Early events in node of Ranvier formation during myelination and remyelination in the PNS

Schafer, Dorothy P.; Custer, Andrew W.; Shrager, Peter; Rasband, Matthew N.

doi:10.1017/s1740925x06000093

Cited by 73 publications

(70 citation statements)

References 40 publications

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“…Experiments are in progress to clarify this point. Meanwhile, the fact that CssII affects Nav1.6, which has been detected during developmental myelination and during remyelination at the node of Ranviers in the peripheral nervous system [30], correlates well with the observation that CssII cause most of the subcutaneous toxicity of the sting of C.s. suffusus.…”

Section: Biological Activitysupporting

confidence: 70%

Four disulfide-bridged scorpion beta neurotoxin CssII: Heterologous expression and proper folding in vitro

Estévez

García

Schiavon

et al. 2007

Biochimica et Biophysica Acta (BBA) - General Subjects

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The gene of the four disulfide-bridged Centruroides suffusus suffusus toxin II was cloned into the expression vector pQE30 containing a 6His-tag and an FXa proteolytic cleavage region. This recombinant vector was transfected into E. coli BL21 cells and expressed under induction with isopropyl thiogalactoside (IPTG). The level of expression was 24.6 mg/L of culture medium, and the His tagged recombinant toxin (HisrCssII) was found exclusively in inclusion bodies. After solubilization the HisrCssII peptide was purified by affinity and hydrophobic interaction chromatography. The reverse-phase HPLC profile of the HisrCssII product obtained from the affinity chromatography step showed several peptide fractions having the same molecular mass of 9,392.6 Da, indicating that HisrCssII was oxidized forming several distinct disulfide bridge arrangements. The multiple forms of HisrCssII after reduction eluted from the column as a single protein component of 9,400.6 Da. Similarly, an in vitro folding of the reduced HisrCssII generated a single oxidized component of HisrCssII, which was cleaved by the proteolytic enzyme FXa to the recombinant CssII (rCssII). The molecular mass of rCssII was 7,538.6 Da as expected. Since native CssII is amidated at the C-terminal residue whereas the rCssII is heterologously expressed in the format of free carboxyl end, there is a difference of 1Da, when comparing both peptides (native versus heterologously expressed). Nevertheless, they show similar toxicity when injected intracranially into mice, and both nCssII and rCssII show the typical electrophysiological properties of beta-toxins in Na v 1.6 channels, which is for the first time demonstrated here. Binding and displacement experiments conducted with radiolabelled CssII confirms the electrophysiological results. Several problems associated with the heterologously expressed toxins containing four disulfide bridges are discussed.

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Section: Biological Activitysupporting

confidence: 70%

Four disulfide-bridged scorpion beta neurotoxin CssII: Heterologous expression and proper folding in vitro

Estévez

García

Schiavon

et al. 2007

Biochimica et Biophysica Acta (BBA) - General Subjects

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“…In the sciatic nerve, paranodes begin to form as early as P0 -P1 (Rios et al, 2000;Schafer et al, 2006). We found that, at approximately P2, the cytoskeletal proteins ␣II spectrin, ␤II spectrin, and protein 4.1B began to accumulate at sites flanking nodal clusters of ␤IV spectrin or Nav channels (Fig.…”

Section: The Paranodal Cytoskeleton Remains Intact After Myelin Retramentioning

confidence: 71%

Spectrins and AnkyrinB Constitute a Specialized Paranodal Cytoskeleton

Ogawa¹,

Schafer²,

Horresh³

et al. 2006

J. Neurosci.

149

169

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Paranodal junctions of myelinated nerve fibers are important for saltatory conduction and function as paracellular and membrane protein diffusion barriers flanking nodes of Ranvier. The formation of these specialized axoglial contacts depends on the presence of three cell adhesion molecules: neurofascin 155 on the glial membrane and a complex of Caspr and contactin on the axon. We isolated axonal and glial membranes highly enriched in these paranodal proteins and then used mass spectrometry to identify additional proteins associated with the paranodal axoglial junction. This strategy led to the identification of three novel components of the paranodal cytoskeleton: ankyrinB, ␣II spectrin, and ␤II spectrin. Biochemical and immunohistochemical analyses revealed that these proteins associate with protein 4.1B in a macromolecular complex that is concentrated at central and peripheral paranodal junctions in the adult and during early myelination. Furthermore, we show that the paranodal localization of ankyrinB is disrupted in Caspr-null mice with aberrant paranodal junctions, demonstrating that paranodal neuron-glia interactions regulate the organization of the underlying cytoskeleton. In contrast, genetic disruption of the juxtaparanodal protein Caspr2 or the nodal cytoskeletal protein ␤IV spectrin did not alter the paranodal cytoskeleton. Our results demonstrate that the paranodal junction contains specialized cytoskeletal components that may be important to stabilize axon-glia interactions and contribute to the membrane protein diffusion barrier found at paranodes.

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“…Spontaneous firing was observed in 41% and 48% of TRG neurons transfected with Met136Val and WT channels, respectively (Met136Val, 20/49 cells; WT, 27/56 cells, p = 0.40; Figure 5C). TRG myelinated fibers of the central and peripheral nervous system (34)(35)(36)(37). Na V 1.6 is also expressed in small DRG neurons that produce unmyelinated C-fibers, and total loss of this channel in DRG neurons slows C-fiber conduction in the sciatic nerve (38), suggesting that it contributes to the transmission of noxious signals in these fibers.…”

Section: Resurgent Current Of Met136val Channel In Trg Neuronsmentioning

confidence: 99%