
both receptor types, and the 100-fold longer mean closed duration for
wild type compared with V249F.
[View Larger Version of this Image (17K GIF file)]
Spontaneous opening of the V249F receptor
In a previous study of an SCCMS, we demonstrated spontaneous
openings attributable to mutations in the M2 domains of the and subunits (Ohno et al., 1995; Engel et al., 1996b). Therefore, we looked
for spontaneous opening of the V249F receptor by recording from
patches in the absence of ACh. Using the presence of green fluorescent
protein as a marker for transfected cells and the increased conductance
as a signature of the V249F receptor, we observed spontaneous
openings in every patch examined (Fig. 10), averaging 1 per second per patch over 25 patches. By contrast, spontaneous openings
of the wild-type receptor were not observed, despite recording from
stable patches for up to 30 min. At our standard voltage of 70 mV,
V249F receptors show a major component of spontaneous openings with
a mean duration of 300 µsec and relative area of 0.85, plus minor
components with means of 60 µsec and 3 msec. Spontaneous and
ACh-induced openings can be distinguished readily because of
differences in burst kinetics and flanking closed intervals; comparison
of closed- and burst-duration histograms indicate that spontaneous
openings are minimized at ACh concentrations of 3 nM and
greater.
Fig. 10.
Spontaneous opening of the V249F receptor.
Current traces obtained in the absence of ACh from a
transfected cell identified by presence of green fluorescent protein.
Burst- and closed-duration histograms are fitted by the sums of
exponentials.
[View Larger Version of this Image (13K GIF file)]
Binding of agonists and antagonists at equilibrium reveal enhanced
desensitization of V249F receptors
V249F receptors exhibit no channel activity at micromolar
ACh concentrations, suggesting enhanced desensitization. Because desensitized receptors bind agonist with high affinity, we tested the
desensitization hypothesis by measuring ACh binding by competition against the initial rate of 125I--bungarotoxin binding.
Under equilibrium conditions, V249F receptors bind ACh 300-fold more
tightly than wild-type receptors (Fig.
11A). The single apparent
dissociation constant of 4.3 nM approaches the two
dissociation constants obtained for the desensitized wild-type
receptor, 4.8 and 41 nM (Ohno et al., 1996). The high affinity, together with lack of channel activity at micromolar ACh
concentrations, suggests that V249F profoundly enhances
desensitization.
Fig. 11.
V249F enhances agonist-binding affinity.
Binding of the specified ligand was determined by competition against
the initial rate of 125I--bgt binding. Intact HEK cells
expressing the indicated receptor type were incubated in the presence
of agonist for 30 min before measuring the initial rate of toxin
binding. The smooth curves are fits to the Hill equation
(A-C) or to an equation
describing the sum of two distinct binding sites
(D). Fitted parameters for each ligand are
acetylcholine, wild type: Kov = 6.9 × 107, n = 1.2; V249F,
Kov = 4.3 × 109, n = 1.0;
carbamylcholine, wild type: Kov = 1.6 × 105, n = 1.6; V249F,
Kov = 4.8 × 108,
n = 0.8; T264P, Kov = 9.5 × 107, n = 1.8. TMA,
wild type: Kov = 1.1 × 104, n = 1.5; V249F:
Kov = 9.5 × 107,
n = 0.8. DMT, wild type:
KA = 4.4 × 108,
KB = 3.8 × 106;
V249F: KA = 6.2 × 108, KB = 6.2 × 107.
[View Larger Version of this Image (24K GIF file)]
To determine whether the marked enhancement of affinity depends on
structure of the agonist, we examined binding of carbamylcholine (CCh),
tetramethylammonium (TMA), and DMT. V249F receptors bind CCh and TMA
300-fold more tightly than wild-type receptors (Figs. 11B,C), as observed for ACh, and
the relative affinities of ACh, CCh, and TMA are preserved. By
contrast, V249F receptors bind DMT only slightly more tightly than
wild-type receptors, with the tighter binding restricted to the
low-affinity binding site (Fig. 11D); this slight
increase in affinity is likely attributable to a shift in equilibrium
from the closed to the open channel state caused by V249F. Thus, the
marked increase in affinity attributable to V249F is specific for
small cholinergic agonists. Because DMT does not appreciably
desensitize the V249F receptor and binds with only slightly greater
affinity, the marked increase in agonist affinity is likely
attributable to enhanced desensitization.
