Colloquium 9: Structural and Molecular Dissection of the Node of Ranvier. Sodium channel β subunits: multifunctional molecules at the node of Ranvier

Voltage‐gated sodium channels are unique in that they combine action potential conduction with cell adhesion. Mammalian sodium channels are heterotrimers, composed of a central, pore‐forming α subunit and two auxiliary β subunits. The α subunits are members of a large gene family containing the voltage‐gated sodium, potassium, and calcium channels. Sodium channel α subunits form a gene subfamily with at least 11 members. Mutations in sodium channel α subunit genes have been linked to paroxysmal disorders such as epilepsy, long QT syndrome (LQT), and hyperkalemic periodic paralysis in humans, and motor endplate disease and cerebellar ataxia in mice. Three genes encode the sodium channel β subunits with at least one alternative splice product. Unlike the pore‐forming α subunits, the sodium channel β subunits are not structurally related to β subunits of calcium and potassium channels. Sodium channel β subunits are multifunctional. They modulate channel gating and regulate the level of channel expression at the plasma membrane. We have shown that β subunits also function as cell adhesion molecules (CAMs) in terms of interaction with extracellular matrix molecules, regulation of cell migration, cellular aggregation, and interaction with the cytoskeleton. A mutation in SCN1B has been shown to cause GEFS + 1 epilepsy in human families. We propose that the sodium channel signalling complex at nodes of Ranvier involves β subunits as channel modulators as well as CAMs, other CAMs such as neurofascin and contactin, RPTPβ, and extracellular matrix molecules such as tenascin.     

Molecular cloning and characterization of LKv1, a novel voltage‐gated potassium channel in leech

We have cloned a novel voltage‐gated K channel, LKv1, in two species of leech. The properties of LKv1 expressed in transiently transfected HEK293 cells is that of a delayed rectifier current. LKv1 may be a major modulator of excitability in leech neurons, since antibody localization studies show that LKv1 is expressed in the soma and axons of all neurons in both the central and peripheral nervous systems. Comparison of the biophysical and pharmacological properties of LKv1 with native voltage‐gated conductances in leech neurons suggests that LKv1 may correspond to the previously characterized delayed rectifier current, I_K. Phylogenetic analysis of LKv1 shows that it is related to the Shaker subfamily of voltage‐gated K channels although it occupies a separate branch from that of the monophyletic Shaker clade composed of the flatworm, Aplysia, Drosophila, and mammalian Shaker homologs as well as from that of two recently identified Shaker‐related K channels in jellyfish. Thus, this analysis indicates that this group of voltage‐gated K channels contains several evolutionarily divergent lineages. © 1999 John Wiley & Sons, Inc. J Neurobiol 38: 287–299, 1999     

Cell‐specific changes in expression of mRNAs encoding splice variants of Aplysia cell adhesion molecule accompany long‐term synaptic plasticity

Aplysia neurons express several splice variants of apCAM, a member of the Ig superfamily of cell adhesion molecules. The major transmembrane isoform is endocytosed in sensory neurons (SNs) during the early phases of long‐term facilitation (LTF) of SN synapses evoked by serotonin (5‐HT) or in the motor neuron L7 during the early phases of long‐term depression (LTD) of SN synapses evoked by Phe‐Met‐Arg‐Phe‐amide (FMRFa). We used single cell RT‐PCR to evaluate whether expression of mRNAs encoding for different apCAM isoforms in SNs and L7 is regulated during LTF produced by 5‐HT, and LTD produced by FMRFa. Single SNs and L7s express mRNAs encoding for all major isoforms, but the proportion of each isoform expressed differs for the two cells. SN expresses more mRNA encoding for GPI‐linked isoforms, while L7 expresses more mRNA encoding for the major transmembrane isoform. The neuromodulators produced significant changes in the proportional levels of mRNAs encoding for specific apCAM isoforms during the first 4 h after treatments without affecting overall levels of apCAM mRNA. 5‐HT evoked changes that exaggerated cell‐specific differences in isoform expression. FMRFa evoked changes that reduced cell‐specific differences in isoform expression. The effects of the neuromodulators on apCAM mRNA expression were not detected when cells were cultured alone or when SNs were cocultured with another motor cell that failed to induce synapse formation (L11). The results suggest that rapid cell‐specific regulation of splice variant expression may contribute to different forms of long‐term synaptic plasticity. © 2000 John Wiley & Sons, Inc. J Neurobiol 45: 152–161, 2000     

