Abstract

Muscle acetylcholine receptor ion channels mediate neurotransmission by depolarizing the postsynaptic membrane at the neuromuscular junction. Inherited disorders of neuromuscular transmission, termed congenital myasthenic syndromes, are commonly caused by mutations in genes encoding the five subunits of the acetylcholine receptor that severely reduce endplate acetylcholine receptor numbers and/or cause kinetic abnormalities of acetylcholine receptor function. We tracked the cause of the myasthenic disorder in a female with onset of first symptoms at birth, who displayed mildly progressive bulbar, respiratory and generalized limb weakness with ptosis and ophthalmoplegia. Direct DNA sequencing revealed heteroallelic mutations in exon 8 of the acetylcholine receptor ε-subunit gene. Two alleles were identified: one with the missense substitution p.εP282R, and the second with a deletion, c.798_800delCTT, which result in the loss of a single amino acid, residue F266, within the M2 transmembrane domain. When these acetylcholine receptor mutations were expressed in HEK 293 cells, the p.εP282R mutation caused severely reduced expression on the cell surface, whereas p.εΔF266 gave robust surface expression. Single-channel analysis for p.εΔF266 acetylcholine receptor channels showed the longest burst duration population was not different from wild-type acetylcholine receptor (4.39 ± 0.6 ms versus 4.68 ± 0.7 ms, n = 5 each) but that the amplitude of channel openings was reduced. Channel amplitudes at different holding potentials showed that single-channel conductance was significantly reduced in p.εΔF266 acetylcholine receptor channels (42.7 ± 1.4 pS, n = 8, compared with 70.9 ± 1.6 pS for wild-type, n = 6). Although a phenylalanine residue at this position within M2 is conserved throughout ligand-gated excitatory cys-loop channel subunits, deletion of equivalent residues in the other subunits of muscle acetylcholine receptor did not have equivalent effects. Modelling the impact of p.εΔF266 revealed only a minor alteration to channel structure. In this study we uncover the novel mechanism of reduced acetylcholine receptor channel conductance as an underlying cause of congenital myasthenic syndrome, with the ‘low conductance’ phenotype that results from the p.εΔF266 deletion mutation revealed by the coinheritance of the low-expressor mutation p.εP282R.

Introduction

Congenital myasthenic syndromes are genetic disorders of neuromuscular transmission (Engel et al., 2010). They are associated with mutations in a series of different proteins that are either directly involved in signal transmission or might be involved in the formation and maintenance of synaptic structure. Prior to the identification of the first congenital myasthenic syndromes-associated mutations it was postulated that mutations of the acetylcholine receptor (AChR) could lead to reduced AChR receptor number at the endplate (Vincent et al., 1981) or altered ion-channel conductance or channel kinetics (Engel et al., 1982, 1990). While mutations that reduce AChR endplate number or alter channel kinetics are common in congenital myasthenic syndromes (Engel et al., 2010), the identification of mutations that significantly alter channel conductance has been elusive. Evidence that changes to ion-channel conductance might readily occur for the AChR came from work that immediately followed the cloning of the mammalian muscle AChR subunit genes. It was demonstrated that the difference between the adult and foetal forms of the AChR was the respective substitution of the adult ε-subunit for the foetal γ-subunit within the AChR pentamer (Mishina et al., 1986). Among the differing properties of the foetal and adult AChR were that foetal receptors had a reduced ion-channel conductance but prolonged channel activations (Mishina et al., 1986). By interchanging residues between the adult and foetal receptors, residues that affected the conductance of the AChR were identified. The studies highlighted that important determinants of conductance were created by residues from the five AChR subunits forming an ‘extracellular ring’ immediately outside the M2 transmembrane domain that lines the channel pore, and a ‘cytoplasmic ring’ and an ‘intermediate ring’ that lie immediately on the cellular side of the M2 region of the pore (Imoto et al., 1988). The results suggested an electrostatic effect in which negatively charged residues at these positions contribute to increased conductance. Subsequent experimentation and modelling has confirmed this initial study but also point to a more complex picture in which there are additional determinants of conductance and ion selectivity in the extracellular lumen of the channel and that conductance is not solely determined through a simple electrostatic mechanism (Konno et al., 1991; Wang and Imoto, 1992; Kienker et al., 1994; Hansen et al., 2008: Sine et al., 2010). Similarly, the amphipathic membrane-associated stretch of the M3–M4 linker of the related 5HT3A receptor ion channel contains three crucial positively charged arginine residues, which influence channel conductance (Kelley et al., 2003). This region of the molecule forms the portals of the inner vestibule of cys-loop cation channels. The analogous region of the AChR ion channel contains neutral or negatively charged residues and contributes to higher conductance (Hales et al., 2006). Of these three determinants of single-channel conductance in the AChR ion channel (M2 rings, extracellular domain and membrane-associated), the rings of charged residues close to and within M2 have the greatest influence, and of the M2 rings the intermediate ring at the entrance to the conduction pore exerts the greatest control over conductance (Cymes et al., 2005).

In congenital myasthenic syndromes the pathogenic mechanism for mutations within the genes that encode the AChR may be via the loss or severe reduction in the number of adult-type receptors at the endplate, or through a change in the kinetics of ion-channel function (Engel et al., 2010). In the slow channel myasthenic syndrome, there are gain-of-function mutations that are dominant and cause prolonged activations of the AChR (Engel et al., 1996). Conversely, in fast channel myasthenic syndromes the activations are abnormally brief (Ohno et al., 1996). Thus, although robust numbers of adult AChR are present at the endplates, these are loss-of-function mutations and the disorder shows recessive inheritance. Some low-expressor mutations may also have ‘slow channel-like’ or ‘fast channel-like’ kinetics (Ohno et al., 1997), but in these cases it is the reduced levels of endplate adult AChR that are the major determinant of phenotype.

Here, we pinpoint a congenital myasthenic syndrome caused by reduced conductance of the AChR ion channel. Surprisingly, the mutation is a deletion within the highly conserved M2 transmembrane domain, which lines the channel pore. Although long considered as a potential pathogenic mechanism this is the first report in which a mutation reducing channel conductance has been found to cause congenital myasthenic syndromes and thus represents a new form of these syndromes.

Materials and methods

Patient DNA analysis

Patient consent was obtained with ethical approval OXREC B: 04.OXB.017 and Oxfordshire REC C 09/H0606/74. DNA was isolated from peripheral blood using the NucleonTM II DNA extraction kit (Nucleon Biosciences). Exons and promoter regions within the AChR subunit genes were screened by bi-direction sequencing of PCR amplicons (Weatherall Institute of Molecular Medicine DNA sequencing facility).

