**3.1.3 Endplate AChR deficiency due to a defect in agrin**

Neural agrin released from the nerve terminal is a key mediator of synaptogenesis at the NMJ. A reported homozygous G1709R agrin mutation, however, did not cause AChR deficiency but mutations in agrin are potential causes of AChR deficiency by interfering with the activation of MuSK and by impeding synaptic maturation.

The patient harboring the G1709R mutation was a 42-year-old woman with right lid ptosis since birth, no oculoparesis, and mild weakness of facial, hip-girdle and anterior tibial muscles, and refractoriness to pyridostigmine or 3,4-diaminopyridine (Huze *et al.*, 2009). The mutation is in the laminin G-like 2 domain, upstream of the neuron-specific y and z exons that are required for MuSK activation and AChR clustering. AChR and agrin expression at the endplate were normal. Structural studies showed endplates with misshaped synaptic gutters partially filled by nerve endings and formation of new endplate regions. The postsynaptic regions were preserved. Expression studies in myotubes using a mini-agrin construct revealed the mutation did not affect MuSK activation or agrin binding to dystroglycan. Forced expression of the mutant mini-agrin gene in mouse soleus muscle induced changes similar to those at patient endplates. Thus, the observed mutation perturbs the maintenance of the endplate without altering the canonical function of agrin to induce development of the postsynaptic compartment.

Congenital Myasthenic Syndromes – Molecular Bases

**3.1.6 Endplate AChR deficiency due to defects in plectin** 

the junctional folds lacking adequate cytoskeletal support.

**3.2 Slow-channel congenital myasthenic syndrome (SCCMS)** 

present early in life and become severely disabled even in the first decade.

**aminotransferase 1 (GFPT1)** 

causing CMS remain elusive.

of Congenital Defects of Proteins at the Neuromuscular Junction 181

Plectin, encoded by *PLEC*, is a highly conserved and ubiquitously expressed intermediate filament-linking protein concentrated at sites of mechanical stress, such as the postsynaptic membrane of the endplate, the sarcolemma, Z-disks in skeletal muscle, hemidesmosomes in skin, and intercalated disks in cardiac muscle. Pathogenic mutations in *PLEC* result in epidermolysis bullosa simplex, a progressive myopathy (Smith *et al.*, 1996), and, in some patients, myasthenic syndrome (Banwell *et al.*, 1999; Selcen *et al.*, 2011). We reported two cases of CMS associated with plectin deficiency (Banwell *et al.*, 1999; Selcen *et al.*, 2011). The dystrophic changes in muscle are attributed to dislocation of the fiber organelles no longer anchored by the cytoskeletal intermediate filaments and to sarcolemmal defects allowing Ca2+ ingress into the muscle fibers. The myasthenic syndrome is attributed to destruction of

**3.1.7 Endplate AChR deficiency due to defects in glutamine-fructose-6-phosphate** 

Glutamine-fructose-6-phosphate transaminase 1, encoded by *GFPT1*, catalyzes transfer of an amino group from glutamine onto fructose-6-phosphate, yielding glucosamine-6-phosphate and glutamate. GFPT1 is a rate-limiting enzyme that controls the flux of glucose into the hexosamine biosynthesis pathway. GFPT1 thus initiates formation of UDP-Nacetylglucosamine (UDP-GlcNAc), which is a source of multiple glycosylation processes including addition of N-acetylglucosamine to serine or threonine residues (O-linked GlcNAc) (Wells *et al.*, 2001). The disease gene was discovered by linkage analysis and homozygosity mapping of 13 kinships with a limb-girdle CMS often associated with tubular aggregates in skeletal muscle (Senderek *et al.*, 2011). Immunoblots of muscle of affected patients revealed decreased expression of O-linked GlcNAc, but the responsible molecule(s)