Profiling of N‐glycosylation gene expression in CHO cell fed‐batch cultures

One of the goals of recombinant glycoprotein production is to achieve consistent glycosylation. Although many studies have examined the changes in the glycosylation quality of recombinant protein with culture, very little has been done to examine the underlying changes in glycosylation gene expression as a culture progresses. In this study, the expression of 24 genes involved in N‐glycosylation were examined using quantitative RT PCR to gain a better understanding of recombinant glycoprotein glycosylation during production processes. Profiling of the N‐glycosylation genes as well as concurrent analysis of glycoprotein quality was performed across the exponential, stationary and death phases of a fed‐batch culture of a CHO cell line producing recombinant human interferon‐γ (IFN‐γ). Of the 24 N‐glycosylation genes examined, 21 showed significant up‐ or down‐regulation of gene expression as the fed‐batch culture progressed from exponential, stationary and death phase. As the fed‐batch culture progressed, there was also an increase in less sialylated IFN‐γ glycoforms, leading to a 30% decrease in the molar ratio of sialic acid to recombinant IFN‐γ. This correlated with decreased expression of genes involved with CMP sialic acid synthesis coupled with increased expression of sialidases. Compared to batch culture, a low glutamine fed‐batch strategy appears to need a 0.5 mM glutamine threshold to maintain similar N‐glycosylation genes expression levels and to achieve comparable glycoprotein quality. This study demonstrates the use of quantitative real time PCR method to identify possible “bottlenecks” or “compromised” pathways in N‐glycosylation and subsequently allow for the development of strategies to improve glycosylation quality. Biotechnol. Bioeng. 2010;107: 516–528. © 2010 Wiley Periodicals, Inc.     

Role of nova‐1 in regulating α2N,a novel glycine receptor splice variant,in developing spinal cord neurons

Inhibitory glycine receptor (GlyR) subunits undergo developmental regulation, but the molecular mechanisms of GlyR regulation in developing neurons are little understood. Using RT‐PCR, we investigated the regulation of GlyR α‐subunit splice forms during the development of the spinal cord of the rat. Experiments to compare the amounts of mRNA for two known splice variants of the GlyR α2 subunit, α2A and α2B, in the developing rat spinal cord revealed the presence of an additional, novel variant that lacked any exon 3, herein named “α2N.” Examination of the RNA from spinal cords of different‐aged rats showed a dramatic down‐regulation of α2N during prenatal development: α2N mRNA formed a significant portion of the α2 subunit pool at E14, but its relative level was reduced by 85% by birth and was undetectable in adults. Two proteins previously implicated in regulating the splicing of GlyR α2 pre‐mRNA, the neurooncological ventral antigen‐1 (Nova‐1) and the brain isoform of the polypyrimidine tract binding protein (brPTB), underwent small changes over the same period that did not correlate directly with the changes in the level of α2N, calling into question their involvement in the developmental regulation of α2N. However, treatment of spinal cord neurons in culture with antisense oligonucleotides designed selectively to knock down one of three Nova‐1 variants significantly altered the relative level of GlyR α2N, showing that Nova‐1 isoforms can regulate GlyR α2 pre‐mRNA splicing in developing neurons. These results provide evidence for a novel splice variant of the GlyR α2 subunit that undergoes dramatic developmental regulation, reveal the expression profiles of Nova‐1 and brPTB in the developing spinal cord, and suggest that Nova‐1 plays a role in regulating GlyR α2N in developing neurons. © 2002 Wiley Periodicals, Inc. J Neurobiol 52: 156–165, 2002     

Down‐regulation of Notch‐1 and Jagged‐1 inhibits prostate cancer cell growth,migration and invasion,and induces apoptosis via inactivation of Akt,mTOR, and NF‐κB signaling pathways