Our previous studies ascribed slow channel myasthenic syndromes to
mutations in the M2 domains of the and subunits, which cause
channel opening in the absence of ACh and prolonged openings in its
presence (Ohno et al., 1995; Engel et al., 1996b). One of these
mutations, T264P, is three residues above the conserved leucine ring
in M2, whereas V249F is two residues below it. Thus, we compared
agonist binding affinities of the two mutations to determine structural
specificity. T264P receptors bind CCh 20-fold more tightly than
wild-type receptors, whereas V249F receptors bind 300-fold more
tightly (Fig. 11B). Thus, the perturbation caused by
V249F is distinct from that caused by T264P, and the enhancement of desensitization is expected to be greater for receptors containing V249F than T264P.
DISCUSSION
The V249F mutation
The slow-channel syndromes are autosomal dominant disorders
(Engel, 1994b), attributable to pathological gain of function by the
mutated receptor. Detection of V249F by allele-specific PCR in the
patient's asymptomatic father at first appeared inconsistent with the
dominant character as well as with the pathogenicity of the identified
mutation. However, sequencing of the entire open reading frames of the
, , , and subunit genes of AChR in the propositus revealed
no other mutations, and failure to detect V249F in paternal DNA by
direct sequencing or restriction analysis suggested that only a small
proportion of paternal DNA harbored the mutation. This assumption was
verified by cloning of paternal DNA, which established that the father
was mosaic for V249F. For the propositus to be affected, the
mutation must have been transmitted by a sperm cell carrying the
mutation. Thus, V249F must have occurred in the father's early
embryonic life to render him both a somatic and a germ-line mosaic
(McGookey-Milewitz et al., 1993).
The SCCMS mutations discovered to date are in the extracellular, M1, or
M2 domains of AChR subunits (Ohno et al., 1995; Sine et al., 1995;
Engel et al., 1996b; Gomez et al., 1996). The M2 mutations identified
previously, however, are in the outer helical region of M2 (T264P,
V266M, and L269F), or at the central leucine ring (L262M) that
may form the channel gate (Unwin, 1993), and the mutated residues are
accessible to labeling reagents when converted to cysteine, predicting
that they face the channel lumen (Akabas et al., 1994). By contrast,
V249F is two residues below the leucine ring, and accessibility
studies indicate that the mutated valine does not face the channel
lumen (Akabas et al., 1994). Thus, M2 mutations can cause a profound
functional disturbance at a variety of locations relative to the
leucine ring or the channel lumen.
Functional consequences of V249F
The expression studies demonstrate that V249F not only
stabilizes the open and desensitized states, but it also enhances affinity of the resting state for ACh. Effects on equilibria between functional states can be explained readily by local perturbation of the
M2 domain, which is a structural endpoint defining each of the three
functional states. Thus, the findings show that the structure of M2 is
essential for correct stabilization of each functional state.
Enhancement of ACh binding affinity, on the other hand, implies spread
of the perturbation from the M2 domain to the binding site, some 30 Å away. Spread of the perturbation suggests reciprocal coupling between
the channel and the binding site through some linkage structure. The
effects of V249F are thus widespread and are expected to affect
receptor function in several ways.