The second class of postsynaptic CMS due to mutations in the AChR subunit genes is SCCMS. SCCMS is an autosomal dominant disorder, in which a gain-of-function mutation on a single allele compromises the neuromuscular signal transduction (Ohno *et al.*, 1995). The mutation causes prolonged AChR channel openings and increases the synaptic response to ACh (Fig. 2). There is a single reported case of autosomal recessive SCCMS, in which an L78P mutation minimally prolongs channel opening events but the mutant channel arising from a single allele is not sufficient to cause disease (Croxen *et al.*, 2002). In general, dominantly inherited disorders, including SCCMS, tend to present after adolescence and have a relatively mild course. Some patients with SCCMS, however,

In SCCMS, neuromuscular transmission is compromised by three distinct mechanisms. First, staircase summation of endplate potentials causes depolarization block of the postsynaptic membrane by rendering the voltage-gated skeletal muscle sodium channel go into an inactivated state and thereby inhibit action potential generation (Maselli & Soliven, 1991). Second, some mutant AChRs are prone to become desensitized (Milone *et al.*, 1997), which reduces the number of AChRs that respond to the released ACh quanta. Third,

### **3.1.4 Endplate AChR deficiency due to defects in MuSK**

MuSK and LRP4 form a heteromeric receptor for agrin. Five *MUSK* mutations have been reported in three papers. The first report describes heteroallelic frameshift (220insC) and missense (V790M) mutations in a patient with respiratory distress in early life, mild ptosis, decreased upward gaze, and fatigable weakness of the cervical and proximal more than distal muscles. The symptoms were worsened by pregnancy. Treatment with pyridostigmine and 3,4-diaminopyridine was ineffective (Chevessier *et al.*, 2004). The frameshift mutation prevents MuSK expression and the missense mutation decreases MuSK expression and impairs its interaction with Dok-7. Forced expression of the mutant protein in mouse muscle decreased AChR expression at the endplate and caused aberrant axonal outgrowth (Chevessier *et al.*, 2004). Interestingly, mice homozygous for MuSK V789M (which corresponds to the human MuSK V790M) are normal but mice hemizygous for V789M are severely affected suggesting that MuSK V790M in humans is a haploinsufficient only when accompanied by a null mutation (Chevessier *et al.*, 2008).

A second report describes heteroallelic M605I and A727V mutations in MuSK in a patient with severe myasthenic symptoms since early life that improved after puberty but worsened after menstrual periods. The MEPP and MEPC amplitudes in anconeus muscle were reduced to about 30% of normal and the EPP quantal content was half-normal. Synaptic contacts were small and electron microscopy showed simplified postsynaptic regions with too few secondary synaptic clefts. The patient failed to respond to pyridostigmine, ephedrine or 3,4-diaminopyuridine but responded partially to albuterol (Maselli *et al.*, 2010).

A third report describes a homozygous P31L mutation in the extracellular domain of MuSK in 5 patients in a consanguineous Sudanese kinship. The findings included ptosis from an early age, partial ophthalmoparesis, and weakness of torso and limb girdle muscles. Pyridostigmine therapy gave only slight benefit (Mihaylova *et al.*, 2009).

#### **3.1.5 Endplate AChR deficiency due to defects in Dok-7**

Phosphorylated MuSK recruits a noncatalytic adaptor protein, Dok-7. Recruited Dok-7 further facilitates phosphorylation of MuSK (Okada *et al.*, 2006). Dok-7 is highly expressed at the postsynaptic region of skeletal muscle and in heart. It harbors an N terminal pleckstrin homology domain (PH) important for membrane association, a phosphotyrosine-binding (PTB) domain, and C-terminal sites for phosphorylation. The PH and PTB domains are required for association with and phosphorylation of MuSK. Phosphorylation of two C terminal residues is a requisite for Dok-7 activation by Crk and Crk-L (Hallock *et al.*, 2010).