Receptor expression

Naturally occurring and artificial mutations were introduced into complementary DNAs encoding the human muscle AChR subunits using the GeneEditor™ in vitro mutagenesis kit (Promega Corp.) Complementary DNAs were sequenced following mutagenesis to confirm the presence of the mutated residue and absence of additional variants. Mutant subunit complementary DNAs, in combination with required wild-type AChR α-, β-, δ- and ε-subunit complementary DNAs were transfected into HEK 293 cells. Surface AChR expression was determined 2 days post-transfection by overlaying the cells in phosphate-buffered saline containing 10 nM 125I-α-bungarotoxin and 1 mg/ml bovine serum albumin for 30 min. Cells were washed four times with phosphate-buffered saline and removed from the plate in 60 mM Tris–HCl (pH 7.4), 100 mM NaCl, 1 mM EDTA, 0.5 mM phenylmethylsulphonyl fluoride and 1.25% Triton X-100. 125I-α-bungarotoxin binding was determined by gamma counter. Levels of surface AChR harbouring the ε-subunit or ε-subunit mutations were measured by immunoprecipitation of the 125I-α-bungarotoxin-labelled surface AChR with a human muscle AChR ε-subunit-specific polyclonal rabbit antiserum (Newland et al., 1995), followed by determining levels of 125I-α-bungarotoxin by gamma counter.

Electrophysiological analysis

Recordings were made from HEK 293 cells 48 h after transfection with AChR complementary DNAs; a 1:10 ratio of δ-subunit complementary DNA was also used to limit possibility of α2βδ2 channels forming. Complementary DNA for enhanced green fluorescent protein was included as a marker of transfection. Recordings were performed in the cell-attached patch configuration (Hamill et al., 1981) at 20–22°C. The cells were bathed in a solution containing (in mM): 150, NaCl; 2.8, KCl; 2, MgCl2; 1, CaCl2, 10, HEPES/NaOH; 10, glucose; pH 7.4. The pipette solution was the same as bath solution, except glucose was omitted and acetylcholine added. Single-channel currents were amplified with an Axopatch 200B amplifier (Molecular Devices), and sampled to hard disk at 100 kHz, initially filtered at 5 kHz (−3 dB, Bessel filter); resolution was set at 50 µs. Burst duration recordings were made with the pipette potential set at +80 mV. Channel transitions were detected by 50% amplitude threshold crossings (pClamp9). Bursts were defined as groups of openings separated by closed intervals longer than a critical duration (tcrit); tcrit was determined for each patch using Equation (1).  

(1)
formula
where amp1 and τ1 refer to the longest intraburst or cluster closed population and amp2 and τ2 refer to the interburst or -cluster closed population.

Histograms of burst duration were fitted to the sum of exponentials by maximum log likelihood. Significant differences between groups were determined by unpaired Student's t-test, with P < 0.05 defined as a significant difference. For single-channel conductance measurements pipette holding potential was adjusted; at least four potentials were tested per patch. Only openings of at least 1 ms were used to determine single-channel amplitude at each potential. Collected amplitudes were fit by Gaussian function to determine mean amplitude of events. Cluster open probability was determined for acetylcholine concentrations ranging from 3 to 500 µM. Clusters were separated by determining tcrit using Equation (1) to define intra- and intercluster closed times for each patch. Cluster channel openings were defined and open probability within each cluster was determined using QUB software (SUNY).

Results

Features of the patient with congenital myasthenic syndrome

The 49-year-old female patient reported onset of generalized muscle weakness, ophthalmoparesis and ptosis from early infancy. She underwent thymectomy aged 4 years without clear benefit. Weakness has gradually progressed through life, with bouts of respiratory insufficiency reported. Eye movements are severely restricted. She showed clear fatigable ptosis, fatigable limb and bulbar weakness, though her facial muscles are strong. A muscle biopsy was non-diagnostic, but electromyography showed decrement and jitter suggesting a disorder of neuromuscular transmission. Treatment with pyridostigmine was beneficial, but required a high dose of 180 mg 3-hourly; 3,4-diaminopyridine appeared to have no effect. She has a reported history of asthma. At the age of 45 years she suffered a respiratory arrest with hypoxic brain injury that left her severely disabled. There was no family history of a muscle disorder. Antibodies to the AChR or MuSK were not present.

Mutational analysis

Sequencing of the coding exons and flanking sequences of the AChR α-, β-, δ- and ε-subunit genes revealed two heterozygous mutations in exon 8 of the ε-subunit gene: first a point mutation c.845C>G that results in the missense substitution p.εP282R, which is located in the extracellular link between transmembrane domains M2 and M3, and a second three nucleotide deletion c.798_800delCTT, which results in the deletion of phenylalanine at position 266, p.εΔF266, but otherwise maintains the reading frame and the primary amino acid sequence. Analysis of the sequence trace showed that the two mutations are on separate alleles. The p.εΔF266 lies within the M2 transmembrane domain approximately one-third into the pore from the extracellular side. p.εP282R lies in the extracellular link between M2 and M3. Both p.εF266 and p.εP282 are conserved across species and in the homologous position in each of the five muscle AChR subunits. Neither mutation was detected in DNA from 100 normal controls, but p.εP282R has been detected as homozygous and heterozygous, respectively in two additional patients with congenital myasthenic syndrome referred to the National Health Service funded National Diagnostic service for congenital myasthenic syndrome, and homozygous in a French patient (Keiko et al., 2006).

Surface expression

Since 1993 we have correlated the levels of expression of mutant AChR obtained on the surface of HEK 293 cells with in vivo muscle biopsies and patient phenotype and observed in each case that the properties and levels of AChR expressed on the surface of HEK 293 cells both predicts the levels in vivo and informs patient phenotype (n > 100). AChR containing the mutant ε-subunits p.εP282R and p.εΔF266 were expressed from respective complementary DNAs transfected into HEK 293 cells. To confirm that the mutant subunits were included in the surface pentamers, ε-subunit-specific antibody was used to precipitate the surface α-bungarotoxin-bound complexes. Surface expression of AChR containing p.εΔF266 in transfected HEK 293 cells was robust, at >60% of wild-type, whereas surface expression of the εP282 mutant AChR channels was depressed at <15% of wild-type (Fig. 1). Although α2βδ2 AChR can be expressed on the surface of HEK 293 cells and bind α-bungarotoxin, as expected these complexes were not precipitated by the anti-ε-subunit antibody.

