MOLECULAR ARCHITECTURE OF THE NEUROMUSCULAR …stan.cropsci.uiuc.edu/people/LSY_teaching/Fall2008/... · 2009-11-06 · discusses neuromuscular transmission and how the physiology

INVITED REVIEW ABSTRACT: The neuromuscular junction (NMJ) is a complex structure thatserves to efficiently communicate the electrical impulse from the motor neuronto the skeletal muscle to signal contraction. Over the last 200 years, techno-logical advances in microscopy allowed visualization of the existence of a gapbetween the motor neuron and skeletal muscle that necessitated the existenceof a messenger, which proved to be acetylcholine. Ultrastructural analysisidentified vesicles in the presynaptic nerve terminal, which provided a beautifulstructural correlate for the quantal nature of neuromuscular transmission, andthe imaging of synaptic folds on the muscle surface demonstrated that special-izations of the underlying protein scaffold were required. Molecular analysis inthe last 20 years has confirmed the preferential expression of synaptic proteins,which is guided by a precise developmental program and maintained by signalsfrom nerve. Although often overlooked, the Schwann cell that caps the NMJ andthe basal lamina is proving to be critical in maintenance of the junction. Geneticand autoimmune disorders are known that compromise neuromuscular trans-mission and provide further insights into the complexities of NMJ function aswell as the subtle differences that exist among NMJ that may underlie thedifferential susceptibility of muscle groups to neuromuscular transmission dis-eases. In this review we summarize the synaptic physiology, architecture, andvariations in synaptic structure among muscle types. The important roles ofspecific signaling pathways involved in NMJ development and acetylcholinereceptor (AChR) clustering are reviewed. Finally, genetic and autoimmunedisorders and their effects on NMJ architecture and neuromuscular transmis-sion are examined.

Muscle Nerve 33: 445–461, 2006

MOLECULAR ARCHITECTURE OF THENEUROMUSCULAR JUNCTION

BENJAMIN W. HUGHES, PhD,1 LINDA L. KUSNER, PhD,1 and HENRY J. KAMINSKI, MD1,2,3

1 Department of Neurology, Case Western University School of Medicine,10900 Euclid Avenue, Cleveland, Ohio 44106, USA2 Department of Neurosciences, Case Western Reserve University School of Medicine,Cleveland, Ohio, USA3 Neurology Service, Louis Stokes Cleveland Veterans Affairs Medical Center,Cleveland, Ohio, USA

Accepted 8 August 2005

Vertebrate skeletal muscle is composed of multinu-cleated cells forming fibers that are innervated bymotor neurons with cell bodies located in the brain-stem or the ventral horn of the spinal cord. Each

motor neuron cell body has a myelinated axon thattravels toward a muscle through a complex of pe-ripheral nerves. The axon enters the muscle andbranches, innervating many individual muscle fibers.As each axon branch approaches its target fiber, itloses its myelin sheath, and further branching orterminal branching occurs until the nerve reachesthe muscle forming the neuromuscular junction(NMJ).121 The NMJ is a specialized synapse designedto transmit electrical impulses from the nerve termi-nal to the skeletal muscle via the chemical transmit-ter, acetylcholine (ACh), and has three major struc-tural elements: the presynaptic region containingthe nerve terminal; the synaptic cleft; and thepostsynaptic surface. All three parts of the synapsecontain organelles and molecules not found in ex-trasynaptic regions, or preferentially expressed when

Available for Category 1 CME credit through the AANEM at www.aanem.org.

Abbreviations: ACh, acetylcholine; AChBP, acetylcholine-binding pro-tein; AChE, acetylcholinesterase; AChR, acetycholine receptor; ChAT, cho-line acetyltransferase; CMS, congenital myasthenic syndromes; CNS, centralnervous system; EOM, extraocular muscle; EPP, endplate potential; MEPP,miniature endplate potential; MG, myasthenia gravis; MuSK, muscle-specifickinase; NMJ, neuromuscular junction; NRG, neuregulinKey words: acetylcholine; acetylcholine receptor; congenital myasthenicsyndromes; Lambert–Eaton myasthenic syndrome; muscle-specific kinase;myasthenia gravis; neuromuscular junction; neuromuscular transmissionCorrespondence to: H. J. Kaminski; e-mail: [email protected]

© 2005 Wiley Periodicals, Inc.Published online 14 October 2005 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/mus.20440

Architecture of the Neuromuscular Junction MUSCLE & NERVE April 2006 445

compared with extrasynaptic regions. This reviewdiscusses neuromuscular transmission and how thephysiology of transmission relates to the architec-tural features of the vertebrate NMJ. The complexi-ties of the developmental program that builds theNMJ are described followed by a review of how dis-orders of neuromuscular transmission compromiseNMJ architecture. We begin with a historical intro-duction to NMJ anatomy.

HISTORICAL REVIEW OF THE NEUROMUSCULARJUNCTION

Through the use of modern technologies, under-standing of NMJ structure has evolved from an ap-parent anatomical fusion of nerve and muscle to theappreciation of distinct boundaries and specializa-tions of nerve terminal, synaptic cleft, and mus-cle.5,126 These regions are also defined by specificprotein localizations, which underpin their anatom-ical features and physiological role. The course inhistory of exploring the NMJ was a debate of philos-ophy as much as it was a search for technical tools,such as the discovery of antagonist and agonist toneuromuscular transmission, the development ofthe microscope, and incorporation of histologicalstaining that allowed the mysteries to be visualized.

The earliest theories of neuromuscular transmis-sion evolved from transportation of gases from thebrain through the nerves that allow for muscle con-traction to the theory of a liquid substance thatcaused the muscle to swell.5,48 In the late 1700s andmid-1800s the idea of an electrical propagationthrough the nerve and muscle first developed itsroots through the work of Galvani and Matteucci.The concept of transferring electrical impulses fromnerve to muscle through a chemical substance wasgreatly advanced by Claude Bernard’s studies of theparalytic agent, curare.36 However, despite appreci-ation that curare acted to prevent muscle contrac-tion if applied to the muscle, but not the nerve,Bernard postulated that curare acted on the nerve,whereas Vulpian suggested the presence of an actualjunction between nerve and muscle.156 John Lan-gley, working in the first decade of the 1900s, inves-tigated the effect of nicotine and curare and was thefirst to suggest a substance could be transmittedfrom the nerve to the muscle that initiated the con-traction.79