Wild-type receptors are designed to open or desensitize with low
probability in the absence of ACh (Jackson, 1989). A stable closed
state minimizes leak of cations and maintains the majority of receptors
ready to be activated. V249F destabilizes the resting closed state
of the receptor in favor of the open channel state, as shown by the
increased rate and extent of opening in both the absence and the
presence of ACh. Stabilization of the open channel state is revealed by
the slower rate of closing of the doubly occupied receptor. V249F
also increases equilibrium affinity for ACh, CCh, and TMA 300-fold, and
the binding affinities for these small quaternary agonists are close to
those for binding to desensitized wild-type receptors (Sine et al.,
1994; Ohno et al., 1996). Such a large increase in apparent affinity
can be explained by a change in the equilibrium between resting and
desensitized states from a wild-type value of 104
to ~0.1 or greater (Sine et al., 1995); thus, V249F destabilizes the resting closed state in favor of the desensitized state. Enhanced opening of the V249F receptor overloads the sarcoplasm with cations, both tonically and during nerve impulse, whereas enhanced
desensitization decreases the number of receptors that can be
activated.
The expression studies also show that V249F markedly enhances ACh
affinity for at least one of the two binding sites in the resting
closed state of the receptor. Evidence for enhanced affinity began with
the observation of three kinetically distinct types of openings, two
singly liganded and one doubly liganded. By monitoring the
concentration dependence of doubly versus singly liganded openings, we
followed the two possible pathways for forming the doubly occupied
receptor. In the first pathway, ACh binds initially to the site with
relatively low affinity, opening in bursts of events of intermediate
durations and then to the site with nanomolar affinity to yield doubly
occupied receptors that open in bursts of prolonged openings. Evidence
for low affinity of the initial site is the fast rate of ACh
dissociation (8650 sec1) obtained from the
kinetics of intermediate bursts, whereas high affinity of the
subsequent site follows from changes of the probabilities of long and
intermediate bursts with nanomolar changes in ACh concentration. In the
second pathway, ACh initially binds to the site with nanomolar affinity
and then to the site with low affinity, again leading to bursts of
prolonged openings. In this pathway, the site with nanomolar affinity
will be occupied until ACh can dissociate, or ~1 sec after nerve
impulse; when bound to this site, ACh elicits single openings of brief
duration that persist throughout occupancy. Moreover, occupancy on the
order of seconds is sufficiently long to allow transition from the
resting activatable to the desensitized state, which at physiological
rates of stimulation may approach the near maximal extent observed in
our equilibrium binding experiments.
Comparison with other studies of M2 mutations
Previous studies revealed changes in activation, desensitization,
ion permeability, and ligand specificity attributable to mutations in
the M2 domain. Both naturally occurring and site-directed mutations
increased channel opening in the absence of ACh and prolonged opening
in its presence (Ohno et al., 1995; Auerbach et al., 1996; Engel et
al., 1996b). Enhanced opening was also inferred from increased apparent
affinity in dose-response studies of M2 mutations in muscle (Filatov
and White, 1995 Labarca et al., 1995) and 7 neuronal
(Revah et al., 1991) receptors. Heterogeneity in the positions of M2
mutated and mutant side chains suggest relatively little structural
specificity in enhancing opening of the channel. Studies of mutant
7 neuronal and 5HT-3 receptors showed either increased
or decreased rates of onset and extents of desensitization (Revah et
al., 1991; Yakel et al., 1993). The profound desensitization we observe
for V249F contrasts with elimination of desensitization reported for
mutations in 7 neuronal and GABAA receptors
(Revah et al., 1991; Clements et al., 1996). Appearance of two
conductance classes of channels was described for mutant
7 neuronal receptors, one of normal conductance and the
other increased approximately twofold. Our findings of altered conductance properties are less striking, because we observe a single
conductance, increased by only ~10%, and infrequent subconductance states associated with it; differences may be attributable to the
presence of five mutant subunits in 7 and only two
mutant subunits in our studies, or to differences in the sites of the mutations and mutant side chains. Activation by competitive antagonists was reported for mutant 7 neuronal receptors (Revah et
al., 1991), similar to our observation of activation of V249F
receptors by DMT. Activation by competitive antagonists is likely
attributable to increase of the exceedingly low opening rate found in
wild-type receptors. The novel functional consequence of V249F is
enhancement of the affinity of the resting state of the receptor for
binding ACh.
