Genes at the junction – candidates for congenital myasthenic syndromes

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Angela Vincent, Claire Newland, Rebecca Croxen and David Beeson The neuromuscular junction is the site of several myasthenic (mys, muscle; aesthenia, weakness) disorders of autoimmune and genetic origin. The acquired autoimmune conditions are mainly adult-onset and caused by antibodies to specific neuronal and muscle ion channels, but can occur neonatally due to placental transfer of maternal antibodies. This review focuses on the rarer genetic conditions, called congenital myasthenic syndromes (CMS), that often present at birth. Mutations have yet to be characterized for familial infantile myasthenia, acetylcholinesterase deficiency and ACh-receptor deficiency; but genes encoding both structural and functional NMJ proteins should be considered. Other syndromes have recently been shown to involve defects in the functioning of the ACh receptor itself. In particular, eight different mutations have been reported in cases of the slow channel syndrome, a dominant condition associated with point mutations that generate single amino acid changes within the ACh receptor and result in prolonged channel activations. These investigations are providing new insights into the structure and function of the ACh receptor. Further studies of CMS should pave the way for analysis and treatment of disorders involving other synapses in the peripheral and central nervous system. Trends Neurosci. (1997) 20, 15–22

T

HE NEUROMUSCULAR JUNCTION (NMJ) is a relatively simple synapse that can be studied in isolated nerve–muscle preparations from muscle biopsies (see Box 1). It lies outside the blood–brain barrier, is accessible to circulating factors and is the target for acquired autoantibody-mediated disorders. These conditions usually present after childhood, are frequently associated with other autoimmune diseases, and sometimes with particular tumours. They are caused by antibodies to specific ion channels and respond to treatments designed to reduce the amount of circulating antibody. The most common is myasthenia gravis in which antibodies to the muscle nicotinic ACh receptor cause muscle weakness and fatigue. Neonatal weakness occurs in about 8% of pregnancies in mothers with myasthenia gravis, due to the transfer of anti-AChreceptor antibodies across the placenta. On rare occasions, transfer of antibodies specific for the foetal ACh receptor may cause severe congenital abnormalities. Table 1 summarizes the main autoimmune disorders. The main focus of this review, however, will be the genetically inherited congenital myasthenic syndromes (CMS). These frequently present at birth or within the first two years of life. Many progress slowly, if at all, throughout life and show little fluctuation in severity from day to day. They do not have detectable autoantibodies to ion channels and do not respond to immunotherapies. Table 2 summarizes the most clearly defined forms of CMS and their characteristic features. Although these syndromes are rare (prevalence <1:500 000), the identification of genetic defects in this heterogeneous group of disorders, as are beginning to be specified for the slow channel syndrome (SCS) (see below), will contribute to the understanding of the development, maintenance and function of the NMJ and other synapses. Copyright © 1997, Elsevier Science Ltd. All rights reserved. 0166 - 2236/97/$17.00

For a brief description of neuromuscular transmission and some of the techniques used to investigate it, see Box 1.

Familial infantile myasthenia This autosomal recessive disorder presents at birth with episodes of apnoea, poor cry, feeding difficulties and respiratory failure, which are often precipitated by stress or infections. It is a potentially fatal condition but responds very well to acetylcholinesterase (AChE) inhibitors and often improves with age. In the few patients studied, miniature endplate potential (MEPP) amplitudes were normal at rest, but became much smaller following 10 Hz nerve stimulation. A similar abnormality can be induced in normal muscle treated with the ACh-uptake inhibitor hemicholinium, and therefore a defect in ACh reuptake, synthesis or packaging has been proposed4. Further studies showed that the number of synaptic vesicles in the resting nerve terminals was normal, but their size was reduced. However, there was no consistent correlation between the size of the synaptic vesicle and the MEPP amplitudes5. In another presynaptic syndrome, ‘paucity of synaptic vesicles’, a patient with a less-episodic clinical picture was described whose muscle biopsy showed a reduction in the numbers of synaptic vesicles and reduced quantal release of neurotransmitter6. Possible candidates for the mutations that underlie these conditions are genes encoding proteins involved in the generation and recycling of ACh or synaptic vesicles.

Acetylcholinesterase deficiency This rare autosomal recessive condition is evident from birth or soon after, and is characterized by moderate to severe generalized muscle weakness that can progress during life, and slowed pupillary responses to PII: S0166-2236(96)10066-7

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Angela Vincent, Claire Newland, Rebecca Croxen and David Beeson are at the Neurosciences Group, Dept of Clinical Neurology, University of Oxford, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, UK OX3 9DU.