Numerous mutations have been identified in *DOK7* (Beeson *et al.*, 2006; Muller *et al.*, 2007; Anderson *et al.*, 2008; Selcen *et al.*, 2008; Vogt *et al.*, 2009; Ben Ammar *et al.*, 2010). Nearly all patients carry a common 1124\_1127dupTGCC mutation in exon 7. This and other mutations upstream of the C-terminal phosphorylation sites abrogate the ability of Dok-7 to associate with Crk1/Crk1L and hence its activation (Hallock *et al.*, 2010; Wu *et al.*, 2010). Mutations disrupting or eliminating the PH and PTB domains of Dok-7 prevent dimerization and association of Dok-7 with MuSK (Bergamin *et al.*, 2010).

MuSK and LRP4 form a heteromeric receptor for agrin. Five *MUSK* mutations have been reported in three papers. The first report describes heteroallelic frameshift (220insC) and missense (V790M) mutations in a patient with respiratory distress in early life, mild ptosis, decreased upward gaze, and fatigable weakness of the cervical and proximal more than distal muscles. The symptoms were worsened by pregnancy. Treatment with pyridostigmine and 3,4-diaminopyridine was ineffective (Chevessier *et al.*, 2004). The frameshift mutation prevents MuSK expression and the missense mutation decreases MuSK expression and impairs its interaction with Dok-7. Forced expression of the mutant protein in mouse muscle decreased AChR expression at the endplate and caused aberrant axonal outgrowth (Chevessier *et al.*, 2004). Interestingly, mice homozygous for MuSK V789M (which corresponds to the human MuSK V790M) are normal but mice hemizygous for V789M are severely affected suggesting that MuSK V790M in humans is a haploinsufficient

A second report describes heteroallelic M605I and A727V mutations in MuSK in a patient with severe myasthenic symptoms since early life that improved after puberty but worsened after menstrual periods. The MEPP and MEPC amplitudes in anconeus muscle were reduced to about 30% of normal and the EPP quantal content was half-normal. Synaptic contacts were small and electron microscopy showed simplified postsynaptic regions with too few secondary synaptic clefts. The patient failed to respond to pyridostigmine, ephedrine or 3,4-diaminopyuridine but responded partially to albuterol

A third report describes a homozygous P31L mutation in the extracellular domain of MuSK in 5 patients in a consanguineous Sudanese kinship. The findings included ptosis from an early age, partial ophthalmoparesis, and weakness of torso and limb girdle muscles.

Phosphorylated MuSK recruits a noncatalytic adaptor protein, Dok-7. Recruited Dok-7 further facilitates phosphorylation of MuSK (Okada *et al.*, 2006). Dok-7 is highly expressed at the postsynaptic region of skeletal muscle and in heart. It harbors an N terminal pleckstrin homology domain (PH) important for membrane association, a phosphotyrosine-binding (PTB) domain, and C-terminal sites for phosphorylation. The PH and PTB domains are required for association with and phosphorylation of MuSK. Phosphorylation of two C terminal residues is a requisite for Dok-7 activation by Crk and Crk-L (Hallock *et al.*, 2010). Numerous mutations have been identified in *DOK7* (Beeson *et al.*, 2006; Muller *et al.*, 2007; Anderson *et al.*, 2008; Selcen *et al.*, 2008; Vogt *et al.*, 2009; Ben Ammar *et al.*, 2010). Nearly all patients carry a common 1124\_1127dupTGCC mutation in exon 7. This and other mutations upstream of the C-terminal phosphorylation sites abrogate the ability of Dok-7 to associate with Crk1/Crk1L and hence its activation (Hallock *et al.*, 2010; Wu *et al.*, 2010). Mutations disrupting or eliminating the PH and PTB domains of Dok-7 prevent dimerization and

**3.1.4 Endplate AChR deficiency due to defects in MuSK** 

only when accompanied by a null mutation (Chevessier *et al.*, 2008).

Pyridostigmine therapy gave only slight benefit (Mihaylova *et al.*, 2009).

**3.1.5 Endplate AChR deficiency due to defects in Dok-7** 

association of Dok-7 with MuSK (Bergamin *et al.*, 2010).

(Maselli *et al.*, 2010).

