Tomoya Kubota

Native Chemical Ligation at Asx-Cys, Glx-Cys: Chemical Synthesis and High-Resolution X-ray Structure of ShK Toxin by Racemic Protein Crystallography.

We have re-examined the utility of native chemical ligation at -Gln/Glu-Cys- [Glx-Cys] and -Asn/Asp-Cys- [Asx-Cys] sites. Using the improved thioaryl catalyst 4-mercaptophenylacetic acid (MPAA), native chemical ligation could be performed at -Gln-Cys- and Asn-Cys- sites without side reactions. After optimization, ligation at a -Glu-Cys- site could also be used as a ligation site, with minimal levels of byproduct formation. However, -Asp-Cys- is not appropriate for use as a site for native chemical ligation because of formation of significant amounts of β-linked byproduct. The feasibility of native chemical ligation at -Gln-Cys- enabled a convergent total chemical synthesis of the enantiomeric forms of the ShK toxin protein molecule. The D-ShK protein molecule was ~50,000-fold less active in blocking the Kv1.3 channel than the L-ShK protein molecule. Racemic protein crystallography was used to obtain high-resolution X-ray diffraction data for ShK toxin. The structure was solved by direct methods and showed significant differences from the previously reported NMR structures in some regions of the ShK protein molecule.

A Kir3.4 mutation causes Andersen–Tawil syndrome by an inhibitory effect on Kir2.1

Objective: To identify other causative genes for Andersen–Tawil syndrome, which is characterized by a triad of periodic paralysis, cardiac arrhythmia, and dysmorphic features. Andersen–Tawil syndrome is caused in a majority of cases by mutations in KCNJ2, which encodes the Kir2.1 subunit of the inwardly rectifying potassium channel. Methods: The proband exhibited episodic flaccid weakness and a characteristic TU-wave pattern, both suggestive of Andersen–Tawil syndrome, but did not harbor KCNJ2 mutations. We performed exome capture resequencing by restricting the analysis to genes that encode ion channels/associated proteins. The expression of gene products in heart and skeletal muscle tissues was examined by immunoblotting. The functional consequences of the mutation were investigated using a heterologous expression system in Xenopus oocytes, focusing on the interaction with the Kir2.1 subunit. Results: We identified a mutation in the KCNJ5 gene, which encodes the G-protein–activated inwardly rectifying potassium channel 4 (Kir3.4). Immunoblotting demonstrated significant expression of the Kir3.4 protein in human heart and skeletal muscles. The coexpression of Kir2.1 and mutant Kir3.4 in Xenopus oocytes reduced the inwardly rectifying current significantly compared with that observed in the presence of wild-type Kir3.4. Conclusions: We propose that KCNJ5 is a second gene causing Andersen–Tawil syndrome. The inhibitory effects of mutant Kir3.4 on inwardly rectifying potassium channels may account for the clinical presentation in both skeletal and heart muscles.

New mutation of the Na channel in the severe form of potassium-aggravated myotonia.

Myotonia manifests in several hereditary diseases, including hyperkalemic periodic paralysis (HyperPP), paramyotonia congenita (PMC), and potassium‐aggravated myotonia (PAM). These are allelic disorders originating from missense mutations in the gene that codes the skeletal muscle sodium channel, Nav1.4. Moreover, a severe form of PAM has been designated as myotonia permanens. A new mutation of Nav1.4, Q1633E, was identified in a Japanese family presenting with the PAM phenotype. The proband suffered from cyanotic attacks during infancy. The mutated amino acid residue is located on the EF‐hand calcium‐binding motif in the intracellular C‐terminus. A functional analysis of the mutant channel using the voltage‐clamp method revealed disruption of fast inactivation, a slower rate of current decay, and a depolarized shift in the voltage dependence of availability. This study has identified a new mutation of PAM with a severe phenotype and emphasizes the importance of the C‐terminus for fast inactivation of the sodium channel. Muscle Nerve 39: 666–673, 2009

A mutation in a rare type of intron in a sodium-channel gene results in aberrant splicing and causes myotonia

Many mutations in the skeletal–muscle sodium‐channel gene SCN4A have been associated with myotonia and/or periodic paralysis, but so far all of these mutations are located in exons. We found a patient with myotonia caused by a deletion/insertion located in intron 21 of SCN4A, which is an AT‐AC type II intron. This is a rare class of introns that, despite having AT‐AC boundaries, are spliced by the major or U2‐type spliceosome. The patients skeletal muscle expressed aberrantly spliced SCN4A mRNA isoforms generated by activation of cryptic splice sites. In addition, genetic suppression experiments using an SCN4A minigene showed that the mutant 5′ splice site has impaired binding to the U1 and U6 snRNPs, which are the cognate factors for recognition of U2‐type 5′ splice sites. One of the aberrantly spliced isoforms encodes a channel with a 35‐amino acid insertion in the cytoplasmic loop between domains III and IV of Nav1.4. The mutant channel exhibited a marked disruption of fast inactivation, and a simulation in silico showed that the channel defect is consistent with the patients myotonic symptoms. This is the first report of a disease‐associated mutation in an AT‐AC type II intron, and also the first intronic mutation in a voltage‐gated ion channel gene showing a gain‐of‐function defect. Hum Mutat 32:1–10, 2011.