Figure 1

Expression of AChR containing mutant ε-subunits on the surface of HEK 293 cells. HEK 293 cells transfected with complementary DNA for wild-type, αβδ AChR alone (n = 7) or with αβδ plus either p.εΔF266 (n = 7) or p.εP282R (n = 3) mutants. 125I-α-bungarotoxin was bound to the surface of intact cells and levels of AChR containing ε-subunits determined following immunoprecipitation with an anti-ε-subunit-specific antibody. Results are normalized against 125I-α-bungarotoxin to wild-type αβδε AChR and represent the mean ± SD (error bars for wild-type are included to show consistency between experiments).

Figure 1

Expression of AChR containing mutant ε-subunits on the surface of HEK 293 cells. HEK 293 cells transfected with complementary DNA for wild-type, αβδ AChR alone (n = 7) or with αβδ plus either p.εΔF266 (n = 7) or p.εP282R (n = 3) mutants. 125I-α-bungarotoxin was bound to the surface of intact cells and levels of AChR containing ε-subunits determined following immunoprecipitation with an anti-ε-subunit-specific antibody. Results are normalized against 125I-α-bungarotoxin to wild-type αβδε AChR and represent the mean ± SD (error bars for wild-type are included to show consistency between experiments).

Burst duration

The functional consequences of these mutations was assessed by analysis of burst duration of channels transiently transfected into HEK 293 cells (Fig. 2). Recordings were made at low acetylcholine concentration (500 nM) when activations occur in well separated, non-desensitizing, bursts. In wild-type (αβδε) AChR channels burst durations occur in three populations (Table 1). The longest of these populations is the most important physiologically, since these represent activations when both binding sites are occupied and are likely to dominate when acetylcholine is released into the neuromuscular synapse at high concentration. The longest burst duration population (τ3) for wild-type channels was 4.68 ± 0.74 ms. For the p.εΔF266 deletion mutation the longest burst duration population (τ3) was unchanged (4.39 ± 0.60 ms), while for the p.εP282R missense mutation burst durations were best fit by only two exponential functions, the longest burst duration mean being only 1.43 ± 0.20 ms. To ascertain that these channel events were not from channels formed without an ε-subunit (i.e. α2βδ2 AChR) recordings were made from cells transfected with only αβδ AChR complementary DNAs. These channel events had mean longest burst duration of 9.32 ± 0.27 ms (n = 3). To determine the efficacy of acetylcholine in activating the mutant channels we measured cluster open probability over a range of desensitizing acetylcholine concentrations (Fig. 3). This showed acetylcholine as effective at activating the mutant channels as it is for wild-type AChR with no change in EC50 (half maximal effective concentration).

Figure 2

Burst duration of the mutant ε-subunit channels. Left: Example traces of AChR activity recorded, 500 nM acetylcholine in patch pipette, from cells transfected with wild-type αβδε AChR (top) or αβδε(ΔF266) AChR (middle) or αβδε(P282R) AChR (bottom). Downward deflections indicate channel opening events; scale bars are identical for each trace. Right: Example burst duration histograms fitted by the sum of two or three exponential functions. For these examples exponential τ values and proportion of burst were as follows: wild-type: τ1: 0.05 ms (39.6%); τ2: 0.99 ms (21.6%); τ3: 4.68 ms (38.8%); 894 bursts. p.εΔF266: τ1: 0.11 ms (30.1%); τ2: 1.00 ms (39.0%); τ3: 4.75 ms (31.0%); 1521 bursts. p.εP282R: τ1: 0.43 ms (59.4%); τ2: 1.29 ms (40.6%); 692 bursts.

Figure 2

Burst duration of the mutant ε-subunit channels. Left: Example traces of AChR activity recorded, 500 nM acetylcholine in patch pipette, from cells transfected with wild-type αβδε AChR (top) or αβδε(ΔF266) AChR (middle) or αβδε(P282R) AChR (bottom). Downward deflections indicate channel opening events; scale bars are identical for each trace. Right: Example burst duration histograms fitted by the sum of two or three exponential functions. For these examples exponential τ values and proportion of burst were as follows: wild-type: τ1: 0.05 ms (39.6%); τ2: 0.99 ms (21.6%); τ3: 4.68 ms (38.8%); 894 bursts. p.εΔF266: τ1: 0.11 ms (30.1%); τ2: 1.00 ms (39.0%); τ3: 4.75 ms (31.0%); 1521 bursts. p.εP282R: τ1: 0.43 ms (59.4%); τ2: 1.29 ms (40.6%); 692 bursts.

Figure 3

Cluster open probability of εΔF266 channels. Cluster open probability (mean ± SD) is plotted versus pipette acetylcholine (ACh) concentration, filled circles are αβδε AChR channels and empty circles are αβδε(ΔF266) AChR channels. Insert is example traces of clusters recorded at 20 µM acetylcholine. Data are from 2 to 6 patches per concentration and are the mean of between 83 and 788 clusters. Data were fit by the logistic function with minimum fixed at zero. EC50 for each channel type was determined as 9.2 ± 0.7 µM for αβδε AChR and 9.0 ± 1.15 µM for αβδε(ΔF266) AChR.

Figure 3

Cluster open probability of εΔF266 channels. Cluster open probability (mean ± SD) is plotted versus pipette acetylcholine (ACh) concentration, filled circles are αβδε AChR channels and empty circles are αβδε(ΔF266) AChR channels. Insert is example traces of clusters recorded at 20 µM acetylcholine. Data are from 2 to 6 patches per concentration and are the mean of between 83 and 788 clusters. Data were fit by the logistic function with minimum fixed at zero. EC50 for each channel type was determined as 9.2 ± 0.7 µM for αβδε AChR and 9.0 ± 1.15 µM for αβδε(ΔF266) AChR.