### **3.1.6 Endplate AChR deficiency due to defects in plectin**

Plectin, encoded by *PLEC*, is a highly conserved and ubiquitously expressed intermediate filament-linking protein concentrated at sites of mechanical stress, such as the postsynaptic membrane of the endplate, the sarcolemma, Z-disks in skeletal muscle, hemidesmosomes in skin, and intercalated disks in cardiac muscle. Pathogenic mutations in *PLEC* result in epidermolysis bullosa simplex, a progressive myopathy (Smith *et al.*, 1996), and, in some patients, myasthenic syndrome (Banwell *et al.*, 1999; Selcen *et al.*, 2011). We reported two cases of CMS associated with plectin deficiency (Banwell *et al.*, 1999; Selcen *et al.*, 2011). The dystrophic changes in muscle are attributed to dislocation of the fiber organelles no longer anchored by the cytoskeletal intermediate filaments and to sarcolemmal defects allowing Ca2+ ingress into the muscle fibers. The myasthenic syndrome is attributed to destruction of the junctional folds lacking adequate cytoskeletal support.

#### **3.1.7 Endplate AChR deficiency due to defects in glutamine-fructose-6-phosphate aminotransferase 1 (GFPT1)**

Glutamine-fructose-6-phosphate transaminase 1, encoded by *GFPT1*, catalyzes transfer of an amino group from glutamine onto fructose-6-phosphate, yielding glucosamine-6-phosphate and glutamate. GFPT1 is a rate-limiting enzyme that controls the flux of glucose into the hexosamine biosynthesis pathway. GFPT1 thus initiates formation of UDP-Nacetylglucosamine (UDP-GlcNAc), which is a source of multiple glycosylation processes including addition of N-acetylglucosamine to serine or threonine residues (O-linked GlcNAc) (Wells *et al.*, 2001). The disease gene was discovered by linkage analysis and homozygosity mapping of 13 kinships with a limb-girdle CMS often associated with tubular aggregates in skeletal muscle (Senderek *et al.*, 2011). Immunoblots of muscle of affected patients revealed decreased expression of O-linked GlcNAc, but the responsible molecule(s) causing CMS remain elusive.

#### **3.2 Slow-channel congenital myasthenic syndrome (SCCMS)**

The second class of postsynaptic CMS due to mutations in the AChR subunit genes is SCCMS. SCCMS is an autosomal dominant disorder, in which a gain-of-function mutation on a single allele compromises the neuromuscular signal transduction (Ohno *et al.*, 1995). The mutation causes prolonged AChR channel openings and increases the synaptic response to ACh (Fig. 2). There is a single reported case of autosomal recessive SCCMS, in which an L78P mutation minimally prolongs channel opening events but the mutant channel arising from a single allele is not sufficient to cause disease (Croxen *et al.*, 2002). In general, dominantly inherited disorders, including SCCMS, tend to present after adolescence and have a relatively mild course. Some patients with SCCMS, however, present early in life and become severely disabled even in the first decade.

In SCCMS, neuromuscular transmission is compromised by three distinct mechanisms. First, staircase summation of endplate potentials causes depolarization block of the postsynaptic membrane by rendering the voltage-gated skeletal muscle sodium channel go into an inactivated state and thereby inhibit action potential generation (Maselli & Soliven, 1991). Second, some mutant AChRs are prone to become desensitized (Milone *et al.*, 1997), which reduces the number of AChRs that respond to the released ACh quanta. Third,

Congenital Myasthenic Syndromes – Molecular Bases

**3.3 Fast-channel congenital myasthenic syndrome (FCCMS)** 

fluoxetine (Harper *et al.*, 2003).

of a quantitative defect in AChR.

to stabilize the open conformation of the AChR.

of Congenital Defects of Proteins at the Neuromuscular Junction 183

SCCMS can be treated with conventional doses of long-lived open channel blockers of AChR, such as the antiarrhythmic agent quinidine (Fukudome *et al.*, 1998; Harper & Engel, 1998) and the antidepressant fluoxetine (Harper *et al.*, 2003). Quinidine reduces the prolonged burst duration of SCCMS to the normal level at 5 µM (Fukudome *et al.*, 1998). As the concentration of quinidine in the treatment of cardiac arrhythmia is 6-15 µM, 5 µM is readily attainable in clinical practice and indeed demonstrates significant effects (Harper & Engel, 1998). Similarly, fluoxetine reduces the prolonged burst duration to the normal level at 10 µM, which is clinically attainable without adverse effects at 80 to 120 mg/day of