In the early 1840s, using the light microscope,Doyere visualized the nerve terminal and muscleinterfaces that had not been possible previously anddemonstrated that the nerve did not directly flowinto the muscle.30,126 The images provided clues that

the separation of the nerve and muscle necessitatedtwo distinct functions but raised a controversy of howthe two areas could communicate.127 The anatomi-cal definition of the NMJ became more detailed withthe development of histological stains that differen-tiated structures to produce a more elaborate imageof the region. Specifically, the use of metals in thestains allowed for a better visualization of the nervebranches and terminals. Gold metal was used first in1878 by Ranvier on the central nervous system tovisualize neuron morphology. Tello first applied sil-ver staining, as developed by Cajal, to visualize theNMJ, and demonstrated structural diversity of junc-tions among various species as well as improving thefine detail of the NMJ.148 Couteaux further definedjunctional architecture utilizing cholinesterase stain-ing methods.22,126

Modern methods of electron and confocal mi-croscopy with specific probes continue to refine thestructure of NMJ and identify subtle differencesamong junctions within the same muscle, as dis-cussed in what follows. Electron microscopy in the1950s defined the ultrastructure of the NMJ126 andremains a valuable method to define disease pathol-ogy as well as to assess structural and functionalcorrelations. Confocal laser scanning microscopy es-sentially eliminates blurring of an image, which al-lows sectioning of an optical image and subsequentthree-dimensional reconstruction, which cannot bedone with conventional microscopy. Fluorescentprobes coupled with confocal microscopy have al-lowed visualization of the NMJ in living tissue andcells.23,85,90 Both electron and confocal microscopymay be coupled with antibody probes to identify thelocation of specific proteins.

NEUROMUSCULAR TRANSMISSION

The architectural and molecular specializations ofthe NMJ are all focused to achieve reliable commu-nication between nerve and muscle. In order toappreciate these specializations, this section providesan overview of neuromuscular transmission and be-gins with the useful organizing concept of the safetyfactor. Several properties of the nerve terminal, basallamina, and postsynaptic muscle surface influencethe safety factor.

The safety factor is defined as the ratio of theendplate potential (EPP) amplitude to the differ-ence between the membrane potential and thethreshold potential for initiating an action poten-tial.7,163 The net postsynaptic depolarization pro-duced by the release of ACh from synaptic vesiclestriggered by a nerve action potential is the EPP. The

446 Architecture of the Neuromuscular Junction MUSCLE & NERVE April 2006

amplitude of the EPP is a function of the miniatureendplate potential (MEPP) amplitude and the quan-tal content, which corresponds to the number oftransmitter vesicles released by a nerve terminal ac-tion potential. At a mammalian NMJ, this is between50 and 300. The MEPP amplitude represents thedepolarization of the postsynaptic membrane pro-duced by the contents of a single vesicle. It should beappreciated that MEPP amplitudes vary in size, andACh packing in synaptic vesicles is not uniform.70,154

Quantal release, AChE, AChR conduction proper-ties and AChR density, density of postsynaptic so-dium channel activity, and the architecture of syn-aptic folds all contribute to the safety factor.

Normally, the nerve terminal releases enoughACh to induce an EPP that is greater than the thresh-old required to initiate an action potential; there-fore, the safety factor is quite large.163 In the verte-brate NMJ, a typical nerve terminal action potentialdoes not fully activate nerve terminal calcium chan-nels, as the duration of the action potential is �1 ms,whereas these calcium channels are activated with atime constant of �1.3 ms.141 Certain drugs may en-hance ACh release from the presynaptic nerve ter-minals. For example, 3,4-diaminopyridine increasesthe nerve terminal action potential duration byblockade of the delayed rectifier potassium channelof the nerve, and quanidine inhibits calcium uptakeand therefore increases the probability of vesicleinfusion. Repetitive stimulation reduces transmitterrelease, which under normal conditions will reduceEPPs, but usually not enough to prevent action po-tential generation, except under extreme condi-tions. In myasthenia gravis (MG), the reduction ofEPPs produced by repetitive stimulation may beenough to produce transmission failure.

The abundance of ACh molecules released fromthe nerve is one factor that promotes consistentcommunication between nerve and muscle. Diffu-sion of ACh across the synaptic cleft after it is re-leased is rapid due to the short distance to be tra-versed and the relatively high diffusion constant forACh.78 The action of acetylcholinesterase (AChE),which is anchored in the basal lamina, along withdiffusion of ACh away from the postsynaptic surfaceterminates the action of ACh, and inhibition ofAChE activity will enhance AChR activation and slowthe decay of the ACh-induced endplate current.69,89

The sarcolemma of the postsynaptic surface con-tains a high density of AChR and voltage-gated so-dium channels. Activation of AChR permits passageof cations through the AChR pore, which leads togeneration of a muscle-cell action potential that pro-duces muscle contraction. Due to its size, muscle-cell

contraction requires the activation of an exception-ally large number of AChRs. Sodium channel densityat the postsynaptic surface and secondary synapticfold structure facilitate the attainment of action po-tential threshold. The narrow secondary fold in-traspace creates a high-resistance path to currentflow. The current flow created by AChR channelopening has its greatest depolarizing effect on themembrane area at the depths of the folds wherevoltage-gated sodium channels are concentrated athigh density. The opening of sodium channels re-sulting from this depolarization would be expectedto increase the effect of the transmitter releasedfrom the nerve, reduce the threshold for actionpotential generation, and thereby enhance the safetyfactor.86,120,162

ANATOMY OF THE NEUROMUSCULAR JUNCTION

The NMJ is composed of three main components:(1) the presynaptic nerve terminal that is capped bya terminal Schwann cell; (2) the synaptic basal lam-ina that occupies the synaptic cleft; and (3) thespecialized postsynaptic membrane of the muscle(Fig. 1). The following sections discuss how thesestructures and their spatial arrangement contributeto NMJ structure and optimal depolarization of thepostsynaptic membrane.

Presynaptic Region. Nerve Terminal Molecular Struc-ture and Function. The hallmark ultrastructural fea-ture of the nerve terminal is the synaptic vesicles,and all proteins of the nerve terminal function, di-rectly or indirectly, to support synaptic vesicle func-tion. Synaptic vesicles are located precisely acrossfrom the AChR-rich synaptic folds and are alignednear release sites called active zones, where calciumchannels are arranged in parallel double rows alongwith a large macromolecular complex designed toaccomplish release of vesicle contents.132,143

When an action potential reaches the nerve ter-minal, calcium channels of the P/Q type (N-typechannels may also localize to the presynaptic mem-brane) are activated, calcium enters the presynapticterminal, and the local calcium concentration risessignificantly, triggering the fusion of the synapticvesicle membrane with the plasma membrane of thenerve terminal. The local internal calcium signal forvesicle fusion3 arises from calcium microdomains136

formed by individual calcium channels, or from theoverlap of calcium microdomains created by severalneighboring calcium channels.8 Physiological stud-ies have demonstrated the existence of functionalpools of vesicles that differ in their probability of


fusion with the plasma membrane upon depolariza-tion, termed the readily releasable and reservepools.113 A study using selective labeling of synapticvesicles has shown that readily releasable vesicles arenot clustered close to the presynaptic membrane,but instead are dispersed almost randomly through-out the vesicle cluster.114 Thus, vesicles are not re-cruited according to proximity to release sites butare mobilized by means that are as yet poorly under-stood.

