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. Endplates (EPs) were localized for electron microscopy by established methods (Engel, 1994). α-Bungarotoxin (α-Bgt) labeled with peroxidase was used for the ultrastructural localization of AChR (Engel et al., 1977). The number of AChRs per EP was measured with ¹²⁵I-labeled α-bgt, as described previously (Engel et al., 1993).

Miniature EP potential (MEPP), miniature EP current (MEPC), and EP potential recordings, and estimates of the number of transmitter quanta released by nerve impulse were performed as described previously (Engel et al., 1993; Uchitel et al., 1993). Patch-clamp recordings from EP AChRs were performed in cell-attached mode by a slight modification of the previously described method (Milone et al., 1994). For all patches, the membrane potential was set to −80 mV. Whenever 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, Inc.) and analyzed at a final bandwidth of 8 kHz (patient EPs) and 12 kHz (control EPs) using the programs PClamp 6 (Axon Instruments, Inc.). 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 a logarithmic abscissa and fitted to the sum of exponentials by maximum likelihood (Sigworth and Sine, 1987).

We directly sequenced the AChR α, β, δ, and ɛ subunit genes using genomic DNA isolated from blood (Ohno et al., 1995). To investigate family members, we traced ɛD175N with allele-specific PCR and ɛN182Y by RsaI restriction analysis of PCR products. ɛN182Y results in gain of an RsaI site. To prove that the patient's two mutations are heteroallelic, we selectively amplified the alleles with and without ɛD175N using mutant and wild-type allele-specific PCR primers, respectively, and digested the products with RsaI that recognizes only the ɛN182Y allele. To exclude that ɛD175N and ɛN182Y represent polymorphisms, we screened for them in 200 normal alleles using allele-specific PCR. To check paternity, we determined the numbers of tri- or tetranucleotide repeats at D17S969, D17S918, and D17S2196 by direct sequencing. The paternity index was calculated using a Bayesian approach (Sozer et al., 2001).

Human AChR α, β, δ, and ɛ subunit cDNA were cloned into the CMV-based expression vector pRBG4 as described elsewhere (Ohno et al., 1996). ɛD175N and ɛN182Y were introduced into the ɛ subunit cDNA in pRBG4 by the QuickChange site-directed mutagenesis kit (Stratagene). The presence of the desired mutations and absence of unwanted artifacts were confirmed by sequencing the entire insert. HEK 293 cells were transfected with AChR subunit cDNAs using calcium phosphate precipitation (Sine et al., 1995; Ohno et al., 1996). In some experiments the BOSC cell line was used, which is a variant of the 293 HEK cell line (Wang et al., 2000).

Recordings were obtained in the cell-attached configuration at a membrane potential of −70 mV and at 22°C. The bath and pipette solutions contained (mM): KCl 142, NaCl 5.4, CaCl₂ 1.8, MgCl₂ 1.7, HEPES 10, pH 7.4. Single channel currents were recorded using an Axopatch 200A amplifier at a bandwidth of 50 kHz, digitized at 5-μs intervals using an ITC-16 AD interface (Instrutech), recorded to hard disk using the program Acquire, and detected by the half-amplitude threshold criterion using the program Tac (Bruxton Corporation) at a final bandwidth of 10 kHz (Wang et al., 1999). To estimate rate constants in Scheme I (see text and Table III) TABLE III

Fits of Scheme I to Dwell Times from Wild-Type and Mutant AChRs

The total number of ¹²⁵I α-bgt sites on the cell surface of transfected HEK cells and ACh competition against the initial rate of ¹²⁵I α-bgt were determined as previously described (Ohno et al., 1996). ACh competition measurements were analyzed according to either the Hill equation (Eq. 1) or the two binding site equation (Eq. 2): (1)\documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{1}}-{\mathrm{Y}}={{\mathrm{1}}}/{\left\{{\mathrm{1}}+\left[{\left({\mathrm{ACh}}\right)}/{{\mathrm{K}}_{{\mathrm{OV}}}}\right]\right\}^{{\mathrm{n}}}}\end{equation*}\end{document} (2)\documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{gather*}{\mathrm{1}}-{\mathrm{Y}}= \\ {\mathrm{fract}}_{{\mathrm{A}}}\left({{\mathrm{1}}}/{{\mathrm{1}}+{\left[{\mathrm{ACh}}\right]}/{{\mathrm{K}}_{{\mathrm{A}}}}}\right)+ \\ \left({\mathrm{1}}-{\mathrm{fract}}_{{\mathrm{A}}}\right)\left({{\mathrm{1}}}/{{\mathrm{1}}+{\left[{\mathrm{ACh}}\right]}/{{\mathrm{K}}_{{\mathrm{B}}}}}\right){\mathrm{,}}\end{gather*}\end{document}where Y is fractional occupancy by ACh, n is the Hill coefficient, K_OV the overall dissociation constant, K_A the dissociation constant for one site, and K_B the dissociation constant for the second site.