The third class of postsynaptic CMS due to mutations in AChR subunit genes is FCCMS. FCCMS is kinetically opposite to SCCMS (Fig. 3). In FCCMS, the closed state of AChR is stabilized compared to the open state which results in abnormally brief channel opening events which, in turn, reduces the amplitude of the endplate potential and impair the safety margin of neuromuscular transmission. The resulting pathophysiology is thus similar to endplate AChR deficiency, but abnormally small endplate potential is a qualitative instead

FCCMS is an autosomal recessive disorder. One allele carries a missense mutation that confers a fast closure of AChRs, and the other allele usually harbors a low-expressor or null mutation, or the fast channel mutation occurs at homozygosity. As in heterozygous healthy parents of endplate AChR deficiency, we humans may completely lack 50% of each AChR subunit without any clinical symptoms. In FCCMS, a low-expressor or null mutation on one allele unmasks the deleterious effect of the fast-channel mutation on the second allele. Detailed kinetic analyses of FCCMS mutations have revealed special insights into the molecular architectures of the AChR subunits. Three such examples are presented here.

The 1254ins18 mutation causes a duplication of STRDQE codons at positions 413 to 418 close to the C-terminal end of the long cytoplasmic loop (LCP) linking the third (M3) and fourth (M4) transmembrane domains of the receptor. 1254ins18-AChR expressed on HEK293 cells opens in three different modes. The opening probabilities of normal AChRs are clustered into a single large peak, whereas the 1254ins18-AChR shows three different peaks (Milone *et al.*, 1998). In all the three modes, the AChR is activated slowly and inactivated rapidly, which gives rise to an inefficient synaptic response to ACh. Another FCCMS mutation, A411P in the LCP also destabilizes the channel opening kinetics. The channel opening probabilities of A411P-AChRs are widely distributed and do not form any discernible peaks (Wang *et al.*, 2000). Our analysis first disclosed that the function of LCP is

N436del is a deletion of Asn at the C-terminal end of the LCP. The deletion shortens the LCP and shifts a negatively charged Asp residue at codon 435 against M4. N436del-AChR decreases the duration of channel opening bursts 2.7-fold compared to the wild type due to a 2.3-fold decrease in gating efficiency and a 2.5-fold decrease in agonist affinity of the diliganded closed state. A series of artificial mutations established that the effects of N436del are not due to juxtaposition of a negative charge against M4 but to the shortening of the LCP. Deletion of the C-terminal residue of the LCP of the and subunits also results in fast-

prolonged opening of AChR causes excessive influx of extracellular calcium, which results in focal degeneration of the junctional folds as well as apoptosis of some of the junctional nuclei (Groshong *et al.*, 2007). In normal adult human -AChR, 7% of the synaptic current is carried by Ca2+, which is higher than that carried by the human fetal -AChR or by muscle AChRs of other species (Fucile *et al.*, 2006). This predisposes endplate to Ca2+ overloading when the channel opening events are prolonged. In addition, at least two SCCMS mutations, T264P (Ohno *et al.*, 1995) and V259F (Fidzianska *et al.*, 2005), increase the Ca2+ permeability 1.5- and 2-fold, respectively (Di Castro *et al.*, 2007).

Fig. 2. Slow channel CMS. (A) Schematic diagram of AChR subunits with SCCMS mutations. (B) Single channel currents from wild-type and slow channel (V249F) AChRs expressed on HEK293 cells. (C) Miniature endplate current (MEPC) recorded from endplates of a control and a patient harboring V249F. The patient's MEPC decays biexponentially (arrows) due to expression of both wild-type and mutant AChRs.