The mechanism of synaptic vesicle fusion in-volves conformational changes in multiple proteinson the vesicle and nerve terminal plasma mem-branes. Although in the last decade some of themolecular mechanisms of this process have beencharacterized, the events leading to synaptic vesiclerelease continue to be defined.7,106,143 The presentconceptualization is that vesicles initially undergo aprocess called docking, where they come into closeproximity with the nerve terminal membrane, andthen undergo priming, which allows them to be-come responsive to the calcium signal.

This review is not designed to describe the com-plex process of synaptic vesicle release, but the majorproteins and interactions are described (readers arereferred to the review by Sudhoff143). During dock-ing, a synaptic core complex forms that is believed tobe composed of syntaxin-1 and synaptic vesicle–asso-

ciated protein 25 (SNAP 25) on the presynapticterminal membrane and synaptobrevin on the syn-aptic vesicle membrane. The association of thesethree proteins, which are SNARE proteins character-ized by the 70-residue SNARE motif, brings theplasma membrane and the vesicle close together,creating an unstable intermediate that has the capa-bility of forming a fusion pore. Munc18-1 controlssynaptic vesicle fusion by allowing the core complexto form when it dissociates from syntaxin while syn-aptophysin dissociates from synaptobrevin. Theidentification of the calcium signal for membranefusion appears to be mediated by synaptotagmins,which are located on the synaptic vesicle membrane.Although the calcium binding affinity of synaptotag-mins is low, it increases significantly when they bindto phospholipid membranes. A reasonable model isthat synaptotagmin binds the SNARE protein com-plex and, as local calcium concentration rises, itmoves to the plasma membrane, which leads to in-sertion in the membrane. The binding of synapto-tagmin produces mechanical stress that destabilizesthe already unstable core complex and leads toopening of a fusion pore and release of synapticvesicle contents.

The recycling of synaptic vesicle membrane ap-pears to occur by three processes.44,45 One involvesthe synaptic vesicle membrane fusing completely

FIGURE 1. Organization of the NMJ. Neuromuscular synapses are composed of primary and secondary specializations in three cells.Primary specializations include a terminal Schwann cell that caps rather than wraps the motor nerve; terminal branches of the motor axon,which accumulate mitochondria (mc) and synaptic vesicles (sv); and an AChR-rich postsynaptic endplate. Secondary specializationsappear at mature NMJs, which enhance neurotransmission and signal transduction. In the presynaptic terminal, active zones (az) formalong the junctional surface, and intraterminal distribution of organelles becomes asymmetric with respect to the synaptic cleft. Theformation of secondary synaptic clefts creates folds in the postsynaptic membrane, and postsynaptic membrane–associated proteins areasymmetrically distributed, with AChR (shown as red portions of the muscle membrane) concentrated at the crests of secondary folds,and voltage-gated sodium channels (green squares) concentrated in the troughs of the folds. Notably, these specializations are preciselycolocalized across a biochemically specialized portion of the muscle fiber basal lamina (BL). Synaptic basal lamina contains components,which organize synaptic specializations in all three cells (detailed in the text). Although the overall morphology of the synapse variesamong muscle types and species, most aspects of synaptic differentiation are present at all vertebrate NMJs.


with the plasma membrane, followed by recycling ofmembrane components via clathrin-dependentmechanisms. The clathrin-coated vesicles then shedtheir coats and translocate to the interior of thenerve terminal. The vesicle membranes fuse withendosomes and new vesicles bud from the endo-some. These vesicles package ACh by active transportand translocate back to the active zone by eitherdiffusion or a cytoskeletal transport process. It hasbeen appreciated that recycling may occur by morerapid methods. The “kiss-and-run” mechanism in-volves endocytosis of fused vesicle membrane, andvesicles are recycled independent of endosomes.The “kiss-and-stay” process involves the transientopening and closing of a fusion pore and recyclingin close proximity to the active zone. These fastrecycling mechanisms have been investigated at cen-tral nervous system (CNS) nerve terminals that arehighly active and may not be as prominent at themajority of neuromuscular junctions. As describedin what follows, ocular motor neurons fire at highrates and the “kiss-and-run” mechanism may be par-ticularly important for these junctions to function.

Abundant mitochondria are present in the nerveterminals, serving as a prominent ultrastructuralmarker. In addition to energy production requiredto power synaptic release, neurotransmitter synthe-sis,106 and transporters of ions and ACh to loadsynaptic vesicles,35 they also participate in bufferingof intracellular calcium. During repetitive action po-tential discharges, cytosolic calcium in nerve termi-nals increases rapidly, then more slowly, but whenuptake of calcium from mitochondria is blocked,cytosolic calcium increases rapidly through the timeof the stimulus.24,25,145 Posttetanic potentiation, theenhancement of synaptic transmission after high-frequency stimulation, is also mediated by the slowrelease over minutes.146

Terminal Schwann Cells. At the NMJ, three tofive Schwann cells form a cap in close apposition tothe nerve terminal, with processes that extend intothe synaptic cleft that may come within a few mi-crons of the active zones.4 Schwann cells have cometo be appreciated as critical in several aspects of NMJfunction and formation, including modulation ofsynaptic transmission, nerve terminal growth andmaintenance, axonal sprouting, and nerve regener-ation.73,111

Schwann cells may influence neuromusculartransmission, but the influence appears to be sub-tle.73 Removal of the terminal Schwann cells by com-plement-induced lysis does not alter MEPP ampli-tude or frequency or EPP amplitude; however, withneuronal stimulation, Schwann cell intracellular cal-

cium concentration increases, indicating that thecell has the ability to sense nerve activity.112 Theinfluence of neuronal activity on the Schwann cell isnot related to a change in extracellular calcium fromrelease of calcium from the stimulated nerve termi-nal,112 but rather to the activation of transmitterreceptors on the Schwann cells. Schwann cells re-spond to muscarinic and purinergic agonists.115,116