Notch signaling is involved in a variety of cellular processes, such as cell fate specification, differentiation, proliferation, and survival. Notch‐1 over‐expression has been reported in prostate cancer metastases. Likewise, Notch ligand Jagged‐1 was found to be over‐expressed in metastatic prostate cancer compared to localized prostate cancer or benign prostatic tissues, suggesting the biological significance of Notch signaling in prostate cancer progression. However, the mechanistic role of Notch signaling and the consequence of its down‐regulation in prostate cancer have not been fully elucidated. Using multiple cellular and molecular approaches such as MTT assay, apoptosis assay, gene transfection, real‐time RT‐PCR, Western blotting, migration, invasion assay and ELISA, we found that down‐regulation of Notch‐1 or Jagged‐1 was mechanistically associated with inhibition of cell growth, migration, invasion and induction of apoptosis in prostate cancer cells, which was mediated via inactivation of Akt, mTOR, and NF‐κB signaling. Consistent with these results, we found that the down‐regulation of Notch‐1 or Jagged‐1 led to decreased expression and the activity of NF‐κB downstream genes such as MMP‐9, VEGF, and uPA, contributing to the inhibition of cell migration and invasion. Taken together, we conclude that the down‐regulation of Notch‐1 or Jagged‐1 mediated inhibition of cell growth, migration and invasion, and the induction of apoptosis was in part due to inactivation of Akt, mTOR, and NF‐κB signaling pathways. Our results further suggest that inactivation of Notch signaling pathways by innovative strategies could be a potential targeted approach for the treatment of metastatic prostate cancer. J. Cell. Biochem. 109: 726–736, 2010. © 2010 Wiley‐Liss, Inc.     

Differential expression of sodium channel β subunits in dorsal root ganglion sensory neurons

The small-diameter (<25 μm) and large-diameter (>30 μm) sensory neurons of the dorsal root ganglion (DRG) express distinct combinations of tetrodotoxin sensitive and tetrodotoxin-resistant Na(+) channels that underlie the unique electrical properties of these neurons. In vivo, these Na(+) channels are formed as complexes of pore-forming α and auxiliary β subunits. The goal of this study was to investigate the expression of β subunits in DRG sensory neurons. Quantitative single-cell RT-PCR revealed that β subunit mRNA is differentially expressed in small (β(2) and β(3)) and large (β(1) and β(2)) DRG neurons. This raises the possibility that β subunit availability and Na(+) channel composition and functional regulation may differ in these subpopulations of sensory neurons. To further explore these possibilities, we quantitatively compared the mRNA expression of the β subunit with that of Na(v)1.7, a TTX-sensitive Na(+) channel widely expressed in both small and large DRG neurons. Na(v)1.7 and β subunit mRNAs were significantly correlated in small (β(2) and β(3)) and large (β(1) and β(2)) DRG neurons, indicating that these subunits are coexpressed in the same populations. Co-immunoprecipitation and immunocytochemistry indicated that Na(v)1.7 formed stable complexes with the β(1)-β(3) subunits in vivo and that Na(v)1.7 and β(3) co-localized within the plasma membranes of small DRG neurons. Heterologous expression studies showed that β(3) induced a hyperpolarizing shift in Na(v)1.7 activation, whereas β(1) produced a depolarizing shift in inactivation and faster recovery. The data indicate that β(3) and β(1) subunits are preferentially expressed in small and large DRG neurons, respectively, and that these auxiliary subunits differentially regulate the gating properties of Na(v)1.7 channels.     

Developmental regulation of the neuronal‐specific isoform of K‐CL cotransporter KCC2 in postnatal rat brains

We examined the expression of the KCC2 isoform of the K‐Cl cotransporter in the developing and adult brain, using an affinity‐purified antibody directed against a unique region of the KCC2 protein. Expression was shown to be limited to neurons at the cell bodies and cell processes in the hippocampus and cerebellum. Expression seemed to be the highest at the end of processes that originated from the CA1 pyramidal cells. Developmental up‐regulation of KCC2 expression was demonstrated in the entire rat brain by Northern and Western blot analyses, and in the hippocampus by immunofluorescence. Level of KCC2 expression was minimal at birth and increased significantly during postnatal development. This pattern of expression was opposite to the one of the Na‐K‐2Cl cotransporter that is highly expressed in immature brain and decreases during development. The up‐regulation of the K‐Cl cotransporter expression is consistent with the developmental down‐regulation of the intracellular Cl⁻ concentration in neurons. The level of intracellular Cl⁻, in turn, determines the excitatory versus inhibitory response of the neurotransmitter γ‐aminobutyric acid in the immature versus mature brain. Finally, KCC2 expression was shown in dorsal root ganglion neurons, demonstrating that expression of the cotransporter is not strictly confined to central nervous system neurons. © 1999 John Wiley & Sons, Inc. J Neurobiol 39: 558–568, 1999     

Brief Report of a New Highly Divergent Variant of Grapevine leafroll‐associated virus 3 (GLRaV‐3)