Slow channel mutations can be divided into two groups. The first group includes mutations at the extracellular domain like G153S (Sine *et al.*, 1995), as well as at the N-terminal part of the first transmembrane domain like N217K (Wang *et al.*, 1997) and L221F (Hatton *et al.*, 2003). These mutations increase the affinity for ACh binding, probably by retarding the dissociation of ACh from the binding site, which gives rise to repeated channel openings after a single event of ACh binding. The second group includes mutations at the second transmembrane domain (M2) that lines the ion channel pore. These mutations mostly introduce a bulky amino acid into the channel lining face, but T264P (Ohno *et al.*, 1995) introduces a kink into the channel pore, whereas V266A (Shen *et al.*, 2003) and εV265A (Ohno *et al.*, 1998) introduce a smaller amino acid into the pore. Mutations in M2 retard the channel closing rate and variably enhance the channel opening rate . Some mutations in M2 also increase affinity for ACh, which include V249F (Milone *et al.*, 1997), L269F (Engel *et al.*, 1996), and T264P (Ohno *et al.*, 1995).

prolonged opening of AChR causes excessive influx of extracellular calcium, which results in focal degeneration of the junctional folds as well as apoptosis of some of the junctional nuclei (Groshong *et al.*, 2007). In normal adult human -AChR, 7% of the synaptic current is carried by Ca2+, which is higher than that carried by the human fetal -AChR or by muscle AChRs of other species (Fucile *et al.*, 2006). This predisposes endplate to Ca2+ overloading when the channel opening events are prolonged. In addition, at least two SCCMS mutations, T264P (Ohno *et al.*, 1995) and V259F (Fidzianska *et al.*, 2005), increase the Ca2+

permeability 1.5- and 2-fold, respectively (Di Castro *et al.*, 2007).

Fig. 2. Slow channel CMS. (A) Schematic diagram of AChR subunits with SCCMS

*et al.*, 1996), and T264P (Ohno *et al.*, 1995).

mutations. (B) Single channel currents from wild-type and slow channel (V249F) AChRs expressed on HEK293 cells. (C) Miniature endplate current (MEPC) recorded from endplates of a control and a patient harboring V249F. The patient's MEPC decays biexponentially (arrows) due to expression of both wild-type and mutant AChRs.

Slow channel mutations can be divided into two groups. The first group includes mutations at the extracellular domain like G153S (Sine *et al.*, 1995), as well as at the N-terminal part of the first transmembrane domain like N217K (Wang *et al.*, 1997) and L221F (Hatton *et al.*, 2003). These mutations increase the affinity for ACh binding, probably by retarding the dissociation of ACh from the binding site, which gives rise to repeated channel openings after a single event of ACh binding. The second group includes mutations at the second transmembrane domain (M2) that lines the ion channel pore. These mutations mostly introduce a bulky amino acid into the channel lining face, but T264P (Ohno *et al.*, 1995) introduces a kink into the channel pore, whereas V266A (Shen *et al.*, 2003) and εV265A (Ohno *et al.*, 1998) introduce a smaller amino acid into the pore. Mutations in M2 retard the channel closing rate and variably enhance the channel opening rate . Some mutations in M2 also increase affinity for ACh, which include V249F (Milone *et al.*, 1997), L269F (Engel

SCCMS can be treated with conventional doses of long-lived open channel blockers of AChR, such as the antiarrhythmic agent quinidine (Fukudome *et al.*, 1998; Harper & Engel, 1998) and the antidepressant fluoxetine (Harper *et al.*, 2003). Quinidine reduces the prolonged burst duration of SCCMS to the normal level at 5 µM (Fukudome *et al.*, 1998). As the concentration of quinidine in the treatment of cardiac arrhythmia is 6-15 µM, 5 µM is readily attainable in clinical practice and indeed demonstrates significant effects (Harper & Engel, 1998). Similarly, fluoxetine reduces the prolonged burst duration to the normal level at 10 µM, which is clinically attainable without adverse effects at 80 to 120 mg/day of fluoxetine (Harper *et al.*, 2003).