Table 1

Burst duration analysis of mutant AChR channels

AChR construct n τ1 τ2 τ3 
αβδε 0.11 ± 0.03 27.4 ± 3.2 1.19 ± 0.22 36.0 ± 4.9 4.68 ± 0.74 36.6 ± 3.5 
αβδε(ΔF266) 0.13 ± 0.02 37.6 ± 5.0 0.94 ± 0.11 32.4 ± 4.2 4.39 ± 0.60 30.0 ± 1.6 
αβδε(P282R) 0.32 ± 0.05 47.8 ± 8.7 1.43 ± 0.20 38.2 ± 8.1 – – 
α(ΔF256)δβε – – – – – – – 
αβ(ΔF268)δε – – – – – – – 
αβδ(ΔF270)ε 0.11 ± 0.04 29.0 ± 5.8 0.86 ± 0.16 33.4 ± 4.3 3.80 ± 0.30 43.0 ± 3.2 
αβδγ 0.14 ± 0.02 23.0 ± 1.2 1.68 ± 0.22 32.7 ± 4.3 7.77 ± 0.33 44.3 ± 3.4 
αβδγ(ΔF265) 0.10 ± 0.01 74.4 ± 2.9 0.51 ± 0.02 25.6 ± 2.91 – – 
α2βδ2 0.10 ± 0.02 46.7 ± 2.2 0.99 ± 0.32 19.0 ± 1.0 9.32 ± 0.27 34.3 ± 3.2 
AChR construct n τ1 τ2 τ3 
αβδε 0.11 ± 0.03 27.4 ± 3.2 1.19 ± 0.22 36.0 ± 4.9 4.68 ± 0.74 36.6 ± 3.5 
αβδε(ΔF266) 0.13 ± 0.02 37.6 ± 5.0 0.94 ± 0.11 32.4 ± 4.2 4.39 ± 0.60 30.0 ± 1.6 
αβδε(P282R) 0.32 ± 0.05 47.8 ± 8.7 1.43 ± 0.20 38.2 ± 8.1 – – 
α(ΔF256)δβε – – – – – – – 
αβ(ΔF268)δε – – – – – – – 
αβδ(ΔF270)ε 0.11 ± 0.04 29.0 ± 5.8 0.86 ± 0.16 33.4 ± 4.3 3.80 ± 0.30 43.0 ± 3.2 
αβδγ 0.14 ± 0.02 23.0 ± 1.2 1.68 ± 0.22 32.7 ± 4.3 7.77 ± 0.33 44.3 ± 3.4 
αβδγ(ΔF265) 0.10 ± 0.01 74.4 ± 2.9 0.51 ± 0.02 25.6 ± 2.91 – – 
α2βδ2 0.10 ± 0.02 46.7 ± 2.2 0.99 ± 0.32 19.0 ± 1.0 9.32 ± 0.27 34.3 ± 3.2 

Conductance

At the pipette holding potential of +80 mV the single-channel amplitude of p.εΔF266 AChR was reduced compared with wild-type channels. This was further investigated by holding the patch at various potentials to investigate the single-channel conductance of these channels (Fig. 4). Single-channel openings were detected and fit by a single Gaussian population at various holding potentials; data for each patch were subjected to a linear fit to give the slope conductance. At each holding potential p.εΔF266 channel amplitude was reduced. Compared to wild-type channels p.εΔF266 channels conductance was reduced by ∼40% from 70.9 ± 1.6 pS to 42.7 ± 1.4 pS.

Figure 4

Single-channel conductance is reduced by deletion mutant p.εΔF266. Top left: Example traces from αβδε AChR and αβδε(ΔF266) AChR transfected cells. Pipette potential was held at potential indicated. Scale bar refers to both sets of example traces. Right: Open channel amplitude histograms at pipette potential of 40, 60, 80 and 100 mV. Red traces are αβδε(ΔF266) AChR. Each set of openings were fit by a single Gaussian population. Bottom left: The relationship between pipette potential and single-channel amplitude. Amplitude of channel events >1 ms in duration were averaged for each potential (40 mV, 80 mV, 100 mV and 120 mV) for each patch, error bars are less than size of symbol. For each patch a linear fit of the data was applied, giving the single-channel conductance for each patch. Mean ± SEM conductance indicated are from six and eight patches, respectively.

Figure 4

Single-channel conductance is reduced by deletion mutant p.εΔF266. Top left: Example traces from αβδε AChR and αβδε(ΔF266) AChR transfected cells. Pipette potential was held at potential indicated. Scale bar refers to both sets of example traces. Right: Open channel amplitude histograms at pipette potential of 40, 60, 80 and 100 mV. Red traces are αβδε(ΔF266) AChR. Each set of openings were fit by a single Gaussian population. Bottom left: The relationship between pipette potential and single-channel amplitude. Amplitude of channel events >1 ms in duration were averaged for each potential (40 mV, 80 mV, 100 mV and 120 mV) for each patch, error bars are less than size of symbol. For each patch a linear fit of the data was applied, giving the single-channel conductance for each patch. Mean ± SEM conductance indicated are from six and eight patches, respectively.

Co-expression of αβδεΔF266 and αβδεP282R in HEK 293 cells

Haploinsufficiency does not play a role in congenital myasthenic syndromes involving mutations in the AChR, where carriers who are heterozygous for a low-expressor congenital myasthenic syndromes mutation in combination with the wild-type allele show no defect in neuromuscular transmission and have normal endplates. However, it is possible that expression of αβδεP282R might compete with αβδεΔF266 at the endplates and severely reduce the level of AChR, thus leading to AChR deficiency. To address this possibility we co-expressed αβδεΔF266 and αβδεP282R in HEK 293 cells and measured the level of cell surface 125I-α-bungarotoxin binding to AChR harbouring the ε-subunits. A similar reduction compared with wild-type was seen when αβδεΔF266 and αβδεP282R were expressed together as is seen when αβδεΔF266 is expressed on its own (Fig. 5A). To investigate further the expression of the two mutants, single-channel recordings were made from the HEK 293 cells expressing the αβδεΔF266 and αβδεP282R combination. Channel expression is shown from one of five patches (Fig. 5B) with similar results seen in patches from another four cells. The recordings show that the great majority of channels are of the lower amplitude and open time characteristic of αβδεΔF266 receptors, whereas there is a very small population of higher amplitude but brief opening channels characteristic of αβδεP282R receptors. The results show that when αβδεΔF266 and αβδεP282R are expressed together, surface expression of ε-subunit-containing AChR remains relatively robust and that the overwhelming majority of the AChR harbour the p.εΔF266 mutation. Thus, it is the characteristics of this mutation that are likely to govern the phenotype of the patient.