Phenotypic consequences
Because of the presence of two subunits in each AChR
macromolecule, AChRs at the SCCMS EPs may harbor either wild-type or mutated (w or m) subunits. If
mutant and wild-type subunits assemble and are expressed as pentameric
receptors with equal efficiency, AChRs containing
ww,
wm, and
mm should be present in a 1:2:1 ratio.
Differences in functional properties among these AChRs would be
expected to introduce multiple components in the MEPC, ACh-induced
current noise, and single-channel recordings, and this likely accounts
for the differences among the values for channel bursts estimated
by the different methods. For example, MEPCs decay with two detectable
time constants of 2.4 msec and 43.9 msec at the SCCMS EP, but
single-channel burst durations show means of 2.95 and 19.7 msec, and
noise analysis shows time constants of 1.4 and 15.1 msec. Because of
the continuous presence of agonist in the single-channel and noise
recordings and the marked tendency of AChRs harboring two mutated subunits to become desensitized, only AChRs with a single mutated subunit are likely to contribute appreciably to the distribution of
burst durations and the noise spectrum. The MEPC, however, which
follows instantaneous application of ACh, provides a more accurate
estimate of the biological activity of AChRs in vivo.
A minor proportion of channel events at the EP had a reduced
conductance of 45 pS and prolonged open durations typical of -AChR.
That a small proportion of these events had an additional longer
component is explained readily by -AChRs harboring one or two
mutated subunits. Because we previously found a low level of
-AChR expression in other slow-channel syndromes with mutations in
the or subunits and a destructive EP myopathy (Ohno et al.,
1995; Engel et al., 1996b), we postulate that the -AChR expression
is secondary to regenerative activity in the postsynaptic region.
-AChR is also expressed in those CMS because of nonsense mutations
in both alleles of the subunit gene, where it is the predominant
species at the EP and may serve as a means of phenotypic rescue (Engel
et al., 1996a).
The widespread functional consequences of V249F are expected to
compromise the safety margin of neuromuscular transmission in several
ways. First, there may be reduced expression of the mutant AChR.
Second, the prolonged channel activation episodes as well as tonic
openings of unliganded receptors in the resting state overload the
junctional sarcoplasm with cations that include calcium (Lester, 1992;
Villarroel and Sakmann, 1996). This results in destruction of
junctional folds with loss of AChR, widening of the synaptic space, and
destruction of organelles in the junctional sarcoplasm (see Fig. 1).
Third, apoptosis of a proportion of junctional nuclei (see Fig.
1E), probably attributable to calcium excess in the
junctional sarcoplasm (McGahon et al., 1995), likely reduces the
transcription of AChR subunit genes. Fourth, the marked tendency of the
mutant AChR to desensitize predicts that an appreciable fraction of the
EP AChR is desensitized even in the resting state, further decreasing
the number of receptors that can be activated. Moreover,
acetylcholinesterase inhibitors, which prolong the life time of ACh in
the synaptic space during activity, may result in desensitization of a
large fraction of the EP AChR, with deleterious clinical effects.
Fifth, the markedly prolonged decay of EP potentials (generally >40
msec; data not shown) predicts their temporal summation and a
depolarization block of transmission during even normal physiological
activity.
FOOTNOTES
Received Feb. 6, 1997; revised April 10, 1997; accepted May 9, 1997.
This work was supported by National Institutes of Health Grants NS6277
to A.G.E. and NS31744 to S.M.S, a Muscular Dystrophy Association (MDA)
research grant to A.G.E., and an MDA postdoctoral fellowship to K.O. We
thank Dr. Susan Iannaccone for the patient referral.
Correspondence should be addressed to Dr. Steven M. Sine, Receptor
Biology Laboratory, Department of Physiology, Mayo Foundation, 200 First Street SW, Rochester, MN 55905.
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