The alignment of the complete genomes of genetic variants of Grapevine leafroll‐associated virus 3 (GLRaV‐3) representing phylogenetic groups I, II, III and VI revealed numerous regions with exceptionally high divergence between group I to III and group VI variants. Oligonucleotide primers universal for all the above groups of the virus were designed in conserved short stretches of sequences flanking the divergent regions in the helicase (Hel) and RNA‐dependent RNA polymerase (RdRP) domains of the replicase gene and the divergent copy of the capsid protein (dCP) gene. Cloning and sequencing of the 549‐bp RT‐PCR amplicon of the helicase domain from grapevine cv. Shiraz lead to the detection of a variant of GLRaV‐3, which shared only 69.6–74.1% nt similarity with other variants, including the recently reported, new, highly divergent variant, isolate 139. This was confirmed by the results of the analysis of 517‐bp amplicon of the HSP70 gene of GLRaV‐3 generated in RT‐nested PCR based on degenerate primers for the simultaneous amplification of members of the Closteroviridae family designed by Dovas and Katis (J Virol Methods, 109, 2003, 217). In this genomic region, the variant shares 72.3–78.7% nt similarity with other variants of GLRaV‐3. This previously unreported, new, highly divergent variant was provisionally named GTG10. From the alignment of the HSP70 sequences primers for the specific RT‐nested PCR amplification of the variant GTG10 and members of group VI, and specific simultaneous amplification of variants of groups I, II and III, were designed. The results obtained from brief testing of various grapevines using all these primers suggest a relatively limited presence of GTG10 variant in vineyards.     

One‐step Multiplex RT‐PCR for Simultaneous Detection of Four Viruses in Tobacco

A one‐step multiplex RT‐PCR method has been developed for the simultaneous detection of four viruses frequently occurring in tobacco (Cucumber mosaic virus, Tobacco mosaic virus, Tobacco etch virus and Potato virus Y). Four sets of specific primers were designed to work with the same reaction reagents and cycling conditions, resulting in four distinguishable amplicons representative of the four viruses independently. This one‐step multiplex RT‐PCR is consistently specific using different combinations of virus RNA as templates, and no non‐specific band was observed. It has high sensitivity compared to single RT‐PCR. Moreover, field samples in China can be tested by this method for virus detection. Our results show that one‐step multiplex RT‐PCR is a high‐throughput, specific, sensitive method for tobacco virus detection.     

A combined RT‐PCR and dot‐blot hybridization method reveals the coexistence of SJNNV and RGNNV betanodavirus genotypes in wild meagre (Argyrosomus regius)

Aims: To detect the possible coexistence of striped jack nervous necrosis virus (SJNNV) and red‐spotted grouper nervous necrosis virus (RGNNV) genotypes in a single fish, a methodology based on the combination of PCR amplification and blot hybridization has been developed and applied in this study. Methods and Results: The degenerate primers designed for the PCR procedure target the T4 region within the capsid gene, resulting in the amplification of both genotypes. The subsequent hybridization of these amplification products with two different specific digoxigenin‐labelled probes resulted in the identification of both genotypes separately. The application of the RT‐PCR protocol to analyse blood samples from asymptomatic wild meagre (Argyrosomus regius) specimens has shown a 46·87% of viral nervous necrosis virus carriers. The combination of RT‐PCR and blot hybridization increases the detection rate up to 90·62%, and, in addition, it has shown the coexistence of both genotypes in 18 out of the 32 specimens analysed (56·25%). Conclusions: This study reports the coexistence of betanodaviruses belonging to two different genotypes (SJNNV and RGNNV) in wild fish specimens. Significance and Impact of the Study: This is the first report demonstrating the presence of SJNNV and RGNNV genotypes in the same specimen. This study also demonstrates a carrier state in this fish species for the first time.     

The human Na,K-ATPase alpha 4 isoform is a ouabain-sensitive alpha isoform that is expressed in sperm