Figure 5

Co-expression of αβδεP282R and αβδεΔF266 does not inhibit αβδε(ΔF266) expression or function. (A) αβδεP282R AChR were co-expressed in HEK 293 cells in combination with either wild-type or αβδε(ΔF266) and 48 h following transfection the level of cell-surface bound 125I-α-bungarotoxin (BuTx) precipitated by an ε-subunit-specific antibody was determined. Results are normalized against 125I-α-bungarotoxin to wild-type αβδε AChR, or wild-type plus αβδεP282R AChR and represent the mean ± SD of three independent transfections (error bars for wild-type are included to show consistency between experiments). (B) Left: Scatter plot of all channel openings >0.1 ms from HEK 293 cells transfected with αβδεP282R and αβδεΔF266 AChR subunits. Two open populations can be identified; the great majority of openings form a population with longer open times (αβδεΔF266) while the minority population has comparatively brief open times (αβδεP282R). Right: Open channel amplitude histogram; data were fit by two Gaussian populations with mean amplitude of 6.06 and 8.53 pA.

Figure 5

Co-expression of αβδεP282R and αβδεΔF266 does not inhibit αβδε(ΔF266) expression or function. (A) αβδεP282R AChR were co-expressed in HEK 293 cells in combination with either wild-type or αβδε(ΔF266) and 48 h following transfection the level of cell-surface bound 125I-α-bungarotoxin (BuTx) precipitated by an ε-subunit-specific antibody was determined. Results are normalized against 125I-α-bungarotoxin to wild-type αβδε AChR, or wild-type plus αβδεP282R AChR and represent the mean ± SD of three independent transfections (error bars for wild-type are included to show consistency between experiments). (B) Left: Scatter plot of all channel openings >0.1 ms from HEK 293 cells transfected with αβδεP282R and αβδεΔF266 AChR subunits. Two open populations can be identified; the great majority of openings form a population with longer open times (αβδεΔF266) while the minority population has comparatively brief open times (αβδεP282R). Right: Open channel amplitude histogram; data were fit by two Gaussian populations with mean amplitude of 6.06 and 8.53 pA.

Equivalent deletions

The deleted phenylalanine at position 266 in the ε-subunit is conserved at the equivalent position within the transmembrane domain in all subunits of the muscle AChR (Fig. 6C). This residue lies in the top third of the M2 transmembrane domain close to the extracellular ring, which has been shown to influence the single-channel conductance of AChR (Imoto et al., 1988). To ascertain if the equivalent residues within the M2 domain from the other AChR subunits play a role in determining conductance in a similar manner to the ε-subunit, the equivalent phenylalanine in each of the four other subunits was deleted and these mutant constructs expressed in HEK 293 cells. Surface expression experiments revealed that the α-subunit deletion did not form surface channels; however, the three other deletion mutations did give surface expression (αΔF256, 2.6 ± 0.8% of wild-type; βΔF268, 34.2 ± 7.1%; δΔF270, 12.6 ± 1.7%; and γΔF265, 79.4 ± 16.5% of wild-type γ-subunit-containing AChR). The levels of surface expression of the AChR incorporating the mutant γ-subunit were determined using a γ-subunit-specific antibody.

Figure 6

Alignment of AChR subunits through the M2 transmembrane domain. Human AChR subunit sequences are aligned and phenylalanine 266, which is conserved in all subunits, is highlighted in black. Cytoplasmic, intermediate and extracellular rings which influence channel conductance are indicated.

Figure 6

Alignment of AChR subunits through the M2 transmembrane domain. Human AChR subunit sequences are aligned and phenylalanine 266, which is conserved in all subunits, is highlighted in black. Cytoplasmic, intermediate and extracellular rings which influence channel conductance are indicated.

The functional consequence of the β-, δ- and γ-subunit deletions was assessed in a similar manner to the p.εΔF266 deletion mutation. Channel activity from αβ(ΔF268)δε AChR was a mixture of conventional openings and more frequently ‘flickery’ openings, which did not have a sustained full amplitude (Fig. 7A). This type of activity was seen in many different patches, although the proportion of normal to abnormal openings did vary. Due to the mixture of channel activity further analysis could not be performed. Channel activity from αβδ(ΔF270)ε AChR appeared similar to wild-type channels (Fig. 7B), with the mean of the longest burst duration population of 3.80 ± 0.30 ms (n = 5) and a single-channel conductance of 67.9 ± 4.5 pS (n = 7). Activity of αβδγ(ΔF265) AChR channels, despite robust surface expression, were very short-lived and rare (Fig. 7C); when sufficient activity was observed the longest burst duration was only 0.51 ± 0.02 ms (n = 3), compared with 7.77 ± 0.32 ms (n = 3) for wild-type γ-containing channels.

Figure 7

Effect of deleting equivalent residues in the M2 transmembrane domain of other AChR subunits. (A) Example traces of αβ(ΔF268)δε AChR recordings, included are example of ‘flickery’ openings and regular full amplitude openings. (B) Example trace of αβδ(ΔF270)ε AChR recordings (left), an example of a burst duration histogram fit by a sum of three exponentials, τ1: 0.12 ms (22.4%); τ2: 1.15 ms (34.1%); τ3: 4.34 ms (43.5%); 1088 bursts (centre) and relationship between single-channel amplitude and pipette holding potential, an example of the open channel amplitude histogram at pipette potential of 20, 40, 80 and 100 mV is shown with the linear fit of the data showing the single-channel conductance (right, n = 7). (C) Example traces of αβδγ(ΔF265) AChR recordings and burst duration histogram fit by the sum of two exponentials; τ1: 0.10 ms (73.7%); τ2: 0.54 ms (26.4%); 653 bursts. Scale bars apply to all example traces.

Figure 7

Effect of deleting equivalent residues in the M2 transmembrane domain of other AChR subunits. (A) Example traces of αβ(ΔF268)δε AChR recordings, included are example of ‘flickery’ openings and regular full amplitude openings. (B) Example trace of αβδ(ΔF270)ε AChR recordings (left), an example of a burst duration histogram fit by a sum of three exponentials, τ1: 0.12 ms (22.4%); τ2: 1.15 ms (34.1%); τ3: 4.34 ms (43.5%); 1088 bursts (centre) and relationship between single-channel amplitude and pipette holding potential, an example of the open channel amplitude histogram at pipette potential of 20, 40, 80 and 100 mV is shown with the linear fit of the data showing the single-channel conductance (right, n = 7). (C) Example traces of αβδγ(ΔF265) AChR recordings and burst duration histogram fit by the sum of two exponentials; τ1: 0.10 ms (73.7%); τ2: 0.54 ms (26.4%); 653 bursts. Scale bars apply to all example traces.