Extracellular potassium ion concentration that in-creases in the circumscribed area of the NMJ couldbe similarly buffered, but whether terminal Schwanncells do so has not been established. Recently, anunsuspected mechanism for glial influence on syn-aptic transmission was identified.139 Utilizing cocul-ture experiments with Lymnaea cholinergic neuronsand glial cells, a train of induced action potentials inthe cholinergic presynaptic neuron produced facili-tatory excitatory postsynapic potentials, if glial cellswere not included. In the presence of glial cells,however, the presynaptically induced action poten-tials failed to elicit facilitatory excitatory potentialsand action potentials in the postsynaptic cell. A glia-derived protein, termed the ACh-binding protein(AChBP), was identified and, under appropriateconditions, was found to cause suppression of cho-linergic synaptic transmission.139 AChBP is a 210-residue protein that has some sequence similaritywith subunits of the Cys-loop family of ligand-gatedion channels.9 The role of AChBP under conditionsof active presynaptic transmitter release might be toeither diminish or terminate the continuing AChresponse, or raise basal AChBP concentration to thepoint that subsequent responses to ACh are de-creased. This could occur by ACh activation of bothpostsynaptic AChRs and the AChRs located on syn-aptic glial cells, which would augment AChBP re-lease, thereby increasing its synaptic cleft concentra-tion and leading to a reduction of ACh available tobind to postsynaptic AChRs.

Schwann cells synthesize and secrete trophic fac-tors, such as neuregulin and nerve growth factor,which promote motor neuron survival, motor neu-ron growth, and Schwann cell process exten-sion.11,51,116,129 For example, studies in whichSchwann cell progenitor cells were destroyed by ge-netic manipulation of neuregulin signaling prior totheir migration into the periphery11,80,160 showedthat, in embryos lacking Schwann cells, axons growtoward muscles and form neuromuscular contacts;however, such contacts quickly disappear, the motorneurons die, and the animals are nonviable at birthor shortly after. Not surprisingly, Schwann cells re-spond to trophic factors of the nerve. Neuregulin


will rescue terminal Schwann cells from degenera-tion in denervated mice.150

Terminal Schwann cells have a function at thetime of nerve-terminal injury; that is, after damage tothe nerve terminal, Schwann cells may becomephagocytic and remove nerve-terminal debris.73 Theremoval process may be important in preparationfor nerve regeneration. During the process of debrisremoval, Schwann cells invade the space between themuscle fiber and the degenerating nerve terminal61

and, as the nerve grows to reinnervate the musclefiber, Schwann cells form paths for axonal growth.61

Synaptic Cleft. A space of �50 nm, termed the syn-aptic cleft, separates nerve and muscle-fiber plasmamembranes and is comprised of basal lamina, whichspans the cleft and may bind receptors on adjacentcell membrane surfaces, providing a means of celladhesion and signaling among NMJ components(Fig. 1). The basal lamina constituents include col-lagen IV, laminin, fibronectin, entactin, and perle-can (Fig. 2),101 and although comparable amountsare found in synaptic and extrasynaptic regions,164

isoform differences exist between the regions. For

example, laminin in the extrasynaptic basal laminacontains primarily the �1 chain, whereas the synap-tic basal lamina contains exclusively the �2 chain,originally called s-laminin, for its synapse-specific dis-tribution.164 Other proteins enriched in the maturesynaptic basal lamina include: agrin; AChE; the lami-nin �4 and �5 chains; the collagen �3 (IV), �4 (IV),and �5 (IV) chains; an isoform of entactin; andneuregulin.128,164 Whereas some synaptic basal lam-ina components are present throughout the cleft,others are localized within the cleft. The restricteddistribution of each component likely is a functionof the adjacent cells, which are responsible for pro-duction of the adjacent basal lamina, and these syn-apse-specific components are likely determinants ofsynapse formation and regulation.

Acetylcholinesterase. AChE, which is concen-trated in the basal lamina, is an asymmetric enzymecomposed of homotetramers of globular catalyticsubunits attached to a collagen tail.75,76 AChE in thesynaptic space is anchored to the basal lamina by twocationic heparan sulfate proteoglycan–binding do-mains within the collagen domain.110 The collagen

FIGURE 2. Schematic of the protein components at the NMJ. The figure illustrates the major structural and signaling proteins located atthe mammalian neuromuscular junction. Details are discussed throughout the article. (Figure used with permission from Dr. AlanPestronk, www.neuro.wustl.edu/neuromuscular/.)


tail subunit is formed by the triple helical associationof three collagen-like strands, encoded by the COLQgene, which are the sites mutated in patients withcongenital myasthenia from AChE deficiency (dis-cussed later). The catalytic subunit, encoded byAChE, has two carboxyl terminal splice variants,AChE7 and AChEH, expressed in muscle and eryth-rocytes, respectively, which explains how patientsmay have cholinesterase deficiencies specific to mus-cle or blood.103 Drugs that inhibit AChE, such aspyridostigmine and edrophonium, prolong the du-ration of action of ACh on the postsynaptic mem-brane and are useful therapies for neuromusculartransmission disorders.

Postsynaptic Surface. The structures located on thepostsynaptic surface are designed to optimize trans-mission of a chemical signal, ACh, to produce anEPP. As will be considered, differences exist amongmuscle fibers and species as to the structure of thesecondary synaptic folds.

Secondary Synaptic Folds. The most striking ul-trastructural features of the mammalian postsynapticmuscle membrane are the deep infoldings of thesarcolemma, termed secondary synaptic folds, at thecrests of which are anchored the AChRs (Fig.1).162,163 The cytoskeletal matrix underlying the syn-aptic folds is largely composed of proteins identicalto those found at the extrasynaptic sarcolemma witha few exceptions. The AChRs are ultimately con-nected to the sarcolemma by �- and �-dystroglycans,two members of a complex of dystrophin-associatedproteins, which are concentrated at the NMJ.88,158,159

The dystroglycans form the core of a larger com-plex of proteins, the dystroglycan complex, whichfunctions to maintain muscle structure and servessignaling functions.56 �-Dystroglycan is located extra-cellularly and binds to �-dystroglycan, which spansthe sarcolemma. Therefore, dystroglycans effectivelyserve to link the basal lamina to the intracellularcytoskeleton. The protein, rapsyn, in turn, serves toanchor �-dystroglycan to the NMJ. �-Dystroglycanbinds to the synapse-specific protein, utrophin, ahomolog of dystrophin, located intracellularly at theNMJ; in contrast, at extrasynaptic regions, dystro-phin binds �-dystroglycan.17 Utrophin is localizedwith AChR at the crests of postsynaptic junctionalfolds, whereas dystrophin is found with sodium chan-nels in the troughs of junctional folds. Utrophin isknown to promote the growth of AChR clusters incultured myotubes, and utrophin-deficient micehave NMJs with only a few synaptic folds, indicatingthat utrophin functions in NMJ maturation. Thesodium channels are present at high density in the

secondary folds, via interaction with ankyrin, thesarcoglycan complex, the dystroglycan complex, dys-trobrevins, and dystrophin. Utrophin and dystrobre-vin also connect to the modular adapter proteins, �1syntrophin and �2 syntrophin, which in turn associ-ate with nitric oxide synthetase.16,77