The Na,K-ATPase generates electrochemical gradients across the plasma membrane that are responsible for numerous cellular and physiological processes. The active Na,K-ATPase is minimally composed of an alpha and a beta subunit and families of isoforms for both subunits exist. Recent studies have identified a physiological role for the rat Na,K-ATPase alpha4 isoform in sperm motility. However, very little is known about the human Na,K-ATPase alpha4 isoform other than its genomic sequence and structure and its mRNA expression pattern. Here, the human alpha4 isoform of the Na,K-ATPase is cloned, expressed, and characterized. Full length cDNAs encoding the putative human alpha4 isoform of the Na,K-ATPase were identified from a number of ESTs and a protein product corresponding to this isoform was shown to be expressed from these cDNAs. The human Na,K-ATPase alpha4 isoform protein was found to be expressed in mature sperm in human testes sections and it is localized specifically to the principle piece of human sperm. In addition, the presence of the Na,K-ATPase alpha4 isoform is absent in immature testes however its expression appears coincident with sexual maturity. And finally, the human Na,K-ATPase alpha4 isoform was shown to be as sensitive to cardiac glycoside inhibition as the human Na,K-ATPase alpha1 isoform. Considering the important role of the rat Na,K-ATPase alpha4 isoform in rat sperm motility, the demonstration that the human alpha4 isoform is a sperm-specific protein localized to the flagellum suggests a role for the human Na,K-ATPase alpha4 isoform in human sperm physiology.     

Seventeen new exon‐primed intron‐crossing polymerase chain reaction amplifiable introns in fish

We used exon‐primed, intron‐crossing polymerase chain reaction (EPIC‐PCR) amplification to assay variation in nuclear loci in some teleost fishes (Carangidae, Centropomidae, Chaetodontidae, Clupeidae, Holocentridae, Moronidae, Mullidae, Pomacentridae, Scombridae, Siganidae). We designed primers in the conserved regions flanking splice sites of consecutive exons of different genes, allowing the amplification of 17 putative introns. Among the satisfactory amplified systems, 14 showed length polymorphism with 2–14 alleles.     

IDENTIFICATION OF PFIESTERIA PISCICIDA (DINOPHYCEAE) AND PFIESTERIA‐LIKE ORGANISMS USING INTERNAL TRANSCRIBED SPACER‐SPECIFIC PCR ASSAYS1

The putative harmful algal bloom dinoflagellate, Pfiesteria piscicida (Steidinger et Burkholder), frequently co‐occurs with other morphologically similar species collectively known as Pfiesteria‐like organisms (PLOs). This study specifically evaluated whether unique sequences in the internal transcribed spacer (ITS) regions, ITS1 and ITS2, could be used to develop PCR assays capable of detecting PLOs in natural assemblages. ITS regions were selected because they are more variable than the flanking small subunit or large subunit rRNA genes and more likely to contain species‐specific sequences. Sequencing of the ITS regions revealed unique oligonucleotide primer binding sites for Pfiesteria piscicida, Pfiesteria shumwayae (Glasgow et Burkholder), Florida “Lucy” species, two cryptoperidiniopsoid species, “H/V14” and “PLO21,” and the estuarine mixotroph, Karlodinium micrum (Leadbetter et Dodge). These PCR assays had a minimum sensitivity of 100 cells in a 100‐mL sample (1 cell·mL^?1) and were successfully used to detect PLOs in the St. Johns River system in Florida, USA. DNA purification and aspects of PCR assay development, PCR optimization, PCR assay controls, and collection of field samples are discussed.     

Independent and joint modulation of rat Nav1.6 voltage-gated sodium channels by coexpression with the auxiliary β1 and β2 subunits

The Na_v1.6 voltage-gated sodium channel α subunit isoform is the most abundant isoform in the brain and is implicated in the transmission of high frequency action potentials. Purification and immunocytochemical studies imply that Na_v1.6 exist predominantly as Na_v1.6 + β1 + β2 heterotrimeric complexes. We assessed the independent and joint effects of the rat β1 and β2 subunits on the gating and kinetic properties of rat Na_v1.6 channels by recording whole-cell currents in the two-electrode voltage clamp configuration following transient expression in Xenopus oocytes. The β1 subunit accelerated fast inactivation of sodium currents but had no effect on the voltage dependence of their activation and steady-state inactivation and also prevented the decline of currents following trains of high-frequency depolarizing prepulses. The β2 subunit selectively retarded the fast phase of fast inactivation and shifted the voltage dependence of activation towards depolarization without affecting other gating properties and had no effect on the decline of currents following repeated depolarization. The β1 and β2 subunits expressed together accelerated both kinetic phases of fast inactivation, shifted the voltage dependence of activation towards hyperpolarization, and gave currents with a persistent component typical of those recorded from neurons expressing Na_v1.6 sodium channels. These results identify unique effects of the β1 and β2 subunits and demonstrate that joint modulation by both auxiliary subunits gives channel properties that are not predicted by the effects of individual subunits.