Joan M. Brengman

Congenital Myasthenic Syndrome Caused by Decreased Agonist Binding Affinity Due to a Mutation in the Acetylcholine Receptor ε Subunit

We describe the genetic and kinetic defects for a low-affinity fast channel disease of the acetylcholine receptor (AChR) that causes a myasthenic syndrome. In two unrelated patients with very small miniature end plate (EP) potentials, but with normal EP AChR density and normal EP ultrastructure, patch-clamp studies demonstrated infrequent AChR channel events, diminished channel reopenings during ACh occupancy, and resistance to desensitization by ACh. Each patient had two heteroallelic AChR epsilon subunit gene mutations: a common epsilon P121L mutation, a signal peptide mutation (epsilon G-8R) (patient 1), and a glycosylation consensus site mutation (epsilon S143L) (patient 2). AChR expression in HEK fibroblasts was normal with epsilon P121L but was markedly reduced with the other mutations. Therefore, epsilon P121L defines the clinical phenotype. Studies of the engineered epsilon P121L AChR revealed a markedly decreased rate of channel opening, little change in affinity of the resting state for ACh, but reduced affinity of the open channel and desensitized states.

Rapsyn Mutations in Humans Cause Endplate Acetylcholine-Receptor Deficiency and Myasthenic Syndrome

Congenital myasthenic syndromes (CMSs) stem from genetic defects in endplate (EP)-specific presynaptic, synaptic, and postsynaptic proteins. The postsynaptic CMSs identified to date stem from a deficiency or kinetic abnormality of the acetylcholine receptor (AChR). All CMSs with a kinetic abnormality of AChR, as well as many CMSs with a deficiency of AChR, have been traced to mutations in AChR-subunit genes. However, in a subset of patients with EP AChR deficiency, the genetic defect has remained elusive. Rapsyn, a 43-kDa postsynaptic protein, plays an essential role in the clustering of AChR at the EP. Seven tetratricopeptide repeats (TPRs) of rapsyn subserve self-association, a coiled-coil domain binds to AChR, and a RING-H2 domain associates with beta-dystroglycan and links rapsyn to the subsynaptic cytoskeleton. Rapsyn self-association precedes recruitment of AChR to rapsyn clusters. In four patients with EP AChR deficiency but with no mutations in AChR subunits, we identify three recessive rapsyn mutations: one patient carries L14P in TPR1 and N88K in TPR3; two are homozygous for N88K; and one carries N88K and 553ins5, which frameshifts in TPR5. EP studies in each case show decreased staining for rapsyn and AChR, as well as impaired postsynaptic morphological development. Expression studies in HEK cells indicate that none of the mutations hinders rapsyn self-association but that all three diminish coclustering of AChR with rapsyn.

Analysis of cytokine expression in muscle in inflammatory myopathies, Duchenne dystrophy, and non-weak controls

We investigated the profiles of cytokine mRNA expression in muscle in 15 cases of inflammatory myopathy (IM) (5 each of polymyositis, inclusion body myositis, and dermatomyositis) and in 10 controls (5 of Duchenne dystrophy and 5 non-weak subjects). Expressions of the predominantly T cell-derived cytokines (interleukin (IL)-2, IL-4, IL-5, and interferon-gamma (IFN-gamma), of the predominantly macrophage-derived cytokines (IL-1, IL-6, and tumor necrosis factor-alpha (TNF-alpha)), as well as cytokines that can be of either T cell or macrophage origin (granulocyte-macrophage colony stimulating factor (GM-CSF) and transforming growth factor beta 1 (TGF-beta 1) and TGF-beta 2), were monitored by the reverse transcriptase-PCR method. The expression of T cell cytokine mRNAs for IL-2, IL-5, and IFN-gamma was generally weak or inconsistent. IL-4 mRNA expression was consistently moderate to strong in polymyositis but generally weak or absent in the other IMs. The expression of macrophage cytokine mRNAs for IL-1 alpha and IL-1 beta was weak or absent in all cases. Variable TNF-alpha mRNA expression was observed in 12 of 15 IM cases and faint or weak expression in 5 of 10 controls. Very strong GM-CSF expression was detected, but only on boosted PCR, in 12 of 15 cases of IM but in none of the controls. IL-6 was expressed only weakly or inconsistently. In contrast to the variable expression of several of the above mentioned cytokine mRNAs, all IM specimens strongly expressed TGF-beta 1 mRNA and 12 of 15 strongly expressed TGF-beta 2 mRNA. Thus, with the exception of IL-4 expression in polymyositis, a similar pattern of cytokine mRNA expression exists in the different types of IMs. Moreover, this pattern resembles that detected in non-weak and DD controls, although expression is generally weaker in the non-weak controls. The findings suggest that in IM muscle a sustained secretion of cytokines by T cells or of IL-1 by macrophages is not a prerequisite for operation of the immune effector response and that muscle may not be the site of ongoing sensitization.