Structural implications of εΔF266 deletion

Jalview (Clamp et al., 2004) and MAFFT (Katoh et al., 2002) were used to align the sequences for the adult (containing the ε-subunit) human AChR with GLIC (PDB identifier: 3EHZ) and Torpedo AChR (PDB: 2BG9). Sequences for the M2 helices, and in the case of the ε-subunit for the M3 helix, were removed. The homology modelling software Modeller (Šali and Blundell, 1993) was used to make 100 attempts at generating human AChR models; the model with the optimal objective-function value was chosen for subsequent investigation. Models for the ε-subunit deletion mutant ΔF266 and for the foetal variant (containing the γ-subunit) were made using the same procedure. In making the homology models for the human AChR, the M2–loop–M3 region of the ε-subunit required special attention. A reasonable model of this region only resulted by removing the template Torpedo structure here and relying solely on the GLIC structure. The modelling failed to detect significant changes in structure that might underlie the observed alteration in conductance.

Discussion

We traced the clinical phenotype of a congenital myasthenic syndrome to the deletion of a phenylalanine residue at position F266 in the M2 transmembrane domain of the nicotinic AChR ε-subunit. The functional consequences of the p.εΔF266 mutation are unmasked by the low-expressor mutation p.εP282R on the second allele of the ε-subunit. Although residue εF266 is located within the highly conserved channel pore-forming domain, ε-subunits with εF266 deleted are incorporated into the AChR pentamer and show robust cell surface expression in HEK 293 cells. Analysis of single-channel recordings from the mutant AChR show p.εΔF266 does not significantly alter the burst duration kinetics but rather reduces the single-channel conductance. The analysis reveals a new mechanism through which human mutations of the AChR ion channel can impair neuromuscular transmission and defines a new subtype of hereditary myasthenia, a low channel-conductance congenital myasthenic syndrome.

p.εΔF266 causes a loss-of-function and therefore, in a similar fashion to fast channel syndrome mutations, an additional mutation on the second allele is required to unmask the phenotype. Here, the second mutation p.εP282R results in severely reduced surface expression when expressed in HEK293 cells and is comparable to levels found with other low-expressor/null ε-subunit mutations (Ealing et al. 2002; Beeson, unpublished data). We were able to demonstrate this by using an ε-subunit-specific antibody to pull down the 125I-α-bungarotoxin-labelled cell surface AChR and thus differentiate between AChR harbouring p.εP282R subunits and AChR consisting of α2βδ2 subunits. This is important, since α2βδ2 AChR pentamers can give levels of cell surface 125I-α-bungarotoxin-binding that are up to 40% of wild-type α2βδε AChR (Ohno et al., 1997; Ealing et al., 2002). Our results suggest that the primary pathogenic effect of p.εP282R is through reduced expression of the AChR, and that the secondary fast channel kinetics of this mutation, that have been previously reported (Keiko et al., 2006) and are shown here, compound this effect. When αβδεP282R and αβδεΔF266 were co-expressed αβδεΔF266 remains well expressed as demonstrated by cell surface expression. When the two mutant forms were co-expressed the functional channels were characteristic of αβδεΔF266 with a very small number of detectable αβδεP282R channels. Thus it is likely that αβδεΔF266 would predominate at the endplate and αβδεP282R would only play a very minor role in determining signal transduction. The deletion of εF266 reduces the single-channel conductance of the AChR (70.9 ± 1.6 pS to 42.7 ± 1.4 pS), which in common with the modest reduction in expression will reduce the current flow at the endplate and should markedly compromise the safety margin of neuromuscular transmission.

The observation that the open probability dose response curve was not shifted by the deletion mutation is consistent with the observation that the burst duration of the longest burst population was unchanged compared with wild-type. Slow-channel type perturbations that enhance channel gating kinetics would be expected to left-shift the dose response curve and also prolong burst durations. Similarly fast-channel type perturbations that reduce the efficiency of channel gating would be expected to cause a right-shift of the dose response curve and shorten burst durations (Shen et al., 2005). These combined observations indicate that the pathophysiological effect of this deletion mutation is unlikely to be via a shift in the gating kinetics of the AChR ion channel. At the highest concentrations of ACh, open probability was reduced compared to wild-type AChR. This is perhaps due to alterations in channel block by acetylcholine or an increased propensity for fast desensitization. However, these effects are unlikely to affect the physiological function of the ion channel at the neuromuscular synapse. Although a detailed kinetic analysis was not performed, acetylcholine was as effective at activating the mutant αβδεΔF266 channels as wild-type, although there was a reduction in the absolute maximal activation at high concentrations. This may have been due to an increased propensity to desensitization within clusters (Jadey et al., 2011).

The molecular mechanism by which the deletion of F266 in the ε-subunit alters channel conductance is not clear. The phenylalanine at position 266 (position 14 in the M2 domain) is conserved both in all the muscle AChR subunits, and in all cys-loop ligand-gated excitatory receptor ion channels (which includes neuronal nicotinic AChR and 5HT3 receptors) suggesting that it must play an important role in channel function. It is therefore somewhat surprising that deletion of this residue from within the crucial M2 functional domain of the ε-subunit does not have a major effect either on levels of surface expression or channel gating.

Adjacent to the p.ε ΔF266 deletion, at position 13 of M2, is a valine residue (invariant in the cationic cys-loop channel family), which is hypothesized to form part of the hydrophobic gate restricting movement of extracellular cations. The hydrophobic pore is thought to be narrow at this point and increased by only a small amount (0.5–1.0 Å), though was sufficient to allow passage of cations when a large transmembrane voltage was applied (Wang et al., 2008). Disruption to the small movement within the pore by deletion of the adjacent F266 residue may hamper translocation of cations manifested by a reduction in overall channel conductance. Similar small changes in pore diameter to allow rapid ion translocation were demonstrated by Cymes and Grosman (2008).

Additionally, εF266 resides six positions below the extracellular ring of charged residues at the upper membrane interface thought to be crucial in conferring a difference in conductance between foetal and adult AChR (Imoto et al., 1988; Bouzat and Barrantes, 1997). Modelling of the structural changes that occur at the top of M2 and the loop to the top of the M3 region does not suggest a large change in the structure or alter the electrostatic interaction with positive ions as they traverse the pore. However, the precise structural changes that occur when the F266 residue is removed are not known; the modelling we performed assumed that adjacent residues moved from both directions. While electrostatic calculations (not shown) could not explain conductance changes in the deletion mutant, they showed qualitative differences consistent with the difference in conductance between the γ- and ε-containing channels. The deleted phenylalanine residue in the ε-subunit did not point into the pore in the model. Since this residue was not charged, the changes in electrostatics could at most be subtle. It should be noted that the structures (Torpedo and GLIC) on which our modelling was based are thought to be of the channel in the closed state (Unwin, 2005; Beckstein and Sansom, 2006; Gonzalez-Gutierrez and Grosman, 2010). The consequence of the deletion on the open conformation may well be more significant but can't be determined by our modelling.