The cytoplasmic extensions of AChRs are associ-ated with rapsyn, a protein that can aggregate tomediate the formation of AChR microclusters.13,39

Rapsyn and AChR colocalize precisely at the NMJ ina 1:1 stoichiometry as soon as clusters form at theadult NMJ, and rapsyn appears to bind directly to theAChR.97,133 Moreover, AChRs are diffusely distrib-uted when expressed in heterologous cells, but formhigh-density clusters when coexpressed withrapsyn.104 Conversely, no AChR clusters form in mus-cles of rapsyn-knockout mice or in myotubes cul-tured from the mutants, even following treatmentwith agrin.46 Rapsyn bears separable domains re-sponsible for association with the membrane, mul-timerization, and interaction with AChR.109 There-fore, rapsyn is necessary for AChR clustering.

Acetylcholine Receptor. From a functional stand-point, AChR is the most important protein in theNMJ and has been the subject of intense study, serv-ing as a model for other ligand-gated ion channelneurotransmitter receptors68 in studies to furtherunderstand their function as well as being investi-gated as the primary antigenic target of the autoim-mune disorder myasthenia gravis.81,82 The receptorspans the membrane and, at the mature mammalianNMJ, is comprised of two � and single �, �, and �subunits; during development and, in certain othersituations discussed in what follows, the � subunitsubstitutes for the � subunit, forming the fetal AChRisoform. High amino acid sequence homology existsamong AChR subunits, with each containing four�-helices, designated M1 to M4, which span theplasma membrane. The extracellular portions of thesubunits consisting of the N- and C-terminal regionsand the region between M2 and M3 form a largeextracellular vestibule that surrounds the channelorifice. The regions between M1 and M2 and be-tween M3 and M4 form a smaller vestibule aroundthe intracellular orifice of the ion channel.81,82,153

Phosphorylation and other posttranslational modifi-cations can alter properties of the AChR and, inparticular, subunit phosphorylation appears to reg-ulate agonist-induced desensitization.

Prior to innervation during muscle development,the fetal AChR is expressed along the entire fibersurface and contributes to the muscle being able tocontract spontaneously, which is critical for normalmaturation of the muscle. Adult and fetal AChR are


distinguished by their electrophysiological charac-teristics. Adult channels have shorter mean opentimes and a single channel conductance that is�50% larger than that found with fetal channels.With innervation, fetal AChR is downregulated andthe adult receptor is expressed at the synapse. Ex-traocular muscle and other bulbar muscles areknown to express fetal AChR protein at the NMJ inadulthood,63 and reexpression of the fetal AChR incertain patients with congenital myasthenia preventslethality.33 Denervated muscle reexpresses the fetalAChR throughout the muscle fiber and this expres-sion contributes to the development of spontaneousaction potentials, in particular, fibrillation poten-tials.

NEUROMUSCULAR JUNCTION FORMATION

NMJ development has focused largely on followingthe formation of concentrations of AChRs acrossfrom the nerve terminal. The processes involved inthis clustering are described in this section.

When the muscle fiber is innervated, the AChRsand other signaling and structural molecules beginto cluster under the overlying nerve terminal alongwith other NMJ-specific proteins. In myoblasts,AChR subunit genes are initially expressed at lowlevels, and then increase expression during fusion aspart of the myogenic program that also forms thecontractile apparatus. AChR subunits are translated,assembled, and inserted in the plasma membrane,where they reach a uniform density of �1000/�m2.6,91 By contrast, the AChR density reaches�10,000/�m2 at the NMJ and falls to �10/�m2

within a few microns from the synapse in maturemuscle. Three distinct processes contribute to thisredistribution: clustering of diffusely distributedAChRs in the postsynaptic membrane; transcrip-tional activation of AChR subunit genes in subsyn-aptic nuclei; and transcriptional repression of AChRsubunit genes in nonsynaptic myonuclei.

Appreciation that neurites organize AChR clus-ters at points of nerve–muscle contact1,38 led to in-vestigation of clustering agents. Fibroblast growthfactor, laminin, and midkine were all found to clus-ter AChRs in myotube cultures.102,103,144,165 However,a heparan sulfate proteoglycan, termed agrin, iso-lated from Torpedo electric organ and subsequentlycloned from mammals and birds, is now appreciatedas the most likely signal for aggregation.117,124 Agrinis synthesized by nerve, transported down motoraxons, released from motor nerve terminals, andincorporated into the basal lamina of the synapticcleft. Recombinant agrin can induce complex

postsynaptic structures, including AChE and AChRson myotubes in the absence of nerve in direct appo-sition to agrin deposits. Further, AChRs in such ec-topic postsynaptic structures possess functionalproperties similar to those in normal NMJs. Addi-tionally, agrin-deficient knockout mice have normaltotal AChR levels but few detectable AChR clustersor other postsynaptic specializations. Motor axonsfail to branch properly on agrin-deficient myotubes.Thus, agrin is necessary and sufficient for postsynap-tic differentiation and can account for the nerve’sability to organize postsynaptic assembly.18,19,27,46,62

Agrin appears to signal the muscle by the trans-membrane muscle-specific kinase (MuSK), althoughit does not bind to agrin directly. MuSK is expressedselectively by skeletal muscle, where it is colocalizedwith AChRs in the postsynaptic membrane.50,83,151

Knockout of the gene encoding MuSK results inmice that die during late embryonic development orare stillborn.26,46 Postsynaptic clusters of ACh recep-tors are completely absent in these MuSK knockoutmice and, in mice treated with RNAi designed toblock MuSK transcription, NMJs do not form.74,84

Interestingly, postsynaptic defects are accompaniedby presynaptic changes, specifically motor neuronaxons fail to differentiate properly and grow unusu-ally long branches along the muscle surface. Purifiedagrin has not been shown to bind either to purifiedMuSK or to MuSK expressed in heterologous cells,which suggests that MuSK may be a portion of amulticomponent receptor, with other subunits beingnecessary for agrin binding.

Another effector for AChR clustering is rapsyn,which is critical for AChR clustering. MuSK is clus-tered at synaptic sites in rapsyn knockout mice,whereas AChR and other synaptic proteins are notconcentrated at the synaptic contact but rather arediffusely distributed.2,47 Although MuSK signals clus-tering, rapsyn is an essential synaptic scaffold com-ponent that maintains clustering of AChR.