Acetylcholine receptor M3 domain: stereochemical and volume contributions to channel gating.

By defining the functional defect in a congenital myasthenic syndrome (CMS), we show that the third transmembrane domain (M3) of the muscle acetylcholine receptor governs the speed and efficiency of gating of its channel. The clinical phenotype of this CMS results from the mutation V285I in M3 of the α subunit, which attenuates endplate currents, accelerates their decay and causes abnormally brief acetylcholine-induced single-channel currents. Kinetic analysis of engineered αV285I receptors demonstrated a predominant effect on channel gating, with abnormally slow opening and rapid closing rates. Analysis of site-directed mutations revealed stereochemical and volume-dependent contributions of αV285 to channel gating. Thus, we demonstrate a functional role for the M3 domain as a key component of the nicotinic acetylcholine receptor channel-gating mechanism.By defining the functional defect in a congenital myasthenic syndrome (CMS), we show that the third transmembrane domain (M3) of the muscle acetylcholine receptor governs the speed and efficiency of gating of its channel. The clinical phenotype of this CMS results from the mutation V285I in M3 of the α subunit, which attenuates endplate currents, accelerates their decay and causes abnormally brief acetylcholine-induced single-channel currents. Kinetic analysis of engineered αV285I receptors demonstrated a predominant effect on channel gating, with abnormally slow opening and rapid closing rates. Analysis of site-directed mutations revealed stereochemical and volume-dependent contributions of αV285 to channel gating. Thus, we demonstrate a functional role for the M3 domain as a key component of the nicotinic acetylcholine receptor channel-gating mechanism.

The spectrum of mutations causing end-plate acetylcholinesterase deficiency.

The end‐plate species of acetylcholinesterase (AChE) is an asymmetric enzyme consisting of a collagenic tail subunit composed of three collagenic strands (ColQ), each attached to a tetramer of the T isoform of the catalytic subunit (AChET) via a proline‐rich attachment domain. The principal function of the tail subunit is to anchor asymmetric AChE in the synaptic basal lamina. Human end‐plate AChE deficiency was recently shown to be caused by mutations in COLQ. We here report nine novel COLQ mutations in 7 patients with end‐plate AChE deficiency. We examine the effects of the mutations on the assembly of asymmetric AChE by coexpressing each genetically engineered COLQ mutant with ACHET in COS cells. We classify the newly recognized and previously reported COLQ mutations into four classes according to their position in ColQ and their effect on AChE expression. We find that missense mutations in the proline‐rich attachment domain abrogate attachment of catalytic subunits, that truncation mutations in the ColQ collagen domain prevent the assembly of asymmetric AChE, that hydrophobic missense residues in the C‐terminal domain prevent triple helical assembly of the ColQ collagen domain, and that other mutations in the C‐terminal region produce asymmetric species of AChE that are likely insertion incompetent. Ann Neurol 2000;47:162–170.

Differential vulnerability of microtubule components in cerebral ischemia

SummaryDifferential vulnerability of the major components of microtubules was examined in ischemic gerbil brains by a light microscopic, immunohisto-chemical method using monoclonal antibodies for microtubule-associated protein (MAP) 1A and MAP2, polyclonal antibody for MAP1 and 2 as well as monoclonal antibody for α-tubulin. Progressive cerebral ischemia during unilateral carotid occlusion for 5, 15 and 120 min and reperfusion for 3, 12 and 48 h following bilateral carotid occlusion for 10 min were studied. Ischemic lesions in the subiculum-CA1 region were visualized by all antibodies after ischemia for 5 min but the antibody for α-tubulin was less sensitive. The antibody for α-tubulin was also less sensitive than antibodies for MAPs for detection of early postischemic lesions. Differential sensitivity was also observed in the cerebral cortex and other brain regions. Microtubules in myelinated axons were more stable than those in dendrites. The observed loss of immunohistochemical reactivities for MAPs and α-tubulin may have been caused by activation of calcium-dependent proteolytic enzymes such as calpains. The discrepancy between MAPs and α-tubulin could be due to differences in affinities or topographic distributions of these proteins within microtubules.

Congenital end-plate acetylcholinesterase deficiency caused by a nonsense mutation and an A-->G splice-donor-site mutation at position +3 of the collagenlike-tail-subunit gene (COLQ): how does G at position +3 result in aberrant splicing?