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Box 1. The neuromuscular junction Neuromuscular transmission depends on the close apposition of nerve and muscle at the neuromuscular junction (NMJ) (see Fig.). The nerve impulse leads to Ca2+ entry through voltage-gated Ca2+ channels (VGCC) in the nerve terminal, resulting in quantal release of ACh from active zones where the VGCC are located, and interaction of ACh with ACh receptors on the postsynaptic membrane. Influx of cations, mainly Na+, through the ACh receptor results in an endplate potential (EPP), activation of voltagegated Na+ channels (VGSC) and generation of an action potential in the muscle fibre. The action of ACh is terminated by its hydrolysis by acetylcholinesterase (AChE), and the nerve terminal membrane potential is restored following closure of the VGSC and opening of the voltagegated K+ channels (VGKC). Spontaneous release of single quanta of ACh results in a miniature EPP (MEPP). In disorders in which there is a reduction in the amount of ACh released per nerve impulse, reduced numbers of ACh receptors, functional impairment of the ACh receptors or abnormalities in the geometry of the NMJ, the EPP might be too small to reach the membrane potential required for activation of the VGSC. This leads to failure of neuromuscular transmission. In humans, the extent to which the EPP normally exceeds the threshold, the ‘safety factor’, is quite small and neuromuscular transmission is

VGSC

Na+

Nerve terminal

K+ ACh

s-laminin

Ca2+

sensitive to pathological changes. By contrast, in mice the safety factor appears higher (for a discussion of some of these issues see Refs a–c), and mouse models of neuromuscular diseases do not always demonstrate obvious weakness. Neuromuscular transmission can be investigated in human muscle fibres, usually from biopsies of intercostal muscle. Intracellular microelectrode techniques can be used to measure the timecourse and amplitude of MEPPs and EPPs or, under voltage clamp, the underlying currents (MEPC and EPC). The number of ACh receptors can be assessed by binding of the snake toxin, ␣-bungarotoxin. Immunohistochemistry and electronmicroscopy are also informative (see Ref. a). In patients with muscle weakness, neuromuscular transmission is investigated by electromyography. The compound muscle action potential (CMAP) is recorded in response to stimulation of the corresponding nerve. Defects can be associated with changes in the initial amplitude of the CMAP, changes in the CMAP during stimulation at different frequencies, or the appearance of more than one CMAP following a single nerve stimulus. The NMJ is highly structured and there is increasing interest in the proteins that help form and maintain the presynaptic and postsynaptic specialization. Among many synapse-specific proteins are those with a role in regulation of the formation of motor nerve terminals (s-laminin), aggregation or clustering of ACh VGKC receptors within the postsynaptic membrane (RAPsyn, agrin, MuSK), or in anchoring the ACh receptors to the cytoskeleton (RAPsyn, utrophin, the syntrophins). Some of these funcVGCC tions are reviewed in Refs d,e. References

ACh receptor RAPsyn Utrophin Syntrophins VGSC

MuSK

ACh receptor AChE

RAPsyn Utrophin Syntrophins

Muscle fibre

VGSC

a Engel, A.G. (1993) Ann. New York Acad. Sci. 681, 425– 434 b Slater, C.R. et al. (1992) Brain 115, 451– 478 c Wood, S.J. and Slater, C.R. (1995) J. Physiol. 486, 401– 410 d Kleiman, R.J. and Reichardt, L.F. (1996) Cell 85, 461– 464 e Hall, Z.W. and Sanes, J.R. (1993) Cell 72/Neuron 10 (Suppl.), 99–121

TABLE 1. Autoimmune neuromuscular disorders Disorder Myasthenia gravis: adult form

Tumour association

Antibody to

Clinical phenotype

Mechanism

Thymoma

ACh receptor

Muscle weakness

ACh receptor

Transient muscle weakness Joint contractures and hypotonia Muscle weakness

Reduction in number of ACh receptors Placental transfer of antibodies to the ACh receptor Placental transfer of antibodies inhibiting foetal ACh receptor Reduction in ACh release from nerve terminal Spontaneous bursts of motor-nerve activity

neonatal form foetal form with congenital abnormalities Lambert–Eaton myasthenic syndrome Acquired neuromyotonia

SCLC

ACh receptor (foetal) VGCC

Thymoma

VGKC

Muscle twitching or cramps, some weakness

Myasthenia gravis and the Lambert–Eaton myasthenic syndrome show clinical improvement with acetylcholinesterase inhibitors and 3,4-diaminopyridine. Acquired neuromyotonia responds to drugs that reduce nerve excitability, for example, carbamazepine. These conditions also respond to immunotherapies. In those cases with tumours, the neurological syndrome often improves after removal or successful treatment of the tumour. For a review, see Ref. 1. Abbreviations: SCLC, small-cell lung cancer; VGCC, voltage-gated Ca2+ channel; VGKC, voltage-gated K+ channel.

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TABLE 2. Main forms of congenital myasthenic syndromes

Recessive

Onset

Typical clinical electromyography

Main biopsy findings

Familial infantile myasthenia

Neonatal

Normal CMAP, decreasing following exercise or 10 Hz stimulation

AChE deficiency

<2 yrs

ACh-receptor deficiency

<2 yrs

Decrement in CMAP at 3 Hz; repetitive response to single stimulus Decrement in CMAP at 3 Hz

Reduced MEPP amplitudes after 10 Hz Improvement stimulation; normal numbers of ACh receptors Prolonged decay of MEPP and MEPC; None absence of AChE at the NMJ Reduced MEPP amplitudes at rest; reduced Improvement numbers of ACh receptors; elongated AChE-stained NMJ