#### **3.3 Fast-channel congenital myasthenic syndrome (FCCMS)**

The third class of postsynaptic CMS due to mutations in AChR subunit genes is FCCMS. FCCMS is kinetically opposite to SCCMS (Fig. 3). In FCCMS, the closed state of AChR is stabilized compared to the open state which results in abnormally brief channel opening events which, in turn, reduces the amplitude of the endplate potential and impair the safety margin of neuromuscular transmission. The resulting pathophysiology is thus similar to endplate AChR deficiency, but abnormally small endplate potential is a qualitative instead of a quantitative defect in AChR.

FCCMS is an autosomal recessive disorder. One allele carries a missense mutation that confers a fast closure of AChRs, and the other allele usually harbors a low-expressor or null mutation, or the fast channel mutation occurs at homozygosity. As in heterozygous healthy parents of endplate AChR deficiency, we humans may completely lack 50% of each AChR subunit without any clinical symptoms. In FCCMS, a low-expressor or null mutation on one allele unmasks the deleterious effect of the fast-channel mutation on the second allele. Detailed kinetic analyses of FCCMS mutations have revealed special insights into the molecular architectures of the AChR subunits. Three such examples are presented here.

The 1254ins18 mutation causes a duplication of STRDQE codons at positions 413 to 418 close to the C-terminal end of the long cytoplasmic loop (LCP) linking the third (M3) and fourth (M4) transmembrane domains of the receptor. 1254ins18-AChR expressed on HEK293 cells opens in three different modes. The opening probabilities of normal AChRs are clustered into a single large peak, whereas the 1254ins18-AChR shows three different peaks (Milone *et al.*, 1998). In all the three modes, the AChR is activated slowly and inactivated rapidly, which gives rise to an inefficient synaptic response to ACh. Another FCCMS mutation, A411P in the LCP also destabilizes the channel opening kinetics. The channel opening probabilities of A411P-AChRs are widely distributed and do not form any discernible peaks (Wang *et al.*, 2000). Our analysis first disclosed that the function of LCP is to stabilize the open conformation of the AChR.

N436del is a deletion of Asn at the C-terminal end of the LCP. The deletion shortens the LCP and shifts a negatively charged Asp residue at codon 435 against M4. N436del-AChR decreases the duration of channel opening bursts 2.7-fold compared to the wild type due to a 2.3-fold decrease in gating efficiency and a 2.5-fold decrease in agonist affinity of the diliganded closed state. A series of artificial mutations established that the effects of N436del are not due to juxtaposition of a negative charge against M4 but to the shortening of the LCP. Deletion of the C-terminal residue of the LCP of the and subunits also results in fast-

Congenital Myasthenic Syndromes – Molecular Bases

loss-of-function mutations cause a CMS.

S246L mutation is relatively benign.

LRP4/MuSK complex at the endplate.

**4.1 Endplate AChE deficiency due to defects in collagen Q** 

**4. Synaptic CMS** 

2004).

of Congenital Defects of Proteins at the Neuromuscular Junction 185

Another class of postsynaptic CMS is due to mutations in skeletal muscle sodium channel, NaV1.4, encoded by *SCN4A* (Tsujino *et al.*, 2003). Dominant gain-of-function mutations in this gene cause hyperkalemic periodic paralysis (Ptacek *et al.*, 1991), paramyotonia congenita (McClatchey *et al.*, 1992; Ptacek *et al.*, 1992), potassium-aggravated myotonia (Lerche *et al.*, 1993), and hypokalemic periodic paralysis type 2 (Bulman *et al.*, 1999). On the other hand,