Congenital end-plate acetylcholinesterase (AChE) deficiency (CEAD), the cause of a disabling myasthenic syndrome, arises from defects in the COLQ gene, which encodes the AChE triple-helical collagenlike-tail subunit that anchors catalytic subunits of AChE to the synaptic basal lamina. Here we describe a patient with CEAD with a nonsense mutation (R315X) and a splice-donor-site mutation at position +3 of intron 16 (IVS16+3A-->G) of COLQ. Because both A and G are consensus nucleotides at the +3 position of splice-donor sites, we constructed a minigene that spans exons 15-17 and harbors IVS16+3A-->G for expression in COS cells. We found that the mutation causes skipping of exon 16. The mutant splice-donor site of intron 16 harbors five discordant nucleotides (at -3, -2, +3, +4, and +6) that do not base-pair with U1 small-nuclear RNA (snRNA), the molecule responsible for splice-donor-site recognition. Versions of the minigene harboring, at either +4 or +6, nucleotides complementary to U1 snRNA restore normal splicing. Analysis of 1,801 native splice-donor sites reveals that presence of a G nucleotide at +3 is associated with preferential usage, at positions +4 to +6, of nucleotides concordant to U1 snRNA. Analysis of 11 disease-associated IVS+3A-->G mutations indicates that, on average, two of three nucleotides at positions +4 to +6 fail to base-pair, and that the nucleotide at +4 never base-pairs, with U1 snRNA. We conclude that, with G at +3, normal splicing generally depends on the concordance that residues at +4 to +6 have with U1 snRNA, but other cis-acting elements may also be important in assuring the fidelity of splicing.

Mutation causing congenital myasthenia reveals acetylcholine receptor β/δ subunit interaction essential for assembly

We describe a severe postsynaptic congenital myasthenic syndrome with marked endplate acetylcholine receptor (AChR) deficiency caused by 2 heteroallelic mutations in the β subunit gene. One mutation causes skipping of exon 8,truncating the β subunit before its M1 transmembrane domain, and abolishing surface expression of pentameric AChR. The other mutation, a 3-codon deletion (β426delEQE) in the long cytoplasmic loop between the M3 and M4 domains, curtails but does not abolish expression. By coexpressing β426delEQE with combinations of wild-type subunits in 293 HEK cells, we demonstrate that β426delEQE impairs AChR assembly by disrupting a specific interaction between β and δ subunits. Studies with related deletion and missense mutants indicate that secondary structure in this region of the β subunit is crucial for interaction with the δ subunit. The findings imply that the mutated residues are positioned at the interface between β and δ subunits and demonstrate contribution of this local region of the long cytoplasmic loop to AChR assembly. J. Clin. Invest. 104:1403‐1410 (1999).

Mutation causing severe myasthenia reveals functional asymmetry of AChR signature cystine loops in agonist binding and gating

We describe a highly disabling congenital myasthenic syndrome (CMS) associated with rapidly decaying, low-amplitude synaptic currents, and trace its cause to a valine to leucine mutation in the signature cystine loop (cys-loop) of the AChR alpha subunit. The recently solved crystal structure of an ACh-binding protein places the cys-loop at the junction between the extracellular ligand-binding and transmembrane domains where it may couple agonist binding to channel gating. We therefore analyzed the kinetics of ACh-induced single-channel currents to identify elementary steps in the receptor activation mechanism altered by the alphaV132L mutation. The analysis reveals that alphaV132L markedly impairs ACh binding to receptors in the resting closed state, decreasing binding affinity for the second binding step 30-fold, but attenuates gating efficiency only about twofold. By contrast, mutation of the equivalent valine residue in the delta subunit impairs channel gating approximately fourfold with little effect on ACh binding, while corresponding mutations in the beta and epsilon subunits are without effect. The unique functional contribution of the alpha subunit cys-loop likely owes to its direct connection via a beta strand to alphaW149 at the center of the ligand-binding domain. The overall findings reveal functional asymmetry between cys-loops of the different AChR subunits in contributing to ACh binding and channel gating.

Quinidine normalizes the open duration of slow-channel mutants of the acetylcholine receptor

QUINIDINE is a long-lived open-channel blocker of the wild-type endplate acetylcholine receptor (AChR). To test the hypothesis that quinidine can normalize the prolonged channel opening events of slow-channel mutants of human AChR, we expressed wild-type AChR and five well characterized slow-channel mutants of AChR in HEK 293 cells and monitored the effects of quinidine on acetylcholine-induced channel currents. Quinidine shortens the longest component of channel opening burst (τ3b) of both wild-type and mutant AChRs in a concentration-dependent manner, and 5 μM quini-dine reduces τ3b of the mutant AChRs to that of wild-type AChRs in the absence of quinidine. Because this concentration of quinidine is attainable in clinical practice, the findings predict a therapeutic effect for quinidine in the slow-channel congenital myasthenic syndrome.