Autosomal Slow channel dominant syndrome or sporadic

Variable

Decrement at 3 Hz; repetitive response to single stimulus

Prolonged decay of EPP and EPC, or MEPP and MEPC

Response to AChE inhibitor

Deterioration

Based on 34th European NeuroMuscular Centre International workshop on congenital myasthenic syndromes. The CMAP was generated by nerve stimulation. In all cases nerve conduction studies were normal. There may also be increased jitter on single-fibre electromyography, due to reduced efficiency of neuromuscular transmission. For a comprehensive review of the different clinical and electrophysiological syndromes, see Refs 2,3. Abbreviations: AChE, acetylcholinesterase; CMAP, compound muscle action potential; EPC, endplate current; EPP, endplate potential; MEPC, miniature endplate current; MEPP, miniature endplate potential; NMJ, neuromuscular junction.

light. Inhibitors of AChE are ineffective but do not cause deterioration (compare with SCS, below). A repetitive muscle response to a single nerve stimulus is usually seen on electromyography, and in vitro microelectrode studies show prolonged decay of both miniature endplate and endplate potentials or currents. This syndrome is thought to be due to loss of AChE at the NMJ as demonstrated by cytochemical and immunocytochemical studies7. The collagentailed forms, which attach to the basal lamina at the NMJ, are reduced or absent. Thus, the total content of AChE in muscle is reduced, but the kinetic properties of the residual AChE appear normal. Morphological changes at the NMJ have been noted, for example, reduced nerve-terminal area and in some cases reduction in the numbers of ACh receptors; but these are probably secondary consequences of prolonged ACh activity (see also SCS, below). Despite extensive analysis

of the genes that encode AChE from five patients, no mutations in the catalytic subunit have been identified8. Thus, defects in the collagen tail of AChE, affecting its association with the catalytic subunit or interaction with the basal lamina, might be responsible.

Acetylcholine-receptor deficiency Many of these patients present at birth or during infancy and have marked ocular muscle weakness as well as limb and respiratory problems. Consanguinity in the parents is common, consistent with recessive inheritance, but in published cases males predominate over females (for example, see Refs 9,10). Muscle biopsies typically show very small MEPPs (20 –50% of control values), small endplate potentials (EPPs) and reduced numbers of ACh receptors that are often distributed abnormally along the muscle fibre, coincident with elongated areas of AChE staining (Fig. 1). These changes

Fig. 1. Acetylcholinesterase (AChE) and ACh receptors at the neuromuscular junctions of patients with ACh-receptor deficiency. Acetylcholinesterase staining (A and C) and localization of ACh receptors, demonstrated by autoradiography following [125I]␣-bungarotoxin binding (B and D) in teased muscle fibres from biopsied intercostal muscles. In the control muscle (A and B), from patients undergoing thoracic surgery for cancer, both AChE and ACh receptors are highly concentrated in discrete oval regions that represent the neuromuscular junctions (NMJs). In the muscle fibres from patients with ACh-receptor deficiency (C and D), AChE staining often shows elongation of the NMJ, sometimes with multiple stained areas (C). The area of ACh-receptor localization is also elongated (D), and the ACh receptors are reduced in density and total number. In some patients the AChE staining is fragmented or diffuse. These results show some similarities to those seen in mice deficient for other NMJ-specific proteins (see Table 3). Scale bar, 35 ␮m.

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TABLE 3. Summary of findings in mice homozygous for deletions of proteins important in the function or structure of the NMJ Presence and distribution at the NMJ of: Knockout

Age at death

ACh-receptor AChE clusters

RAPsyn, utrophin NMJ morphology

Ref.

s-laminin

Days to weeks

Normal

Present

13

Agrin

Stillborn

Reduced in Diffuse, barely size or density detectable

Co-localized with ACh receptors

MuSK

Neonatal

Not detectable

RAPsyn Utrophin

Hours >100 days

Not detectable Diffuse, barely detectable Not detectable Reduced Normal Normal

Present

Not detectable Normal

Reduced nerve terminal branching; reduced active zones and junctional folds Extensive axon growth; reduced terminal branching; some noninnervated clusters Extensive axon growth; reduced terminal branching Excessive axon growth Reduced junctional folds

14 15 16 *

In all cases, heterozygotes were normal. Electrophysiological studies have only been reported in s-laminin and utrophin knockouts. A fuller description of some of these knockouts is given in Ref. 17. Abbreviations: AChE, acetylcholinesterase; NMJ, neuromuscular junction. *A. Deconinck, A. Potter, J. Tinsley, S. Wood, R. Vater, C. Young, L. Metzinger, A. Vincent, C. Slater and K.E. Davies, unpublished observations).