Failure of normal-amplitude endplate potential depolarizing the resting potential to -40 mV in intercostal muscle of a CMS patient with episodes of apnea and myasthenic symptoms since birth prompted us to search for mutations in *SCN4A*. We identified two heteroallelic missense mutations, S246L and V1442E (Tsujino *et al.*, 2003). Activation kinetics of the mutant NaV1.4 was normal for both S246L and V1442E, but the fast inactivation curves were shifted to hyperpolarization by 7.3 mV for S246L and 33.2 mV for V1442E, indicating that both mutations enhance fast inactivation of the NaV1.4 immediately after it is activated. Moreover, a high proportion of the V1442 channel was in the inactivated state even at a normal resting membrane potential. Recovery from the fast-inactivated state was slowed for both mutations. This was in contrast to gain-of-function mutations in other diseases, which shift the fast inactivation curves to depolarization. Neither S246L nor V1442E affected slow inactivation. Analysis of use-dependent inactivation in HEK293 cells by stimulating at 50 Hz for 3 ms revealed that wild-type and S246L channels decreased the peak current only by 5% and V1442E channel decreased it by 30% during the first few pulses and suggested that the

Defects in three components of the synaptic basal lamina, AChE, 2 laminin and neural agrin, are associated with CMS. The CMS caused by mutations in agrin was discussed above under the postsynaptic CMS (Section 3.1.3) because the site of action of agrin is the

Three tetramers of catalytic AChE subunits are linked by a triple helical collagen Q (ColQ) to constitute an asymmetric ColQ-tailed AChE (Krejci *et al.*, 1997). ColQ carries three domains (i) an N-terminal proline-rich attachment domain (PRAD) that organizes the catalytic AChE subunits into a tetramer, (ii) a collagenic domain that forms a triple helix, and (iii) a Cterminal domain enriched in charged residues and cysteines. ColQ-tailed AChE is organized in the secretory pathway, excreted, and anchored into the synaptic basal lamina using two domains of ColQ (Fig. 4). First, the collagen domain harbors two heparan sulfate proteoglycan (HSPG) binding domains (Deprez *et al.*, 2003) that bind to HSPG, such as perlecan (Peng *et al.*, 1999). Second, the C-terminal domain binds to MuSK (Cartaud *et al.*,

Endplate AChE deficiency is caused by congenital defects of ColQ (Donger *et al.*, 1998; Ohno *et al.*, 1998; Ohno *et al.*, 2000). Congenital defects of ColQ cause endplate AChE deficiency. No mutations have been detected in a gene encoding the catalytic subunit of AChE in CMS

**3.4 CMS due to defects in skeletal muscle sodium channel, NaV1.4** 

channel kinetics, but that in the subunit dictates slow-channel kinetics. Thus, the LCPs of four AChR subunits contribute in an asymmetric manner to optimize the activation of AChRs through allosteric links to the channel and to the agonist binding sites (Shen *et al.*, 2005).

The mutation V285I introduces a bulky amino acid into the M3 transmembrane domain and causes FCCMS (Fig. 3). Kinetic studies demonstrate that the mutation slows the channel opening rate and speeds the channel closing rate , resulting in a 15.1-fold reduction in the channel gating equilibrium constant (= /). On the other hand, the mutation minimally affects affinity for ACh. The probability of channel openings decreased when we introduced Leu, a bulky amino acid, at position V285, but rather increased when we introduced smaller amino acids such as Thr and Ala. We observed similar effects when we introduced similar substitutions into the , , and subunits. Thus, introduction of bulky amino acids narrows the channel pore, while introduction of smaller amino acids widens the channel pore. Our analysis thus revealed that the M3 domain backs up the channel-lining pore lined by the M2 transmembrane domains and has stereochemical effects on channel gating kinetics (Wang *et al.*, 1999).

FCCMS can be effectively treated with anticholinesterases and 3,4-diaminopyridine. The pharmacologic effects of these drugs were discussed in the section of endplate AChR deficiency (Section 3.1.2).

Fig. 3. Fast channel CMS. (A) Schematic diagram of AChR subunits with FCCMS mutations. (B) Single channel currents from wild-type and fast channel (V285I) AChRs expressed on HEK293 cells. (C) Miniature endplate current (MEPC) recorded from endplates of a control and a patient harboring V285I. The patient's MEPC decays faster than that of the normal control.