A 5-yr-old male had decreased movements in utero, eyelid ptosis since early infancy, and delayed motor development. He fatigued rapidly on mild exertion and could not keep up with his peers in physical activities. He had a decremental electromyographic response on stimulation of motor nerves, negative tests for anti-AChR antibodies, and responded partially to cholinesterase inhibitors. He has a high-arched palate, mild scoliosis, slight limitation of the ocular ductions, and fluctuating eyelid ptosis. He can extend his arm horizontally for only 30 s. The parents and a younger sibling are unaffected.

The configuration of the EPs, evaluated from the cytochemical reaction for acetylcholinesterase on 40 teased single muscle fibers, was normal on 33 fibers and consisted of 2–3 closely spaced small EP regions on 7 fibers. The number of ¹²⁵I-α-bgt sites per EP was normal for the patient's age (Table I) TABLE I

Endplate Studies

Quantal release by nerve impulse was normal. Although the density and distribution of AChR on the junctional folds appeared normal, the amplitude of the MEPPs was reduced to 32%, and that of the MEPCs to 40% of normal. The MEPCs decayed abnormally fast, so that τ_MEPC was only 26% of normal (Table I).

To confirm the kinetic defect of AChR predicted by the rapidly decaying MEPCs, we compared single channel currents recorded from control and patient EPs. In contrast to control EPs, only few channel openings could be recorded from patient EPs with ACh concentrations lower than 5 μM, suggesting a decreased probability of channel opening. At patient EPs, 96% of all channel openings had a normal conductance of 60 pS, but they showed abnormally brief open durations; by contrast, 4% (∼1% at three EPs and 16% at one EP) of channel openings had a conductance of ∼44 pS and four- to fivefold longer than normal open durations, typical of fetal receptors that harbor the γ instead of the ɛ subunit. Analysis of the 60 pS channel events at patient and control EPs resolved two components both in bursts of openings and single openings (τ₁ and τ₂), but in the patient the briefer component, τ₁, was dominant, whereas at control EPs the longer component, τ₂, was dominant (Table II TABLE II

Open Intervals at Seven Control and Four Patient EPs

To determine the basis of the observed kinetic abnormalities, we proceeded with mutation analysis. Direct sequencing of all exons and flanking regions of the AChR α, β, δ, and ɛ subunit genes revealed two heterozygous mutations in the extracellular domain of the AChR ɛ subunit: a G-to-A substitution at nucleotide 523 in ɛ exon 6, predicting an aspartate-to-asparagine substitution at codon 175 (ɛD175N), and an A-to-T substitution at nucleotide 544 in ɛ exon 7, predicting an asparagine-to-tyrosine substitution at codon 182 (ɛN182Y). Aspartate at ɛ codon 175 is conserved in the human α, δ, and γ subunits, and in ɛ subunits of all species. Asparagine at ɛ codon 182 is conserved in the human β, δ, and γ subunits, and in ɛ subunits of all species (Fig. 2 Figure 2.

(A) Allele-specific PCR (ASP) for ɛD175N and RsaI restriction analysis for ɛN182Y of genomic DNA from patient (arrow) and family members. The patient carries ɛD175N and ɛN182Y; patient, mother, and brother harbor ɛN182Y. Open and closed arrowheads indicate wild-type and mutant fragments, respectively. (B) The ɛD175N and ɛN182Y mutations are heteroallelic. Allele-specific PCR selectively amplifies the ɛD175N allele (left), and the ɛD175N allele (right). PCR product amplified from each allele is digested with RsaI that only cuts the ɛN182Y allele. That only allele “b” is cut by RsaI indicates that ɛN182Y is on allele “b”. (C) Aspartate at ɛ codon 175 (D175) is conserved in human α, δ, and γ subunits, and in ɛ subunits of all species. Asparagine at ɛ codon 182 (N182) is conserved in human β, δ, and γ subunits, and in ɛ subunits of all species.