A neuronally derived synaptic regulatory factorcalled neuregulin (NRG-1) also promotes the accu-mulation of AChR at the NMJ (Fig. 3). NRG, similarto agrin, acts through distinct receptor signalingpathways promoting an increase of AChR density atthe NMJ, but its activity appears more complex thanthat of agrin.12,37 NRG is a transmembrane proteinand acts as a ligand for the ErbB tyrosine kinases.161

NRG increases the transcriptional rates of AChRgenes in vitro, and at synaptic nuclei in vivo. Adultmice heterozygous for a mutation of NRG expressroughly half the AChR at the NMJ and, as expected,have smaller MEPP amplitudes, but the EPP is nor-mal because of compensatory increased quantal con-


tent. Mice targeted with ablations of ErB-2 andErbB-3 express broadened NMJs during develop-ment and reduced AChRs, but these mutations alsolead to significant motor neuron degeneration andabnormalities of Schwann cell development, compli-cating understanding of specific effects on NMJ for-mation.83

The administration of NRG to cultured myocytesalso stimulates the tyrosine phosphatase SHP2,147

which results in increased levels, and phosphoryla-tion of the Ets domain transcription factors increasestranscription of the � and � subunits of theAChR.40,130,131 Several groups have independentlyidentified a DNA element (N-box) that is bound byEts transcription factors and may account for thesynaptic transcription of AChR � and � subunitgenes. A patient with a congenital myasthenia hasbeen identified who had a mutation in the � subunitpromoter with reduced AChR expression. The utro-phin gene, which is expressed at the NMJ and regu-

lated by NRG, also requires this specific DNA ele-ment for its expression.54,72 Consistent with thesefindings, inhibition of Ets domain transcription fac-tors in vivo results in reduced expression of theAChR � subunit, utrophin, and AChE—all compo-nents of the NMJ.10 These results indicate that theregulation of Ets transcription factors by NRG regu-lates NMJ-specific expression of certain genes.

DIVERSITY OF NEUROMUSCULAR JUNCTIONSTRUCTURE AND PHYSIOLOGY

The NMJ demonstrates structural and functionalvariations across species and between muscle-fibertypes, and is being appreciated to have more subtledifferences among muscles. Thus far, this review hasfocused on the NMJ of singly innervated musclefibers with en plaque morphology, which is the normin mature mammalian muscle. However, amphibian,reptile, and certain mammalian muscles also contain

FIGURE 3. Model for interactions between the agrin/MuSK and the NRG/ErbB signaling pathways. Synapse-specific gene transcriptionin subsynaptic nuclei is initiated by MuSK either as a result of auto-activation, or by the action of neuronal agrin, and initiates pathwaysthat regulate AChR subunit transcription and aggregation, respectively. Neuregulins then accumulate in the basal lamina and on theirErbB receptors on the muscle cell plasma membrane. Activated MuSK and ErbB receptors may act cooperatively and trigger signalingpathways that involve Ras, Rac, and Cdc42 along with their downstream effectors, ERK and JNK. This leads to phosphorylation of theEts-related transcription factor, GABP, which induces synapse-specific gene transcription via binding to the N-box. ACh released from thenerve terminal and its binding to the AChR evokes electrical activity on the muscle fibers that represses AChR transcription at bothsynaptic and extrasynaptic nuclei.


fibers with multiple synaptic contacts, with the NMJhaving en grappe synapses. En grappe endings aredispersed as small, surface terminals along thelength of individual muscle fibers with each engrappe NMJ being about 10–16 �m in diameter, ascompared with en plaque endplates being on theorder of 50 �m in diameter. The en grappe end-plates have few or no postjunctional folds; thus, thepostjunctional membrane follows the contour of thenerve terminal membrane.95,122,126

En grappe endplates are found on tonic or inter-mediate muscle fibers, which occur primarily in non-mammalian muscles where they constitute a signifi-cant fraction of the fiber population. The term“tonic” refers to the maintained contracture thesefibers develop in depolarizing solution. Tonic mus-cle fibers do not respond to a single nerve impulseand do not propagate action potentials, but ratherare activated by passive spread of depolarizationfrom many small nerve endings. This is in contrast tothe twitch muscle fibers, which produce a synchro-nized contraction (twitch) in response to a singlenerve stimulus and propagate action potentials. In-termediate fibers have en plaque and en grappeendplates and appear to have twitch characteristicsin the region of the en plaque endplate, but contractin a tonic fashion in the area of an en grappe NMJ.Although the size of an individual en grappe contactis smaller than the en plaque endplate, a study oflizard twitch and tonic NMJs found that the summedsynaptic area on tonic and twitch fibers is equal.157

The total number of presynaptic active zones, how-ever, is less in tonic than in twitch fibers, and eachhas only one rather than two rows of particles, sug-gesting that lower density in calcium channels attonic junctions could reduce local calcium influxand, therefore, the probability of neurotransmitterrelease. The en grappe junctions may have lowerAChE concentrations than en plaque NMJs.95,157

Tonic fiber NMJs can continuously release transmit-ter by almost an order-of-magnitude longer than cantwitch terminals.20,21

At snake tonic fibers, NMJs differ from those attwitch junctions in that their gating is less voltagesensitive, they have slightly lower unit channel con-ductance, and their channel open time is longer,causing the EPP to decay more slowly.28,29 By elec-trophysiological criteria, the snake tonic muscle fi-bers express both fetal- and adult-type AChRs at theNMJ. The fetal-type channels may facilitate tonicforce generation, as described earlier for the engrappe endplates. Also, the small conductance chan-nel at tonic synapses is relatively resistant to desen-sitization. Such a property would enable tonic fibers

to better respond to repeated or prolonged nervestimulation.

The extraocular muscles (EOMs) of mammalsdemonstrate the greatest structural diversity ofNMJs. In addition to twitch muscle fibers, they pos-sess multi-innervated fibers that have either only engrappe endplates or possess both en grappe and enplaque endplates.67,107,140 Tensor tympani, stapedius,laryngeal muscles, and the tongue have also beenfound to contain multi-innervated fibers.55 Manytwitch junctions of EOMs also have sparse secondarysynaptic folds, which would suggest a lower safetyfactor (Fig. 4). Despite the rapid motor neuron fir-ing frequencies that these NMJs undergo, they per-form reliably within a narrow range of tolerance. Incontrast to all other mature mammalian muscle,certain en grappe and en plaque NMJs express thefetal isoform of the AChR in the maturestate.58,65,66,93 The function of the fetal AChRs atEOM junctions has not been studied. Despite theultrastructural uniqueness of the EOM junctions, theprotein scaffold that underlies the EOM NMJ,whether en grappe or en plaque, does not differsignificantly from that of other skeletal muscles.71