can be similar to those found in autoimmune myasthenia gravis, but in congenital ACh-receptor deficiency immunohistochemistry does not detect antibody bound to the NMJ (Ref. 2). These cases are almost certainly heterogeneous, with reduced density or altered pattern of AChE staining evident in some10. Similarly, in certain cases, electronmicroscopy has demonstrated a reduction in the number of postsynaptic folds as well as reduced ACh-receptor density11,12. It should be pointed out that a moderate reduction in the number of ACh receptors at the NMJ can also be found in AChE deficiency and the SCS, but in these cases it is thought to be a secondary phenomenon. A primary reduction in the numbers and density of ACh receptors could stem from mutations in the genes encoding the ACh receptor, leading to reduced synthesis, assembly, membrane insertion or stability. Mutations in the ACh-receptor genes have been detected in a few cases but their involvement in causing a reduced number of ACh receptors has not yet been demonstrated. Although there is no direct evidence, the recent data emphasizing the role of other synaptic proteins in orchestrating and maintaining the structure of the NMJ (see Box 1) suggest that mutations in these proteins could be involved in AChreceptor deficiency. The changes to the NMJ that are observed in mice deficient in expression of these proteins (‘knockouts’; see Table 3) show certain similarities to those observed in patients’ muscle biopsies. For example, mice deficient in nerve-derived agrin, RAPsyn, and the muscle-specific kinase, MuSK, show extensive axon outgrowth and reduced terminal nerve branches with marked changes in ACh-receptor and AChE density and clustering (see Table 3). Utrophin appears absent (or is very reduced) at the NMJ in RAPsyn knockouts, and utrophin is also reduced in AChreceptor deficiency (C.R. Slater, S. Wood, C. Young and A. Vincent, unpublished observations). These findings are in keeping with the possibility that recessive mutations in the genes encoding RAPsyn or utrophin could be responsible for some cases of ACh-receptor deficiency. However, the RAPsyn knockout mice have a far more-severe phenotype than the patients, so any mutations within the gene encoding RAPsyn in the human condition would need to modify rather than inactivate its function. 18

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Moreover, utrophin knockout mice show only minimal phenotypic changes up to five months of age (A. Deconinck, A. Potter, J. Tinsley, S. Wood, R. Vater, C. Young, L. Metzinger, A. Vincent, C. Slater and K.E. Davies, unpublished observations), but it is possible that greater effects might be seen at a later age. It will be of interest to examine the phenotypes of transgenic mice with less-severe RAPsyn mutations, and mice heterozygous for mutations in more than one candidate protein. In the meantime, immunohistochemical studies on the amount and localization of these proteins in muscle biopsies, although not distinguishing between primary and secondary effects, might nevertheless help in the identification of candidates.

Functional abnormalities of the ACh receptor A number of CMS appear to result from functional abnormalities of the ACh receptor, principally agonist binding, channel gating and ion conduction6. Functional defects might arise from mutations in the ACh-binding site, the channel pore, or structures involved in coupling ACh binding to channel opening, or from altered subunit composition or posttranslational modifications. To date, mutations affecting ACh-receptor function have been identified in only two syndromes. Most of these mutations underlie the ‘slow channel syndrome’ (SCS; see below), but more recently a mutation that gives rise to another syndrome, described as a ‘low-affinity fast channel syndrome’, has been identified18.

The slow channel syndrome This syndrome is inherited in an autosomal dominant fashion, although many cases appear to be sporadic. There is weakness of cervical and scapular muscles and often distal wasting and weakness, particularly of the finger extensors19. The onset of weakness can occur neonatally, or might not arise until adolescence, adulthood or during pregnancy. Moreover, family members sometimes show characteristic electrophysiological changes without clinical symptoms, suggesting that other factors might be involved in the full expression of the disease20. Inhibitors of AChE cause clinical deterioration. Slow channel syndrome is characterized by a repetitive response to a single nerve stimulus on electromyography.