To establish that ɛN182Y and ɛD175N cause the fast-channel CMS, we recorded single channel currents from 293 human embryonic kidney (293 HEK) cells transfected with wild-type α, β, and δ plus either wild-type or mutant ɛ subunit cDNAs. For each wild-type or mutant receptor, we applied a range of high concentrations of ACh (1–1000 μM) to elicit clustering of the currents into epochs during which only one channel is active. Recording individual clusters of channel activity allows subsequent removal of intercluster closed periods due to desensitization, allowing analysis of receptor activation independent of desensitization (Sine et al., 1990). Inspection of current pulses within clusters reveals briefer openings and prolonged closings for both ɛN182Y and ɛD175N receptors compared with wild-type receptors (Fig. 3) Figure 3.

Kinetics of activation of wild-type and mutant receptors. Left column displays individual clusters of single channel currents recorded at the indicated ACh concentrations from cells expressing adult human AChRs containing the indicated mutant ɛ subunits. Currents are displayed at a bandwidth of 10 kHz, with channel openings shown as upward deflections. Center and right columns display the corresponding closed and open duration histograms with the fits for Scheme I superimposed. Fitted rate constants are given in Table III.

To uncover the mechanistic consequences of ɛN182Y and ɛD175N, we constructed dwell time histograms from open and closed intervals within individual clusters of single channel activity (see materials and methods). Histograms of open dwell times show a major peak, or exponential component at all ACh concentrations (Fig. 3). A second brief component, visible as a rise in the distribution to the left of the major component, is present at low but not high ACh concentrations. The second component is readily discerned in the data for wild-type and ɛD175N receptors selected for display. However, because the ɛN182Y mutation increases agonist affinity, as described below, the second component is present only at ACh concentrations <10 μM (unpublished data). Histograms of closed dwell times show two predominant components, the longer of which moves to briefer time as the ACh concentration increases. These concentration dependencies of open and closed dwell times are observed for wild-type and each mutant AChR, and are characteristic of an activation process driven by ACh concentration.

To identify elementary kinetic steps altered by ɛN182Y and ɛD175N, we analyzed the closed and open dwell times according to the following standard description of receptor activation, Figure

where A is the agonist, R is the closed receptor, R* is the open receptor, k₊₁ and k₊₂ are association rate constants, and k₋₁ and k₋₂ are dissociation rate constants. Singly occupied receptors AR open with rate constant β₁ and close with rate constant α₁, whereas doubly occupied receptors A₂R open with rate constant β₂ and close with rate constant α₂. A blocked state A₂R_B is included to account for brief channel closures elicited by high ACh concentrations.

We estimated rate constants for each state transition in Scheme I by computing the likelihood of the experimental series of dwell times, given a set of trial rate constants, and then changing the rate constants to maximize the likelihood (see materials and methods). For wild-type and each mutant receptor, we fitted Scheme I simultaneously to open and closed dwell times obtained for the entire range of ACh concentrations. Use of a range of ACh concentrations optimizes the contribution of dwells in each receptor state to the cumulative likelihood. The fitted rate constants yield probability density functions that superimpose upon the open and closed duration histograms (Fig. 3), showing that Scheme I describes activation of wild-type, ɛN182Y, and ɛD175N receptors.

The fitted rate constants show that ɛN182Y markedly increases ACh affinity for one of the two binding sites in the resting closed state of the receptor, but does not affect ACh affinity for the second binding site (Table III). The dissociation constant for the altered site falls from 138 μM for the wild-type receptor to 2 μM for the mutant, owing to a 30-fold slowing of the rate constant for ACh dissociation and a twofold increase of the rate constant for ACh association. Because the mutation is present in the ɛ subunit, the binding site with increased ACh affinity corresponds to the αɛ site. The increased affinity conferred by ɛN182Y defines the sequence in which ACh occupies the binding sites of the mutant receptor in the course of opening the channel; the αɛ site is occupied first, whereas the αδ site is occupied second.

The ɛN182Y mutation not only alters ACh binding, but it also alters gating of the channel. The gating equilibrium constants for singly occupied (θ₁) and doubly occupied (θ₂) receptors are reduced by an order of magnitude. Reduction of θ₂ likely follows directly from the reduction in θ₁ because ACh occupancy of the second binding site begins with a singly occupied receptor less prone to opening. Thus, although the second occupancy step in the mutant receptor promotes channel gating as well as the second occupancy step in wild-type (θ₂/θ₁ = 1,080 for the mutant and θ₂/θ₁ = 1,083 for wild-type; see Table III), gating of the doubly occupied mutant receptor is reduced by an order of magnitude.

Mechanistic underpinnings for the reduced θ₁ can be understood by inspection of the following expanded form of Scheme I (Monod et al., 1965): Figure

where K₁* and K₂* are dissociation constants for ACh binding to the open states and θ₀ is the gating equilibrium constant in the absence of agonist. According to Scheme II, θ₁ could decrease due to (a) a decrease of K₁ relative to K₁* or (b) a decrease of θ₀ without a change in K₁ relative to K₁*. Because neither K₁* nor θ₀ are defined by the data, both alternatives are formal possibilities.