Known signaling pathways that control developmentand maintenance of the junction also do not differbetween EOMs and other muscles.71

Structural and physiological differences existamong singly innervated NMJs as well. For example,the safety factor for neuromuscular transmission offast-twitch mammalian skeletal muscle fibers is largerthan that of slow-twitch fibers, which is likely second-ary to several properties.49 Fast-twitch fibers havegreater quantal contents than slow-twitch fibers,49,149

and greater postsynaptic sensitivities than do slow-twitch fibers; fast-twitch fiber NMJs exhibit greaterdepolarization than NMJs of slow-twitch fibers inresponse to quantitative iontophoresis of ACh.142

Fast-twitch fibers also have increased sodium currentin the region of the NMJ, which may serve an adap-tive function because fast-twitch fibers require largerdepolarizations to initiate contraction than slow-twitch fibers.92,118,119 Structurally, the extent ofpostsynaptic folding is greater at NMJs of fast fibersthan those of slow fibers, which would serve to ele-vate the EPP.162

Differences in development of NMJs can be ap-preciated among muscles, which are maintained forat least part of maturity. Pun and colleagues identi-fied “fast-synapsing muscle” and “delayed-synapsing”muscle during mouse myogenesis, which was notrelated to the content of fast or slow fibers.108 Infast-synapsing muscles, as the tibialis anterior wasfound to be, focal AChR clustering, alignment of the


presynaptic nerve with AChR clusters, and the align-ment of Schwann cells with the presynaptic nerveterminal occurred over 1 day. In contrast, in a slow-synapsing muscle, such as the lateral gastrocnemius,the focal organization process occurred over 5 days.In the absence of agrin, the NMJs on fast-synapsingmuscles could form for a few days, but they could notin the delayed-synapsing muscles. In young adultmice, AChR clusters of slow-synapsing muscle wouldbe lost with denervation or application of botulinumtoxin, whereas clusters would be retained in thefast-synapsing muscles. These experiments demon-strated that the intrinsic NMJ properties vary amongmuscles in a manner not previously appreciated.Such differences may influence the susceptibility ofmuscle groups to neuromuscular transmission disor-ders.

PATHOLOGY OF THE NEUROMUSCULAR JUNCTION

Neuromuscular transmission in humans can be com-promised by pathology at the presynaptic, synapticcleft, and postsynaptic regions of the NMJ. A discus-

sion is provided here of genetic and acquired disor-ders that alter NMJ architecture.

Congenital Myasthenic Syndromes. Congenital my-asthenic syndromes (CMSs) are genetic disorders ofthe NMJ that compromise neuromuscular transmis-sion, and causal defects in presynaptic, synaptic cleft,and postsynaptic apparatus have been identified.31

Clinically, they share a similar phenotype of fatigu-ing weakness presenting usually from birth, al-though patients with adult onset have been de-scribed. Electrodiagnostic studies often demonstratedecremental responses with repetitive stimulation.We focus briefly on CMSs in which a gene defect hasbeen identified, providing insight into structure–function correlations at the NMJ.

Presynaptic abnormalities attributable to defectsof choline acetyltransferase deficiency (ChAT) havebeen identified.15 The ChAT-deficient phenotype isdistinguished by sudden episodes of severe respira-tory distress and bulbar weakness leading to apnea,precipitated usually by a stress, such as infection,

FIGURE 4. Electron micrographs of (A) mouse extraocular muscle NMJ. Note the sparseness of synaptic folds compared to diaphragmNMJ in (B).


fever, or excitement, against a background of vari-able myasthenic symptoms. Some patients are nor-mal at birth and develop apneic attacks and myas-thenic manifestations during infancy or childhood.The apneic crises can be treated with AChE inhibi-tors.15 Investigation of the NMJ reveals normal AChRand postsynaptic structure; however, the synaptic ves-icles are smaller than normal in rested muscle andincrease or remain the same size after stimulation.94

MEPP and EPP amplitudes are normal in restedmuscle but decrease significantly during 10-Hz stim-ulation for 5 minutes, and then recover slowly over10–15 minutes while the quantal content of the EPPis essentially normal.15 MEPP and EPP amplitudesdecline abnormally when neuronal impulse flow isincreased and then recover slowly, which points to adefect in resynthesis or vesicular packaging of ACh.ChAT, vesicular ACh transporter, and the vesicularproton pump all could explain such a defect, and amutation in ChAT has been identified.15

Mutations in the collagen tail of AChE occur,which leads to an absence of AChE from the synapticcleft.34 The presynaptic terminals are small and of-ten encased by Schwann cells, reducing quantal re-lease, which may serve a protective function by lim-iting desensitization and cationic overloading fromexcess AChR opening. In spite of reduced quantalrelease, secondary synaptic folds degenerate, leadingto a loss of AChRs, which explains the myasthenicmanifestations observed in patients. The junctionalsarcoplasm contains apoptotic nuclei and degener-ating membranous organelles.60 Neuromusculartransmission is impaired by reduced quantal release,desensitization of AChR by prolonged exposure toACh (i.e., a depolarization block at physiologic ratesof stimulation), and by an “endplate myopathy”caused by cationic overloading of the postsynapticregion.52,87

Mutations have been discovered in all AChR sub-units and in different subunit domains.34,96,155 Themutations fall into two major groups: those thatreduce expression of AChR, and those that alter itskinetic properties. Kinetic mutations can be furtherdivided into two types: slow-channel mutations thatincrease, and fast-channel mutations that decreasethe synaptic response to ACh. The slow- and fast-channel mutations are physiologically opposites intheir phenotypic consequences and alterations ofunderlying AChR activation steps.

Morphological studies in slow-channel CMSs re-veal junctional fold degeneration with loss of AChRs,and degenerating membranous organelles, apopto-tic nuclei, and small vacuoles in junctional fiberregions.52,53,96 Postsynaptic degeneration is attrib-

uted to excessive calcium influx into the postsynapticregion during prolonged episodes of synaptic activa-tion and an endplate myopathy similar to the AChEdeficiency. The slow decay of the synaptic potentialswas attributed to prolonged openings of the AChRchannel. Normal activity of AChE at the NMJ differ-entiates this condition from the AChE-deficiency dis-order. The slow-channel CMS described at the mo-lecular level was a threonine-to-proline mutation in aportion of the AChR known to form an �-helix thatcontributes to the channel portion.100 The mutationdramatically slows the closing rate-constant govern-ing the active receptor channel and enhances theexceptionally rare spontaneous normal receptorchannel openings to produce a steady stream ofchannel activity in the absence of ACh. Another typeof slow channel was identified due to a mutation atthe ACh binding site that caused abnormally highaffinity of the receptor for the agonist, which againprolongs the time the channel allows ion passage.137

Phenotypically, this syndrome is recognized by itsdistinct features: dominant inheritance; selectiveweakness of cervical, scapular, and finger extensormuscles; mild ophthalmoparesis; and variable weak-ness of other muscles.