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Microelectrode studies on biopsied A B intercostal muscle show prolonged 10 ms 10 ms decay of miniature endplate poten2 nA 2 nA tials or currents, with normal AChE activity, suggesting prolonged activations of the ACh receptor. In some patients this has been con40 ms 40 ms firmed by direct measurement of single-channel currents from the 10 pA 10 pA muscle biopsies (for example, see Refs 21–23; Fig. 2). The channels also showed normal conductance, ruling out the possibility that the prolonged ACh-receptor activations were due to the substitution of foetal for adult ACh receptor at the NMJ (see Box 2, Fig. A). There is Fig. 2. Comparison of miniature endplate currents (MEPCs) and single-channel currents recorded from control and sometimes an associated reduction slow channel syndrome (SCS) endplates. Upper traces, MEPCs; lower traces, single-channel currents. (A) Control. in the numbers of ACh receptors, (B) Slow channel syndrome endplate. Note the markedly prolonged decay of the MEPC and the presence of both with moderately reduced MEPP normal and very prolonged channel events in the SCS muscle. The MEPC decay is best fitted by the sum of two expoamplitudes20–24. nentials (vertical arrows indicate MEPC decay time constants). Reproduced, with permission, from Ref. 21. Eight mutations of the ACh receptor have now been reported that cause the physio- although this and other rates (for example, agonist logical abnormality (Fig. 3A), although not all the cases dissociation rate) were not determined. In addition, showed typical repetitive responses to a single nerve ⑀L269F (located in M2) and ␣N217K (located in M1) stimulus. It is useful to consider the location of these enhanced densensitization. Kinetic analysis of SCS mutations is likely to promutations and their effect on ACh-receptor function in light of our current knowledge of the structure and vide new insights into the structural determinants of function of the ACh receptor (see Box 2). The identi- binding affinity and channel gating. Although some fied SCS mutations are in different subunits (␣, ␤ and ⑀) of the mutations identified to date lie within or close and in diverse regions, including M1, M2, in the extra- to the ACh-binding pocket or regions thought to be cellular loop between M2 and M3, and near residues important in channel gating, they actually pinpoint that are known to be involved in ACh binding21–26. residues not previously investigated (the exception Homozygous expression of mutant ACh receptors in being ␤L262 whose functional significance had human embryonic kidney fibroblast cells (HEK 293) or already been determined from structure–function Xenopus oocytes shows that these mutations prolong studies27,28). Other mutations (notably ␣S269I) are in ACh-receptor activations, confirming their role in pro- regions not previously identified as being involved in ducing the electrophysiological phenotype21–24,26 (Fig. 3B). ACh binding or channel gating. Studies of other The mutations are heterozygously expressed at the syndromes that are thought to result from abnormal NMJs of SCS patients. Therefore, the number of differ- functioning of the ACh receptor are also likely to ent ACh-receptor types in the patient’s muscle will identify additional residues involved in agonist depend on whether the mutation is in one of the binding, channel gating and ion conduction18. non-␣ subunits, which are singly represented in each Furthermore, as the functional consequence of these pentamer, or in the doubly represented ␣ subunit. In mutations is the alteration of the synaptic response, the latter case, four receptor types will be present, but they might also shed light on determinants of synapthey might not all be kinetically distinct. tic transmission. Indeed, Sine and colleagues18,21 have The mechanism by which a particular SCS mutation shown the importance of the ACh-receptor affinity for prolongs receptor activations has been determined in ACh in governing the timecourse of the synaptic several cases21–24. The most comprehensive investi- response. gation was of mutation ␣G153S (Ref. 21), which is in An animal model of SCS a region thought to contribute to the ACh-binding site on the ␣ subunit. This mutation prolongs receptor As yet there is no obvious correlation between the activations primarily by reducing the rate of dissoci- mutations found within the genes encoding the ACh ation of ACh from the receptor (slower k–2; see Box 2), receptor and the severity of the disease phenotype, thereby increasing the number of openings per burst, and it is not entirely clear how prolonged channel with no change in the duration of individual open- activations cause muscle weakness. It has been proings. The mutation also increased the propensity for posed that excess Ca2+ ions flow through the ACh desensitization. receptor, leading to Ca2+ ‘overload’ at the NMJ, resultThe mutations in other regions of the ACh receptor ing in inhibition of mitochondrial respiration, and prolong receptor activations through different mecha- activation of intracellular enzymes with focal damage nisms. The M1 and M2 mutations all slow the rate of to the NMJ (‘endplate myopathy’)19. channel closure, indicating stabilization of the open One means to investigate pathological mechanisms state, and resulting in longer individual openings22–24. in SCS is to study a mouse transgenic model expressReceptors carrying some of the M2 mutations (⑀T264P, ing the mutant ACh receptor. Mice overexpressing ⑀L269F and ␤V266M) also exhibited an unusually ␦S262T, ␣C418W and ␣L251T, each of which had high rate of spontaneous openings in the absence of been shown to prolong channel activations in in vitro ACh, suggesting an increased rate of channel opening, mutagenesis experiments, and ⑀L269F, which has TINS Vol. 20, No. 1, 1997

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Box 2. The ACh receptor – structure and function ACh-receptor structure The ACh receptor (see Fig. A) is a pentameric transmembrane glycoprotein that exists in two main types in mammalian muscle. The foetal form consists of ␣ (two copies), ␤, ␦ and ␥ subunits, whereas in the adult form the ␥ subunit is replaced by the ⑀ subunit. The adult form has a greater single-channel conductance but a briefer channel open time than the foetal form. All five subunits are homologous, each with four transmembrane domains (M1 to M4). The topology of the extracellular domain (around 210 amino acids) is unknown, but there is a highly conserved disulphide loop between amino acids 128 and 142 (human ␣ subunit numbering). Each ACh receptor binds two molecules of ACh, and binding of the neurotoxin ␣-bungarotoxin overlaps these sites. For detailed review see Refs a,b.

Mechanisms of ACh-receptor activation To understand how slow channel syndrome (SCS) mutations cause a change in the function of the ACh receptor it is worthwhile considering the mechanisms of channel activation. A standard mechanism for the activation of the ACh receptor is shown in Fig. B. Closed resting states of the receptor are indicated as R, open states are represented as R*, and D indicates a desensitized closed state. As there is some debate about which states can lead directly to the desensitized state (or states), we have arbitrarily included only one desensitized state connected to the bi-liganded closed state (A2R). Agonist (A) binds to the two sites on the closed receptor, R, with association and dissociation rates of k+1, k+2, k–1 and k–2. For simplicity, the two binding sites are assumed to be equivalent before ACh binds, and when two agonist molecules are bound they have equal rates of dissociation (although it is more likely that these two sites are not equivalent c ). The doubly occupied closed receptor (A2R) opens with a rate of ␤2, and the doubly occupied open receptor (A2R*) closes with a rate of ␣2, while the singly occupied closed receptor (AR) opens with a rate of ␤1, and AR* closes with a rate of ␣1. Rates into and out of the desensitized state (D) are assigned as f and b. During neuromuscular transmission at the normal neuromuscular junction there is a transient saturating concentration of ACh in the synaptic cleft. The high concentration ensures