Aberrantly spliced α-dystrobrevin alters α-syntrophin binding in myotonic dystrophy type 1

Background: Myotonic dystrophy type 1 (DM1) is a multisystemic disorder caused by a CTG repeat expansion in the DMPK gene. Aberrant messenger RNA (mRNA) splicing of several genes has been reported to explain some of the symptoms in DM1, but the cause of muscle wasting is still unknown. By contrast, many forms of muscular dystrophy are caused by abnormalities of the dystrophin–glycoprotein complex (DGC). α-Dystrobrevin is a key component of the DGC in striated muscle and plays important roles in maturation and signal transduction by interacting with α-syntrophin. The goal of this study was to investigate alternative splicing of α-dystrobrevin in DM1 and examine α-syntrophin binding of different α-dystrobrevin splice isoforms. Methods: Splicing patterns of α-dystrobrevin in DM1 muscle were studied by reverse-transcriptase PCR. Expression of the variant splice isoform was examined by immunoblotting and immunohistochemistry. Alternatively spliced isoforms were expressed in cultured cells to investigate interaction with α-syntrophin. α-Syntrophin expression was examined by immunoblotting. Results: α-Dystrobrevin mRNA including exons 11A and 12 was increased in both skeletal and cardiac muscle of DM1 patients. The aberrantly spliced α-dystrobrevin isoform was localized to the sarcolemma, and showed increased binding with α-syntrophin. Furthermore, levels of α-syntrophin associated with the DGC were increased in DM1 muscle. Conclusion: Alternative splicing of α-dystrobrevin is dysregulated in myotonic dystrophy type 1 (DM1) muscle, resulting in changes in α-syntrophin binding. These results raise the possibility that effects on α-dystrobrevin splicing may influence signaling in DM1 muscle cells. GLOSSARY: α-DB = α-dystrobrevin; α-syn = α-syntrophin; ALS = amyotrophic lateral sclerosis; β-DG = β-dystroglycan; CC = coiled-coil domain; cDM = congenital myotonic dystrophy type 1; cDNA = complementary DNA; Cont = control; DAPC = dystrophin-associated protein complex; DBS = dystrophin binding site; DGC = dystrophin–glycoprotein complex; DM1 = myotonic dystrophy type 1; EF = EF hand region; GAPDH = glyceraldehyde 3-phosphate dehydrogenase; IP = immunoprecipitation; LGMD = limb-girdle muscular dystrophy; MDRS = muscular disability rating scale; mRNA = messenger RNA; NA = not available; NMJ = neuromuscular junction; NT = not tested; P = postnatal day; PM = polymyositis; RT-PCR = reverse-transcriptase PCR; SBS = syntrophin binding site; SDS-PAGE = sodium dodecyl sulfate polyacrylamide gel electrophoresis; TBS = Tris-buffered saline; WCL = whole-cell lysate; vr = variable region; Y = unique tyrosine kinase substrate domain; ZZ = zinc-binding domain.

Total Chemical Synthesis of Biologically Active Fluorescent Dye‐Labeled Ts1 Toxin

Ts1 toxin is a protein found in the venom of the Brazilian scorpion Tityus serrulatus. Ts1 binds to the domain II voltage sensor in the voltage-gated sodium channel Nav and modifies its voltage dependence. In the work reported here, we established an efficient total chemical synthesis of the Ts1 protein using modern chemical ligation methods and demonstrated that it was fully active in modifying the voltage dependence of the rat skeletal muscle voltage-gated sodium channel rNav1.4 expressed in oocytes. Total synthesis combined with click chemistry was used to label the Ts1 protein molecule with the fluorescent dyes Alexa-Fluor 488 and Bodipy. Dye-labeled Ts1 proteins retained their optical properties and bound to and modified the voltage dependence of the sodium channel Nav. Because of the highly specific binding of Ts1 toxin to Nav, successful chemical synthesis and labeling of Ts1 toxin provides an important tool for biophysical studies, histochemical studies, and opto-pharmacological studies of the Nav protein.