The name fast-channel syndrome originates fromthe abnormally fast decay of the synaptic responsecaused by abnormally brief channel openingevents.152 Morphological studies show that synapticvesicles are of normal size, eliminating the possibilityof reduced vesicle ACh content, and AChE overac-tivity is unlikely, because synaptic response is brief,not prolonged, and cholinesterase inhibitors do notdiminish the synaptic response.152 Endplate studiesrevealed normal endplate fine structure, no AChE orAChR deficiency, and normal quantal release bynerve impulse, but very small MEPPs.100,138 Analysisof ACh-induced current noise demonstrated normalconductance and abnormally short-lived openings ofthe AChR channel. The findings are consistent withan AChR kinetic defect in which opening of thereceptor channel was impaired and closing was en-hanced. AChR mutations were revealed in patientswith identical proline-to-leucine mutations in the �subunit, coupled with a null mutation in the secondallele of the � subunit.100 Other fast-channel muta-tions have been identified since the first discovery offast-channel CMS, and a variety of AChR mutationswere found.138 Common to all fast-channel muta-tions is a dramatically diminished postsynaptic re-sponse to ACh.

Most CMSs are caused by homozygous or het-erozygous low-expressor mutations in AChR subunitgenes, and these mutations are concentrated in the


� subunit. Morphological studies of endplates withlow expressor mutations have shown an increasednumber of endplate regions distributed over an in-creased span of individual muscle fibers. Junctionalfold integrity is preserved, but some endplate re-gions have a paucity of synaptic folds and are smallerthan normal. AChR distribution on the junctionalfolds is patchy, and the AChR density is diminished.Immunostaining for rapsyn, an AChR cross-linkermolecule, is decreased in proportion to decreasedAChR expression. The EPP is reduced, but AChrelease by nerve impulse is frequently higher thannormal, suggesting an adaptation to postsynaptic in-jury.34,96

In a subset of CMS patients with endplate AChRdeficiency but no mutations in AChR, rapsyn muta-tions have been identified.14,98 Morphological stud-ies in rapsyn-deficient individuals have shownpostsynaptic regions that are sparse, with synapticfolds suggesting impaired postsynaptic morphologicdevelopment. Expression studies in HEK cells re-vealed that no mutations diminished rapsyn self-association but they did diminish coclustering ofAChR with rapsyn. Mutations in the promoter regionof the rapsyn gene leading to reduced expression ofAChR at the NMJ have been found.99

Acquired Neuromuscular Transmission Disorders.

Lambert–Eaton myasthenic syndrome is an acquiredcondition resulting from autoantibodies directedagainst voltage-gated calcium channels in the motornerve terminal.32 The condition is characterized bymuscle weakness and fatigability during activity. Theunderlying autoimmune response is often associatedwith a small-cell lung carcinoma in which the tumorcells have calcium channels that resemble thosefound at the nerve terminal. Generally, the autoan-tibodies do not have a direct pharmacological block-ing effect on the calcium channel but bind to thecalcium channels on the nerve terminal, leading totheir downregulation via endocytosis. The only struc-tural alteration of the NMJ observed is the antibody-induced reduction in the number and size of activezones.41–43 The net effect is that fewer calcium ionsenter the terminal during each nerve action poten-tial; therefore, fewer quanta of transmitter are re-leased. This, in turn, leads to a reduction in thesafety factor that may result in transmission failure.During sustained muscle activity, there is a transient,yet dramatic, increase in muscle strength from aninitially very low level. This postexercise facilitation islikely a consequence of a temporary calciumbuild-up in the nerve terminal, which results in cat-

ion channel formation in the nerve terminal mem-brane, leading to neurotransmission block.121

Myasthenia gravis (MG) is the most commonpostsynaptic neuromuscular transmission disorderand is an autoimmune condition.59,64,82 The primaryautoantigen is the AChR, and autoantibodies inducethe disorder by induction of complement-mediatedlysis of postsynaptic membrane producing a dra-matic structural alteration with loss of postsynapticfolds.125 Consequently, fewer AChR are opened byeach quantum of ACh and the amplitude of theMEPP is reduced. This effect, together with the lossof secondary postsynaptic folds and the voltage-gatedsodium channels at the NMJ, leads to a markedreduction of the safety factor and compromised neu-romuscular transmission.123 The NMJ may attemptto compensate for the compromised postsynapticfunction by an increase in neurotransmitter releasefrom the nerve terminal.105 This effect compensates,in part, for the loss of AChR, and prevents anyfurther decline of the safety factor.

In 2001, autoantibodies directed against MuSKwere identified in patients with MG who were sero-negative for autoantibodies to AChR, but not inpatients with autoantibodies to AChR.57 Patient serawere found to disrupt aggregation of AChR on myo-tubes, but absolute confirmation that MuSK autoan-tibodies are responsible for MG has not been con-firmed by induction of disease in an animalmodel.134 In the only study of muscle biopsies ofMuSK-positive patients, AChR reductions at the NMJwere not identified, compared with AChR antibody–positive NMJ, and complement was detected in onlytwo of eight MuSK antibody–positive patients. Inves-tigators concluded that MuSK antibodies do notcause substantial AChR loss, complement deposi-tion, or morphological alterations in NMJ struc-ture.135

CONCLUSION

About 100 years ago, a receptor was hypothesized toexist on the muscle surface. Now, this receptor, itsactivation, supportive cytoskeleton, and the sur-rounding cellular and molecular environment havebeen defined. As in all areas of science, an increasein understanding has only led to further questions.Neuromuscular transmission disorders demonstratea propensity for involvement of certain musclegroups, suggesting heterogeneity in properties thatare not immediately obvious from present molecularstudies, as discussed by Pun et al.108 The details ofhow the high density of AChR is established andmaintained at the NMJ remains to be elucidated.


Also, how the extremely rapid fusion of nerve termi-nal and synaptic vesicle plasma membranes is accom-plished remains to be defined. Finally, the neglectedfield of the Schwann cell in NMJ physiology andjunction maintenance is young and likely to producesurprising discoveries. The authors’ intent in thisreview has been to provide a snapshot of how theNMJ is structured and develops, and how pathologycompromises its architecture and function.

This study was supported by National Institutes of Health grantsR24 EY014837, R01 EY-015306, and R01 EY013238 to H.J.K. GrantP30 EY11370 supported preparation of the figures.

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