rapid binding of ACh to the two binding sites (reaching A2R), and the fast opening rate (␤2) ensures rapid subsequent channel opening (reaching A2R*), so that the miniature endplate current (MEPC) takes about 100 ␮s to reach its peak. At this point the concentration of free ACh in the synaptic cleft has fallen to near zero [due to rapid hydrolysis by acetylcholinesterase (AChE) and the binding of ACh to receptors], so there will be no re-occupancy of either binding site once ACh has dissociated. In this review, the entire period of openings and closings that occur from the start of the first opening, following binding of one or two molecules of ACh, to the end of the last opening (that which precedes dissociation of the last agonist molecule) is termed an activationd, but it is also known as a burst. The time constant for the decay of the MEPC is dictated by the duration of an activation. The occupancy, and therefore the contribution, of various states to the duration of the activation depends on the particular set of rate constants. Although in principle both desensitized states (D) and mono-liganded open states (AR*) could affect the duration of activations at the synapse, in practice they probably contribute very little to the the activation of wild-type ACh receptor during neuromuscular transmissiond,e. Thus, at the synapse, the duration of the activation of wildtype ACh receptor is dictated largely by the agonist dissociation rate (k–2), the channel opening rate (␤2) and the channel closing rate (␣2). Because k–2 and ␤2 are comparable, the receptor oscillates several times between the doubly occupied closed (A2R) and open (A2R*) states, before ACh dissociates. In the slow channel syndrome (SCS), the duration of channel activations is prolonged, resulting in a prolonged decay of the MEPC. This could result from an increase in the duration of each individual bi-liganded opening (A2R*) or an increase in the number of these openings per activation, or both. An increase in the number of openings per activation could result from a mutation that decreases the rate of dissociation of ACh from the receptor (slower k–2) or increases the channel opening rate (␤2), although this is already fast for the wild-type ACh receptor. The duration of individual openings within an activation could be increased by a mutation that stabilizes the open state, that is, reducing the rate of channel closure (slower ␣2). Although desensitized states and mono-liganded open states probably occur

A

B NH2

R

k –1

2k +1

Extracellular β1

Membrane M1

M2 M3

M4

AR

Intracellular 2k –2

ACh-receptor subunit

γ

ε

α δ

α β

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AR*

2k *–2

k *+2

β2

α

Foetal

β

ACh or α-bungarotoxin binding site

20

k +2

A2R

α δ

α1

Adult

b

f

D

α2

A2R*

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A rarely during the normal endplate current, this is not necessarily the case for mutant channels. For example, mutant channels might have a much higher tendency (fast f relative to ␤2 and k–2) to enter ‘short-lived’ (b comparable to ␣2) desensitized states during an activation. Thus, the receptor will oscillate between A2R and D and between A2R and A2R* before the agonist finally dissociates, thus prolonging the total duration of the activation after transient exposure to agonist. Desensitization of other ligand-gated ion channels has been proposed to shape the timecourse of other fast synaptic currentse. In principle, the duration of channel activations during synaptic transmission can be affected by any of the rates shown in the scheme, apart from the association rates (k+1, k+2, k*+2). Thus from a mechanistic point of view, one might predict that mutations in SCS would be in the ACh-binding pocket, or in regions involved in channel gating. This is discussed in detail below.

Structure–functional relationship of ACh receptor Recent studies have indicated the role of different parts of the receptor in ACh binding, channel gating and ion conductiona,b. The two ACh-binding sites are formed at the interface between the ␣ subunits and the adjacent ␦ and ␥ (or ⑀) subunits. Residues from several different regions of the ␣, ␦ and ␥ subunit N-terminus extracellular domains have been shown to contribute to the ligand-binding pocketa,b. The second transmembrane region (M2) of each subunit is thought to line the pore, although residues toward the extracellular end of M1 also contribute. Far less is known about the coupling between ACh binding and channel gating. Since the ACh-binding sites are distant from the pore itself, ACh binding must trigger channel opening via propagated conformational changes. Indeed Unwinf has shown that ACh binding results in small rotations of the subunits in the extracellular domain, leading to a change in configuration of the ␣-helices that line the pore (presumed to be M2). Conformational changes of M2 (and M1) during channel gating have also been deduced from differences in the accessibilities of residues in these domains in the presence and absence of ACh (Ref. a). Channel gating is altered by mutations engineered in membrane-spanning domains, including M1 (Ref. g), M2 (Refs h,i) and M4 (Refs j–l). However, participation in channel gating is not just restricted to extracellular and transmembrane domains. A direct involvement in channel gating has been observed for an intracellularly located segment of 30 amino acids between M3 and M4 of the ␥ and ⑀ subunitsl. This region is the main determinant of the difference in gating between the adult (␣2␤␦⑀) and foetal (␣2␤␦␥) forms of the receptor, the foetal form exhibiting longer open times. References a Karlin, A. and Akabas, M.H. (1995) Neuron 15, 1231–1244 b Hucho, F., Tsetlin, V.I. and Machold, J. (1996) Eur. J. Biochem. 239, 539–557 c Lingle, C.J., Maconochie, D. and Steinbach, J.H. (1992) J. Membr. Biol. 126, 195–217 d Edmonds, B., Gibb, A.J. and Colquhoun, D. (1995) Annu. Rev. Physiol. 57, 469– 493 e Jones, M.V. and Westbrook, G.L. (1996) Trends Neurosci. 19, 96–101 f Unwin, N. (1995) Nature 373, 37– 43 g Lo, D.C., Pinkham, J.L. and Stevens, C.F. (1991) Neuron 6, 31– 40 h Filatov, G.N. and White, M.M. (1995) Mol. Pharm. 48, 379–384 i Labarca, C. et al. (1995) Nature 376, 514–516 j Li, L. et al. (1992) Biophys. J. 62, 61–63 k Lee, Y-H. et al. (1994) Biophys. J. 66, 646–653 l Bouzat, C., Bren, N. and Sine, S.M. (1994) Neuron 13, 1395–1402