Comparison of ACh binding steps for wild-type and ɛN182Y mutant receptors shows that the second binding site in the ɛN182Y receptor is similar to the first binding site in the wild-type receptor; both the association and dissociation rate constants are similar between the two types of receptors (Table III). A common unaltered site is also observed for the site-directed mutations ɛN182F and ɛN182D described below. The most likely explanation for a common site is ɛN182Y affects the αɛ binding site without altering the αδ binding site. Thus, mutations at ɛN182 not only reveal a new determinant of ACh binding affinity, but also provide independent estimates of the rate constants underlying ACh occupancy of the wild-type αδ site.

To determine the structural basis for the effect of ɛN182Y on receptor activation, we constructed mutations with electron-rich side chains, ɛN182F and ɛN182D, and examined single channel kinetics of the resulting mutant receptors (Fig. 4) Figure 4.

Kinetics of activation of receptors with site-directed mutations. Left column displays individual clusters of single channel currents recorded at the indicated ACh concentrations from cells expressing adult human AChRs containing the indicated mutant subunits, as in Fig. 3.

We constructed the analogous mutation in the δ subunit, δN187Y, to determine whether it affects ACh binding to one of the two binding sites, as observed for ɛN182Y, and to independently estimate rate constants underlying ACh occupancy of the nonmutant αɛ site. Single channel currents and the corresponding fitted rate constants show that the δN187Y mutation enhances ACh affinity for one binding site in the resting closed state, but does not affect ACh affinity for the second site (Fig. 4; Table III). The rate constant for ACh association accelerates 1.3-fold, and the rate constant for ACh dissociation slows 1.7-fold; the resulting dissociation constant for the altered site falls from 26 μM for wild-type AChR to 12 μM for the mutant. Because the mutation is present in the δ subunit, the binding site with increased agonist affinity corresponds to the αδ site. Moreover, rate constants underlying ACh occupancy of the nonmutant αɛ site are very similar to those of the low affinity binding site in the wild-type receptor (Table III). Thus, the ɛN182Y and δN187Y mutations locally affect ACh affinity of the corresponding αɛ or αδ sites, providing independent estimates of rate constants underlying ACh binding to nonmutant αδ and αɛ sites. In the wild-type human AChR, the αδ subunit interface forms the binding site with high affinity for ACh, whereas αɛ interface forms the site with low affinity.

Single channel kinetic analysis of currents through receptors containing ɛD175N suggests that ACh binding is impaired at both binding sites (Table III). However, because the mutation is present in the ɛ subunit, we tested the idea that ɛD175N affects the affinity of only one binding site. A mutational effect restricted to the αɛ site is likely because four mutations, ɛN182Y, ɛN182F, ɛN182D, and δN187Y, had local effects at the corresponding α–ɛ or α–δ sites. Furthermore, the protein chain harboring ɛN182 and ɛD175 is not part of a secondary structural element and lodges in a crevice on the periphery of the subunit (Sine et al., 2002). Thus, we constrained rate constants for one binding site to the average of those obtained for the nonmutant α–δ site from analyses of the three mutations at ɛN182, and allowed rate constants for the other binding site to vary. The resulting fits provided well-defined rate constants for both wild-type and ɛD175N receptors (Table III); probability density functions computed from the fitted rate constants superimpose upon the dwell time histograms for both types of receptors (Fig. 3), and were indistinguishable from those obtained from the unconstrained fitting (not shown). Moreover, the resulting fitted parameters reveal that ɛD175N slows the rate constant for ACh association ∼10-fold, and accelerates the rate constant for ACh dissociation twofold; the mutation increases the dissociation constant for ACh binding to the αɛ site from 111 to 1,980 μM (Table III).

The fitting analysis also reveals that ɛD175N reduces gating of singly and doubly occupied receptors by about an order of magnitude (Table III). The gating equilibrium constant θ₁ is particularly well discerned in the data where a predominant brief component of openings, present at low ACh concentrations, is replaced by a longer-lived component at high concentrations (Fig. 3). The decrease of θ₂ is likely a direct consequence of the decrease of θ₁ because binding of the second ACh starts with a singly occupied receptor less prone to opening. Thus, although ACh occupancy of the mutant binding site promotes channel gating nearly as well as occupancy of the second binding site in wild-type (θ₂/θ₁ = 677 for ɛD175N and θ₂/θ₁ = 1,083 for wild-type), gating of the doubly occupied mutant receptor is reduced by an order of magnitude.