One possible mechanism for the influence of the εF266 deletion on channel conductance comes from a comparison of the γ- and ε-subunit composition of the extracellular ring. For the γ-subunit there is positive lysine while for ε-subunit there is a neutral glutamine. However, if the deletion of F266 causes the helix to shift downwards the glutamine is replaced by lysine, analogous to the γ-subunit. This shift causes a +1 change in net charge within the ring, similar to interchanging the γ- and ε-subunits and might explain why the εΔF266 channel conductance is converted to ∼45 pS, similar to the conductance of the γ-containing foetal AChR channel.

To test this hypothesis we engineered equivalent phenylalanine deletions in each of the muscle AChR subunits and assessed the effect on channel function. A change in the composition of the extracellular ring, and consequently its net charge, might have predictable consequences for channel activity. For the α-subunit the predicted shift would result in a negative glutamic acid being replaced by neutral leucine at two positions in the ring, with a net +2 change in the ring charge. However, this channel did not express on the cell surface and so we cannot determine what influence this might have had on channel function and/or conductance. In the β-subunit a negatively charged aspartic acid would be replaced by a positive lysine in the extracellular ring, resulting in a +2 change in net charge. This resulted in a complex change in channel activity, many channel activations were flickery and did not maintain a full amplitude opening for the duration of the opening, although there were some full amplitude openings. This mixture of channel activities prevented any further analysis of burst duration or conductance. However, it is possible to determine that a simple change in channel conductance did not result from this deletion mutation in the β-subunit. In the δ-subunit, deletion of F270 results in a conservative change in the extracellular ring, lysine to an arginine, with no change in the net charge within the ring. The activity of channels containing this subunit was relatively unaffected; burst duration and conductance was similar to wild-type. However in the γ-subunit, where deletion of F265 does not result in a change of residue in the extracellular ring, the function of the channel is severely disrupted. Only very brief channel openings are detected. In the case of the β-subunit, where flickery channels were observed, this could be the result of forcing the polar aspartic acid residue deeper into the membrane environment and destabilizing the pore structure and interfering with channel gating. Thus, equivalent mutations to the α-, β- and γ-subunits disrupted channel function to the extent that alterations to conduction could not be ascertained. The data from the δ-subunit where AChR function was maintained is consistent with the hypothesis that the shift in register of the M2 helix so that the positively charged lysine replaces the glutamine at the extracellular ring is the key factor governing the observed change in AChR conductance.

The clinical features of the patient share some characteristics with the fast channel syndrome. Symptoms are present from birth; there is generalized weakness with marked ptosis, ophthalmoparesis and some bulbar difficulties. As is common in the fast channel syndrome, bouts of respiratory insufficiency that can be life-threatening were noted in the patient with low conductance congenital myasthenic syndrome. Anti-cholinesterase medication shows some beneficial effects. In both syndromes reduced current through the adult endplate receptors is likely to restrict activation of the postsynaptic voltage-gated sodium channels. Some modest effects on channel conductance have been reported for pathogenic mutations affecting gating in cys-loop ion-channel receptors, but here we provide the first example of reduced ion-channel conductance as a primary pathogenic molecular mechanism of the muscle AChR. There is the potential that mutations altering channel conductance in related cys-loop ion channels may similarly impair synaptic transmission in the CNS.

Funding

Medical Research Council, UK (G0701521); the Muscular Dystrophy Campaign (RA722). Additional support from the Myasthenia Gravis Association, UK.