NH2 COOH Extracellular

Intracellular

α Gly 153 Ser Val 156 Met Asn 217 Lys Ser 269 Ile

M1

Refs 21,26 26 23 26

M2

M3

β

M4

ε

Refs

Leu 262 Met Val 266 Met

24 23

Refs

Thr 264 Pro Leu 269 Phe

22 23,25

B α

αS269I

4 pA 200 ms Fig. 3. Location and functional effects of slow channel syndrome (SCS) mutations. (A) Schematic representation of the location of SCS mutations in ACh-receptor subunits. Note that the amino acid numbers do not correspond to exactly the same position in each subunit, due to the slight variation in subunit length. (B) Homozygous expression of an SCS mutation in Xenopus oocytes. Single-channel currents were recorded from oocytes injected with cRNAs encoding wild-type human ␣ or ␣S269I subunits, together with human ␤, ␦ and ⑀ subunits. Channel openings are downward. Patches held at –100 mV, with 200 nM ACh and at 19–21°C, 1.5 kHz. Adapted from Ref. 26.

been identified in an SCS patient, have been generated29–31. The different transgenic mice demonstrated prolonged MEPPs (three- to tenfold depending on the mutation), and repetitive compound muscle action potentials after a single nerve stimulus. Some NMJ myopathy was evident in ␣L251T and ⑀L269F. Although weakness was not observed, the mouse has a high ‘safety factor’ for neuromuscular transmission (Box 1), and the lack of clinical signs does not in itself invalidate the model, which might prove useful for examining the link between prolonged receptor activations and neuromuscular dysfunction.

Concluding remarks The CMS are rare and heterogeneous. Many are sporadic or show reduced penetration, making it difficult to do genetic studies. Their study is complicated TINS Vol. 20, No. 1, 1997

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PERSPECTIVES

Acknowledgements We are very grateful to the Muscular Dystrophy Group/ Myasthenia Gravis Association (CN, DB, RC), and the Medical Research Council, for support, and to John Newsom-Davis and Alasdair Gibb for their helpful comments.

ON DISEASE

A. Vincent et al. – Genetic disorders of the NMJ

by the heterogeneity of the condition, with different mutations causing similar phenotypes, as now clearly seen in SCS. Nevertheless, with the existing state of our knowledge of the functional and structural proteins at the NMJ it should be possible to test for mutations in candidate genes for both presynaptic and postsynaptic conditions. Lessons learnt from the study of CMS should be of general relevance to genetic disorders of morecomplex synapses in the autonomic and central nervous systems. For example, the deg-3 (u662) mutation of Caenorhabditis elegans, which leads to the degeneration of a small subset of neurones, lies within the M2 region of a neuronal nicotinic ACh-receptor ␣ subunit, and is thought to generate excitotoxic prolonged receptor activations32. Similarities between SCS and the deg-3 mutant suggest a similar mechanism, and this in turn might be related to other excitotoxic conditions. Understanding how increased activations of ACh receptors can lead to an endplate myopathy might provide a basis for devising therapeutic intervention not only for SCS, but also for some morecommon excitotoxic neurodegenerative disorders. Selected references 1 Vincent, A. et al. (1995) J. Physiol. Paris 89, 129–136 2 Engel, A.G. (1993) Ann. New York Acad. Sci. 681, 425– 434