The possibility that ɛD175N preferentially affects ACh affinity for the open relative to the closed state of the receptor was assessed by applying detailed balancing to the following cycle taken from Scheme II: Figure

Here K₂* is the dissociation constant for agonist binding to the open state of the receptor, which equals K₂θ₁/θ₂. For the wild-type receptor, K₂* calculated from the present data is 168 nM, whereas K₂* for the ɛD175N mutant is 2,920 nM. Thus, ɛD175N increases K₂* by 17-fold, which is close to its 18-fold increase of K₂, indicating that the mutation does not preferentially affect ACh affinity for the open relative to the closed state of the receptor.

If the ɛD175N mutation maintains a large K₂/K₂* ratio, providing a normal increase of θ₂ relative to θ₁, how does ɛD175N impair overall gating of the channel? The earliest discernable effect in Scheme I is reduction of θ₁, but this observation leads to the following paradox: how does a mutation at the αɛ site affect the gating equilibrium constant associated with ACh occupancy of the nonmutant αδ site? A likely explanation is that residues at the binding site not only mediate recognition of ACh, but also set the trigger point for opening the channel. Thus, the results suggest that residues at the binding site may contribute to stability of closed relative to open states of the channel even when agonist is not bound.

To illustrate the overall consequences of ɛD175N, ɛN182Y, and ɛN182D, and to confirm our estimated rate constants, we determined the fraction of receptors activated as a function of ACh concentration, and compared it with the dose–response relationship calculated from the fitted rate constants in Table III. Our measure of the fraction of receptors activated is the mean open probability (P_open) within identified clusters due to single AChRs (see traces in Fig. 3). The ɛN182Y mutation reduces the maximum channel open probability, but does not significantly shift the curve along the concentration axis (Fig. 5) Figure 5.

Dependence of channel open probability (P_open) on ACh concentration for receptors containing the indicated mutant ɛ subunits plus complementary α, β, and δ subunits. The mean fraction of time the channel was open during a cluster (P_open) was determined at the indicated concentrations of ACh. The theoretical P_open was calculated from Scheme I using the fitted rate constants in Table III, and superimposed on the P_open measurements. Error bars indicate standard deviations of P_open for the population of clusters at the specified ACh concentration.

Our single channel kinetic analysis reveals that ɛN182Y enhances, whereas ɛD175N reduces, ACh affinity for receptors in the resting closed state. To determine whether these mutations affect ACh affinity for receptors in the desensitized state, we converted receptors to the desensitized state using the local anesthetic proadifen (Sine and Taylor, 1982), and then measured binding of ACh (Ohno et al., 1996). We incubated intact 293 HEK cells expressing recombinant receptors with specified concentrations of ACh, with or without proadifen, and then measured binding of ACh by competition against the initial rate of ¹²⁵I-α-bgt binding. For the wild-type receptor, the presence of proadifen increases ACh affinity ∼100-fold due to high affinity of the desensitized state (Fig. 6) Figure 6.

ACh binding to receptors in the presence of the desensitizing agent proadifen. (A) Comparison of ACh binding to wild-type and ɛN182Y receptors. (B) Comparison of ACh binding to wild-type and ɛD175N receptors. Binding of ACh was determined by competition against the initial rate of ¹²⁵I-α-bgt binding. Intact HEK cells expressing the indicated receptor type were incubated in the presence of ACh, with or without 100 μM proadifen (pro), 30 min before measuring the initial rate of α-bgt binding. Smooth curves are fits to the Hill equation (measurements in the absence of proadifen) or to an equation describing the sum of two binding sites (measurements in the presence of proadifen). Fitted parameters (A): wild-type, K_ov = 7.9 × 10⁻⁷, n = 1.4; wild-type plus proadifen, K_A = 3.3 × 10⁻⁸, K_B = 2.1 × 10⁻⁹; ɛN182Y, K_ov = 1.4 × 10⁻⁷, n = 0.9; ɛN182Y plus proadifen, K_A = 2.3 × 10⁻⁸, K_B = 1.2 × 10⁻⁹. Fitted parameters (B): wild-type, K_ov = 1.5 × 10⁻⁶, n = 1.1; ɛD175N, K_ov = 1.2 × 10⁻⁵, n = 1.1; wild-type plus proadifen, K_A = 2.9 × 10⁻⁸, K_B = 8.9 × 10⁻⁹; ɛD175N plus proadifen, K_A = 2.7 × 10⁻⁸, K_B = 1.7 × 10⁻⁶.