**4. Synaptic CMS**

184 Neuromuscular Disorders

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* 

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

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

*al.*, 1999).

control.

deficiency (Section 3.1.2).

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 LRP4/MuSK complex at the endplate.

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

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.*, 2004).

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

Congenital Myasthenic Syndromes – Molecular Bases

**4.2 CMS due to a defect in 2 laminin** 

sulfate 8 to 16 mg per day also shows benefit (Liewluck *et al.*, in press).

nephrotic syndrome that requires renal transplantation (Zenker *et al.*, 2004).

pyridostigmine but were improved by ephedrine.

Maselli and coworkers reported a 20-year-old woman with Pierson syndrome caused by two heteroallelic frameshifting mutations (1478delG and 4804delC) in *LAMB2* who also had a severe CMS (Maselli *et al.*, 2009). The nephrotic syndrome was corrected by a renal transplant at age 15 months. The patient had respiratory distress in infancy, delayed motor milestones, a decremental EMG response, limited ocular ductions, bilateral ptosis, severe proximal limb weakness, scoliosis, and required assisted ventilation at night and sometimes during the day. AChE activity was spared at the NMJ. Electron microscopy of the NMJ showed small axon terminal size and encasement of nerve endings by the Schwann cell, widening of the primary synaptic clefts with invasion of the synaptic space by processes of Schwann cells, moderate simplification of postsynaptic membranes, and decreased number of synaptic vesicles. Both morphological and microelectrode studies were similar to those observed in *Lamb2*-mice (Noakes *et al.*, 1995). Notably, symptoms were worsened by

of Congenital Defects of Proteins at the Neuromuscular Junction 187

Anticholinesterase medications have no effect on neuromuscular transmission and can cause excessive muscarinic side effects. Quinidine (Fukudome *et al.*, 1997; Harper & Engel, 1997) and fluoxetine (Harper *et al.*, 2003), which shorten the open duration of the AChR channel and benefit the slow-channel syndrome, can increase muscle weakness. A respirator dependent infant with severe endplate AChE deficiency was improved by intermittent blockade of AChR by atracurium, an agent that protects AChR from overexposure to ACh (Breningstall *et al.*, 1996). Ephedrine sulfate at a dose of 150 to 200 mg per day in adults is effective for myasthenic symptoms (Bestue-Cardiel *et al.*, 2005; Mihaylova *et al.*, 2008). Although high concentrations of ephedrine are able to block AChR openings (Milone & Engel, 1996), molecular bases of ephedrine effects in clinical practice remain elusive. As an alternative to ephedrine, albuterol

Laminins are cruciform heterotrimeric glycoproteins composed of , , and chains and are assembled from products of five , four , and three genes. The laminin molecules are named according to their chain composition. For example, laminin-321 contains 3, 2, and 1 chains (Aumailley *et al.*, 2005). Three laminins are present at the synaptic basal lamina, laminin-221, laminin-421, and laminin-521. Each contains the 2 subunit. Laminin-421 is restricted to the primary synaptic cleft and promotes the precise alignment of pre- and postsynaptic specializations. Laminin-521 lines the primary and secondary clefts, promotes presynaptic differentiation, and prevents Schwann cells from entering the synaptic cleft. The synaptic laminins provide a stop signal for axons at developing endplates and organize presynaptic differentiation (Sanes, 1997). Mice deficient for *Lamb2* that encodes 2 laminin show reduced terminal branching of presynaptic motor axons, with a decreased number of active zones, no clustering of the synaptic vesicles above the active zones, and extension of Schwann cell processes into the primary synaptic cleft, and decreased spontaneous and evoked quantal release (Noakes *et al.*, 1995; Patton *et al.*, 1998). In addition to its presence at the endplate, 2 laminin is also highly expressed in renal glomeruli and the eye. *LAMB2* mutations in humans cause Pierson syndrome characterized by ocular malformation including small non-reactive pupils, loss of accommodation, and abnormalities of the lens, cornea and retina and by fatal

or in any other disease. There are three classes of ColQ mutations. First, mutations in the proline-rich attachment domain (PRAD) hinder binding of ColQ to AChE. Sedimentation analysis of AChE species of the patient muscle and transfected cells shows complete lack of ColQ-tailed AChE. Second, mutations in the collagen domain, most of which are truncation mutations, hinder formation of triple helix of ColQ. Sedimentation analysis of muscle and transfected cells demonstrate a truncated single-stranded ColQ associated with a homotetramer of AChE. Third, the mutations in the C-terminal domain have no deleterious effect on formation of the asymmetric ColQ-tailed AChE, but they compromise anchoring of ColQ-tailed AChE to the synaptic basal lamina as elegantly shown in vitro overlay binding of mutant and wild-type human recombinant ColQ-tailed AChE to the frog endplate (Kimbell *et al.*, 2004).

Fig. 4. ColQ anchors to the synaptic basal lamina by binding to perlecan and MuSK.

EMG studies show a decremental response as in other CMS. In addition, most patients have a repetitive CMAP response on a single nerve stimulus. The repetitive CMAP decrements faster than the primary CMAP. It can be overlooked unless a well rested muscle is tested by single nerve stimuli. The prolonged dwell time of unhydrolyzed ACh in the synaptic space prolongs the endplate potential; when this exceeds the absolute refractory period of the muscle fiber action potential, it elicits a repetitive CMAP. As mentioned above, a repetitive CMAP also occurs in slow channel syndrome.

Some aspects of the pathophysiology of endplate AChE deficiency resemble those of the SCCMS. As in the SCCMS, neuromuscular transmission is compromised by three distinct mechanisms. First, staircase summation of endplate potentials causes a depolarization block, which inactivates a proportion the voltage-gated skeletal sodium channel, NaV1.4. (Maselli & Soliven, 1991). Second, prolonged exposure of AChR to ACh during physiologic activity desensitizes a fraction of the available AChRs (Milone *et al.*, 1997). Third, repeated openings of AChR cause calcium overloading to the endplate, which culminates in an endplate myopathy (Groshong *et al.*, 2007). Unlike in the SCCMS, the nerve terminals are abnormally small and often encased by Schwann cells. This decreases the quantal content and hence the amplitude of the endplate potential (Engel *et al.*, 1977).

Anticholinesterase medications have no effect on neuromuscular transmission and can cause excessive muscarinic side effects. Quinidine (Fukudome *et al.*, 1997; Harper & Engel, 1997) and fluoxetine (Harper *et al.*, 2003), which shorten the open duration of the AChR channel and benefit the slow-channel syndrome, can increase muscle weakness. A respirator dependent infant with severe endplate AChE deficiency was improved by intermittent blockade of AChR by atracurium, an agent that protects AChR from overexposure to ACh (Breningstall *et al.*, 1996). Ephedrine sulfate at a dose of 150 to 200 mg per day in adults is effective for myasthenic symptoms (Bestue-Cardiel *et al.*, 2005; Mihaylova *et al.*, 2008). Although high concentrations of ephedrine are able to block AChR openings (Milone & Engel, 1996), molecular bases of ephedrine effects in clinical practice remain elusive. As an alternative to ephedrine, albuterol sulfate 8 to 16 mg per day also shows benefit (Liewluck *et al.*, in press).