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3 Shillito, P., Vincent, A. and Newsom-Davis, J. (1993) Neuromuscular Disord. 3, 183–190 4 Engel, A.G. et al. (1981) Ann. New York Acad. Sci. 377, 614–639 5 Mora, M., Lambert, E.H. and Engel, A.G. (1987) Neurology 37, 206–214 6 Engel, A.G. et al. (1990) Prog. Brain Res. 84, 125–137 7 Hutchinson, D.O. et al. (1993) Brain 116, 633–653 8 Camp, S. et al. (1995) J. Clin. Invest. 95, 333–340 9 Vincent, A. et al. (1981) Muscle Nerve 4, 306–318 10 Vincent, A. et al. (1993) Ann. New York Acad. Sci. 681, 451– 460 11 Smit, L.M.E. et al. (1984) J. Neurol. Neurosurg. Psychiatry 47, 1091–1097 12 Wokke, J.H. et al. (1989) Neurology 39, 648–654 13 Noakes, P.G. et al. (1995) Nature 374, 258–262 14 Gautam, M. et al. (1996) Cell 85, 525–535 15 DeChiara, T.M. et al. (1996) Cell 85, 501–512 16 Gautam, M. et al. (1995) Nature 377, 232–236 17 Kleiman, R.J. and Reichardt, L.F. (1996) Cell 85, 461– 464 18 Ohno, K. et al. (1996) Neuron 17, 157–170 19 Engel, A.G. et al. (1982) Ann. Neurol. 11, 553–569 20 Oosterhuis, H.J. et al. (1987) Brain 110, 1061–1079 21 Sine, S.M. et al. (1995) Neuron 15, 229–239 22 Ohno, K. et al. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 758–762 23 Engel, A.G. et al. (1996) Hum. Mol. Genet. 5, 1217–1227 24 Gomez, C.M. et al. (1996) Ann. Neurol. 39, 712–723 25 Gomez, C.M. and Gammack, J.T. (1995) Neurology 45, 982–985 26 Newland, C. et al. (1996) J. Physiol. 495, 79P 27 Filatov, G.N. and White, M.M. (1995) Mol. Pharmacol. 48, 379–384 28 Labarca, C. et al (1995) Nature 376, 514–516 29 Gomez, C.M. et al. (1996) Muscle Nerve 19, 79–87 30 Gomez, C.M. et al. (1995) Soc. Neurosci. Abstr. 21, 1490 31 Gomez, C.M. et al. (1996) Neurology 46 (Suppl. 2), A310 32 Treinin, M. and Chalfie, M. (1995) Neuron 14, 871–877

TO THE EDITOR

Another variety of vision Petra Stoerig’s1 link between the loss of awareness in blindsight and lesions in the primary visual cortex and its connections with the extravisual cortex finds support in another kind of blindsight: the inhibition of visual processing during the disruption to visual input during blinks2, eye vergences3 and scans4. This temporary ‘blindness’ induced by visual masking1, possibly of efferent copies from eye movements5 and tactile reflexes6, stops the processing of visual blur during scans and eye vergences, and the onset and offset of occultation during blinks. This ‘functional lesioning’ of vision parallels blindsight in three ways. First, it is a real visual impairment (though we are unaware of our blindness – see below). During scans, eye vergences and eye blinks (even if occultation is prevented) vision is reduced greatly. Look in a mirror: because of this we cannot see our own eye movements even though we can readily see them in other people. It is significant – at least when these milliseconds of lost vision are added together. The average person makes 12–15 or so spontaneous blinks each minute, which block the pupil for 40–200 milliseconds7; given a 16-hour waking day this amounts to somewhere between 10–40 minutes. On top of this, the average person makes two to 22

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three eye scans each second, blurring vision for about 30 milliseconds: Irwin and Carson-Radvansky8 estimate that during a 16-hour waking day this occupies 60–90 minutes. So, even ignoring the time spent in vergence movements, people spend roughly one to two hours each day effectively blind. Second, as in blindsight some visual tasks are preserved during eye scans, such as the ability to point at displaced targets9 or hit targets10, yet, in contrast to such behavioural measures, subjects profess they are inaccurate11. For example, subjects that accurately point at targets, report, with echoes to cases of blindsight, that the targets have not moved1. Third, this blindsight also depends upon ‘lesions’ but of a functional kind to the visual system. Higher visual processes use areas of visual cortex. The suppression of sight during eye movements blocks activity in these areas. For example, during eye scans the priming of the mental rotation of Shepard’s stimuli stops8, as does the memory scanning in a Sternberg characterclassification task12, perceptual classifications13 and a counting task14. Furthermore, as David Irwin and Laura CarlsonRadvansky8 note, the sum of fixations upon a written word, gaze duration (an

important dependent variable in reading research), does not include saccade durations, suggesting that word processing stops as well. The main difference to blindsight is that we are not only blind but cannot even with deliberate attention bring this blindness into awareness. We are doubly unaware: unaware of being blind and unaware that in spite of this we can see. This resembles the case of the nonfoveal visual field, which is only illusionarily present in its detail. Part of the reason is also functional: when we stop visual processing in the visual cortex the posterior parietal cortex keeps us up to date with the retinal coordinates of visual space15. Likewise, during eye blinks, the posterior parietal cortex becomes active, and so maintains a stable visual world16. Stoerig suggests awareness links to ‘the integrity of the pathways and structures that are responsible for the many uses we can make of visual information’. This blocking of higher visual cognition during functional blindsight would fit Stoerig’s suggestion that awareness is not linked to visual processing but is linked to higher cognitive faculties. In the case of lesion blindsight this would be due to tissue damage to the primary visual cortex and its connections with the extravisual cortex; in the alternative functional blindsight described above it would be due to the suppression of visual cortex.