Abbreviation

    Abbreviation
  • AChR

    acetylcholine receptor

References

Beckstein
O
Sansom
MS
A hydrophobic gate in an ion channel: the closed state of the nicotinic acetylcholine receptor
Phys Biol
 , 
2006
, vol. 
3
 (pg. 
147
-
59
)
Bouzat
C
Barrantes
FJ
Assigning functions to residues in the acetylcholine receptor channel region (review)
Mol Membr Biol
 , 
1997
, vol. 
14
 (pg. 
167
-
77
)
Clamp
M
Cuff
J
Searle
SM
Barton
GJ
The Jalview Java alignment editor
Bioinformatics
 , 
2004
, vol. 
20
 (pg. 
426
-
7
)
Cymes
GD
Grosman
C
Pore-opening mechanism of the nicotinic acetylcholine receptor evinced by proton transfer
Nat Struct Mol Biol
 , 
2008
, vol. 
15
 (pg. 
389
-
96
)
Cymes
GD
Ni
Y
Grosman
C
Probing ion-channel pores one proton at a time
Nature
 , 
2005
, vol. 
438
 (pg. 
975
-
80
)
Ealing
J
Webster
R
Brownlow
S
Abdelgany
A
Oosterhuis
H
Muntoni
F
, et al.  . 
Mutations in congenital myasthenic syndromes reveal an epsilon subunit C-terminal cysteine, C470, crucial for maturation and surface expression of adult AChR
Hum Mol Genet
 , 
2002
, vol. 
11
 (pg. 
3087
-
96
)
Engel
AG
Lambert
EH
Mulder
DM
Torres
CF
Sahashi
K
Bertorini
TE
Whitaker
A
A newly recognized congenital myasthenic syndrome attributed to a prolonged open time of the acetylcholine-induced ion channel
Ann Neurol
 , 
1982
, vol. 
11
 (pg. 
553
-
69
)
Engel
AG
Ohno
K
Milone
M
Wang
HL
Nakano
S
Bouzat
C
, et al.  . 
New mutations in acetylcholine receptor subunit genes reveal heterogeneity in the slow-channel congenital myasthenic syndrome
Hum Mol Genet
 , 
1996
, vol. 
5
 (pg. 
1217
-
27
)
Engel
AG
Shen
XM
Selcen
D
Sine
SM
What have we learned from the congenital myasthenic syndromes
J Mol Neurosci
 , 
2010
, vol. 
40
 (pg. 
143
-
53
)
Engel
AG
Walls
TJ
Nagel
A
Uchitel
O
Newly recognized congenital myasthenic syndromes: I. Congenital paucity of synaptic vesicles and reduced quantal release. II. High-conductance fast-channel syndrome. III. Abnormal acetylcholine receptor (AChR) interaction with acetylcholine. IV. AChR deficiency and short channel-open time
Prog Brain Res
 , 
1990
, vol. 
84
 (pg. 
125
-
37
)
Gonzalez-Gutierrez
G
Grosman
C
Bridging the gap between structural models of nicotinic receptor superfamily ion channels and their corresponding functional states
J Mol Biol
 , 
2010
, vol. 
403
 (pg. 
693
-
705
)
Hales
TG
Dunlop
JI
Deeb
TZ
Carland
JE
Kelley
SP
Lambert
JJ
, et al.  . 
Common determinants of single channel conductance within the large cytoplasmic loop of 5-hydroxytryptamine type 3 and alpha4beta2 nicotinic acetylcholine receptors
J Biol Chem
 , 
2006
, vol. 
281
 (pg. 
8062
-
71
)
Hamill
OP
Marty
A
Neher
E
Sakmann
B
Sigworth
FJ
Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches
Pflugers Arch
 , 
1981
, vol. 
391
 (pg. 
85
-
100
)
Hansen
S
Wand
H-L
Taylor
P
Sine
S
An ion selectivity filter in the extracellular domain of cys-loop receptors reveals determinants for ion conductance
J Biol Chem
 , 
2008
, vol. 
283
 (pg. 
36066
-
70
)
Imoto
K
Busch
C
Sakmann
B
Mishina
M
Konno
T
Nakai
J
, et al.  . 
Rings of negatively charged amino acids determine the acetylcholine receptor channel conductance
Nature
 , 
1988
, vol. 
335
 (pg. 
645
-
8
)
Jadey
S
Purohit
P
Bruhova
I
Gregg
TM
Auerbach
A
Design and control of acetylcholine receptor conformational change
Proc Natl Acad Sci USA
 , 
2011
, vol. 
108
 (pg. 
4328
-
33
)
Katoh
K
Misawa
K
Kuma
K-I
Miyata
T
MAFFT: a novel method for rapid multiple sequence alignment based on fast fourier transform
Nucleic Acids Res
 , 
2002
, vol. 
30
 (pg. 
3059
-
66
)
Keiko
I
Hun
Y
Takeshi
S
Roland
B
Chantal
L
Liu
P
, et al.  . 
Congenital myasthenic syndrome caused by decreased receptor channel openings
Neuromuscul Disord
 , 
2006
, vol. 
16
  
S151–152 (NMJ-O-2.02 Abstract)
Kelley
SP
Dunlop
JI
Kirkness
EF
Lambert
JJ
Peters
JA
A cytoplasmic region determines single-channel conductance in 5-HT3 receptors
Nature
 , 
2003
, vol. 
424
 (pg. 
321
-
4
)
Kienker
P
Tomaselli
G
Jurman
M
Yellen
G
Conductance mutations of the nicotinic acetylcholine receptor do not act by a simple electrostatic mechanism
Biophys J
 , 
1994
, vol. 
66
 (pg. 
325
-
34
)
Konno
T
Busch
C
Von Kitzing
E
Imoto
K
Wang
F
Nakai
J
, et al.  . 
Rings of anionic amino acids as structural determinants of ion selectivity in the acetylcholine receptor channel
Proc Biol Sci
 , 
1991
, vol. 
244
 (pg. 
69
-
79
)
Mishina
M
Takai
T
Imoto
K
Noda
M
Takahashi
T
Numa
S
, et al.  . 
Molecular distinction between fetal and adult forms of muscle acetylcholine receptor
Nature
 , 
1986
, vol. 
32
 (pg. 
406
-
11
)
Newland
CF
Beeson
D
Vincent
A
Newsom-Davis
J
Functional and non-functional isoforms of the human muscle acetylcholine receptor
J Physiol
 , 
1995
, vol. 
489
 (pg. 
767
-
78
)
Ohno
K
Quiram
PA
Milone
M
Wang
HL
Harper
MC
Pruitt
JN
II
, et al.  . 
Congenital myasthenic syndromes due to heteroallelic nonsense/missense mutations in the acetylcholine receptor epsilon subunit gene: identification and functional characterization of six new mutations
Hum Mol Genet
 , 
1997
, vol. 
6
 (pg. 
753
-
66
)
Ohno
K
Wang
HL
Milone
M
Bren
N
Brengman
JM
Nakano
S
, et al.  . 
Congenital myasthenic syndrome caused by decreased agonist binding affinity due to a mutation in the acetylcholine receptor epsilon subunit
Neuron
 , 
1996
, vol. 
17
 (pg. 
157
-
70
)
Šali
A
Blundell
TL
Comparative protein modelling by satisfaction of spatial restraints
J Mol Biol
 , 
1993
, vol. 
234
 (pg. 
779
-
815
)
Shen
XM
Ohno
K
Sine
SM
Engel
AG
Subunit-specific contribution to agonist binding and channel gating revealed by inherited mutation in muscle acetylcholine receptor M3-M4 linker
Brain
 , 
2005
, vol. 
128
 (pg. 
345
-
55
)
Sine
S
Wang
H-L
Hansen
S
Taylor
P
On the origin of ion selectivity in the cys-loop receptor family
J Mol Neurosci
 , 
2010
, vol. 
40
 (pg. 
70
-
6
)
Unwin
N
Refined structure of the nicotinic acetylcholine receptor at 4A resolution
J Mol Biol
 , 
2005
, vol. 
346
 (pg. 
967
-
89
)
Vincent
A
Cull-Candy
SG
Newsom-Davis
J
Trautmann
A
Molenaar
PC
Polak
RL
Congenital myasthenia: end-plate acetylcholine receptors and electrophysiology in five cases
Muscle Nerve
 , 
1981
, vol. 
4
 (pg. 
306
-
18
)
Wang
HL
Cheng
X
Taylor
P
McCammon
JA
Sine
SM
Control of cation permeation through the nicotinic receptor channel
PLoS Comput Biol
 , 
2008
, vol. 
4
 pg. 
e41
 
Wang
F
Imoto
K
Pore size and negative charge as structural determinants of permeability in the Torpedo nicotinic acetylcholine receptor
Proc R Soc Lond B
 , 
1992
, vol. 
250
 (pg. 
11
-
7
)