Mapping of voltage sensor positions in resting and inactivated mammalian sodium channels by LRET

Significance Physical activities of our body and extremities are achieved by the propagation of electrical signals called action potentials from our brain, through nerves, to skeletal muscles. Voltage-gated sodium channel (Navs) play essential roles in the generation and propagation of action potentials in such excitable cells. Although mammalian Nav function has been studied comprehensively, the precise structural basis for the gating mechanisms has not been fully clarified. In this study, we have used lanthanide-based resonance energy transfer to obtain dynamic structural information in mammalian Nav gating. Our data define a geometrical map of Navs with the bound toxins and reveal voltage-induced structural changes related to channel gating, which lead us further toward an understanding of the gating mechanism in mammalian Navs. Voltage-gated sodium channels (Navs) play crucial roles in excitable cells. Although vertebrate Nav function has been extensively studied, the detailed structural basis for voltage-dependent gating mechanisms remain obscure. We have assessed the structural changes of the Nav voltage sensor domain using lanthanide-based resonance energy transfer (LRET) between the rat skeletal muscle voltage-gated sodium channel (Nav1.4) and fluorescently labeled Nav1.4-targeting toxins. We generated donor constructs with genetically encoded lanthanide-binding tags (LBTs) inserted at the extracellular end of the S4 segment of each domain (with a single LBT per construct). Three different Bodipy-labeled, Nav1.4-targeting toxins were synthesized as acceptors: β-scorpion toxin (Ts1)-Bodipy, KIIIA-Bodipy, and GIIIA-Bodipy analogs. Functional Nav-LBT channels expressed in Xenopus oocytes were voltage-clamped, and distinct LRET signals were obtained in the resting and slow inactivated states. Intramolecular distances computed from the LRET signals define a geometrical map of Nav1.4 with the bound toxins, and reveal voltage-dependent structural changes related to channel gating.

Probing α-310 Transitions in a Voltage-Sensing S4 Helix

The S4 helix of voltage sensor domains (VSDs) transfers its gating charges across the membrane electrical field in response to changes of the membrane potential. Recent studies suggest that this process may occur via the helical conversion of the entire S4 between α and 310 conformations. Here, using LRET and FRET, we tested this hypothesis by measuring dynamic changes in the transmembrane length of S4 from engineered VSDs expressed in Xenopus oocytes. Our results suggest that the native S4 from the Ciona intestinalis voltage-sensitive phosphatase (Ci-VSP) does not exhibit extended and long-lived 310 conformations and remains mostly α-helical. Although the S4 of NavAb displays a fully extended 310 conformation in x-ray structures, its transplantation in the Ci-VSP VSD scaffold yielded similar results as the native Ci-VSP S4. Taken together, our study does not support the presence of long-lived extended α-to-310 helical conversions of the S4 in Ci-VSP associated with voltage activation.

A novel mutation in SCN4A causes severe myotonia and school-age-onset paralytic episodes

Mutations in the pore-forming subunit of the skeletal muscle sodium channel (SCN4A) are responsible for hyperkalemic periodic paralysis, paramyotonia congenita and sodium channel myotonia. These disorders are classified based on their cardinal symptoms, myotonia and/or paralysis. We report the case of a Japanese boy with a novel mutation of SCN4A, p.I693L, who exhibited severe episodic myotonia from infancy and later onset mild paralytic attack. He started to have apneic episodes with generalized hypertonia at age of 11 months, then developed severe episodic myotonia since 2 years of age. He presented characteristic generalized features which resembled Schwarz-Jampel syndrome. After 7 years old, paralytic episodes occurred several times a year. The compound muscle action potential did not change during short and long exercise tests. Functional analysis of the mutant channel expressed in cultured cell revealed enhancement of the activation and disruption of the slow inactivation, which were consistent with myotonia and paralytic attack. The severe clinical features in his infancy may correspond to myotonia permanence, however, he subsequently experienced paralytic attacks. This case provides an example of the complexity and overlap of the clinical features of sodium channel myotonic disorders.

Elucidation of the Covalent and Tertiary Structures of Biologically Active Ts3 Toxin

Ts3 is an alpha scorpion toxin from the venom of the Brazilian scorpion Tityus serrulatus. Ts3 binds to the domain IV voltage sensor of voltage-gated sodium channels (Nav ) and slows down their fast inactivation. The covalent structure of the Ts3 toxin is uncertain, and the structure of the folded protein molecule is unknown. Herein, we report the total chemical synthesis of four candidate Ts3 toxin protein molecules and the results of structure-activity studies that enabled us to establish the covalent structure of biologically active Ts3 toxin. We also report the synthesis of the mirror image form of the Ts3 protein molecule, and the use of racemic protein crystallography to determine the folded (tertiary) structure of biologically active Ts3 toxin by X-ray diffraction.