Modulation of the cardiac sodium channel Nav1.5 by fibroblast growth factor homologous factor 1B

We have previously shown that fibroblast growth factor homologous factor 1B (FHF1B), a cytosolic member of the fibroblast growth factor family, associates with the sensory neuron-specific channel Na(v)1.9 but not with the other sodium channels present in adult rat dorsal root ganglia neurons. We show in this study that FHF1B binds to the C terminus of the cardiac voltage-gated sodium channel Na(v)1.5 and modulates the properties of the channel. The N-terminal 41 amino acid residues of FHF1B are essential for binding to Na(v)1.5, and the conserved acidic rich domain (amino acids 1773-1832) in the C terminus of Na(v)1.5 is sufficient for association with this factor. Binding of the growth factor to recombinant wild type human Na(v)1.5 in human embryonic kidney 293 cells produces a significant hyperpolarizing shift in the voltage dependence of channel inactivation. An aspartic acid to glycine substitution at position 1790 of the channel, which underlies one of the LQT-3 phenotypes of cardiac arrythmias, abolishes the interaction of the Na(v)1.5 channel with FHF1B. This is the first report showing that interaction with a growth factor can modulate properties of a voltage-gated sodium channel.     

FGF13 modulates the gating properties of the cardiac sodium channel Nav1.5 in an isoform-specific manner

FGF13 (FHF2), the major fibroblast growth factor homologous factor (FHF) in rodent heart, directly binds to the C-terminus of the main cardiac sodium channel, Na_V1.5. Knockdown of FGF13 in cardiomyocytes induces slowed ventricular conduction by altering Na_V1.5 function. FGF13 has five splice variants, each of which possess the same core region and C terminus but differing in their respective N termini. Whether and how these alternatively spliced N termini impart isoform-specific regulation of Na_V1.5, however, has not been reported. Here, we exploited a heterologous expression to explore the specific modulatory effects of FGF13 splice variants FGF13S, FGF13U and FGF13YV on Na_V1.5 function. We found these three splice variants differentially modulated Na_V1.5 current density. Although steady-state activation was unaltered by any of the FGF13 isoforms (compared to control cells expressing Nav1.5 but not expressing FGF13), open-state fast inactivation and closed-state fast inactivation were markedly slowed, steady-state availability was significantly shifted toward the depolarizing direction, and the window current was increased by each of FGF13 isoforms. Most strikingly, FGF13S hastened the rate of Na_V1.5 entry into the slow inactivation state and induced a dramatic slowing of recovery from inactivation, which caused a large decrease in current after either low or high frequency stimulation. Overall, these data showed the diversity of the roles of the FGF13 N-termini in Na_V1.5 channel modulation and suggested the importance of isoform-specific regulation.     

Nav1.5/SCN5A基因新的变构体编码人脑组织Nav1.5 Na+通道

河豚毒-抵抗性(TTX-R)Nav1.5 Na 通道是心肌的特异性Na 通道,虽然研究发现神经元中也存在河豚毒-抵抗性Na 电流及Nav1.5/SCN5A mRNA的表达,但其确切的cDNA序列尚不清楚.采用RT-PCR法对人脑组织Nav1.5/SCN5A基因cDNA进行克隆发现:人脑组织Nav1.5/SCN5A基因cDNA有2种变构体,hB1和hB2(accession number EF629346,EF629347),其中hB1全长6201个碱基,其开放读码框架(ORF)参与编码2016个氨基酸,和人心肌Nav1.5 Na 通道氨基酸序列相同率高达98%,共有28个不同的氨基酸,其中7个集中位于第6A外显子与第6外显子编码区.与人心肌Nav1.5/SCN5A基因cDNA不同的是,在对人脑组织Nav1.5/SCN5A基因cDNA的克隆中未发现该基因第18外显子的选择性剪接,但却发现其第24外显子的选择性剪接,2种选择性剪接体(hB1和hB2)在脑组织中基本同时表达,表达比率接近1∶1,但在心脏中二者的表达比率却与年龄有关.人Nav1.5/SCN5A基因的第24外显子定位于染色体3P21区,共有54个碱基,参与编码18个氨基酸.RT-PCR法证实第24外显子的选择性剪接也可发生在大鼠心脑之外的其他组织中,竞争性PCR法证明,不同组织中2种选择性剪接体的表达比率不同,且随着周龄的增加,2种选择性剪接体在各组织中表达的变化趋势不同.此外,RT-PCR法还发现Wistar大鼠全身16种组织中均可检测到Nav1.5/SCN5A mRNA的表达.上述实验结果说明,Nav1.5 Na 通道在全身组织中分布广泛,但编码人脑组织Nav1.5 Na 通道与心肌组织该离子通道的cDNA序列不同,是Nav1.5/SCN5A基因的2种变构体,这为深入研究不同组织中Nav1.5 Na 通道的功能提供了基础.     

The distribution and function of alternatively spliced insertions in hDlg.

hDlg is the human homolog of the Drosophila Discs-large tumor suppressor. As a member of the MAGUK (membrane-associated guanylate kinase) family of scaffolding proteins, hDlg is composed of three PDZ (PSD-95, Dlg, and ZO-1) repeats, an SH3 (Src homology 3) motif, and a GUK (guanylate kinase-like) domain. Additionally, hDlg contains two regions of alternative splicing. Here we identify a novel insertion, I1B, located N-terminal to the PDZ repeats. We further analyze the tissue-specific combinations of insertions and correlate those results with the distribution of protein isoforms. We also identify the functions of the two alternatively spliced regions. The N-terminal alternatively spliced region is capable of binding several SH3 domains and also moderates the level of protein oligomerization. Insertions in the second region are responsible for determining the localization of hDlg, with insertion I3 targeting the protein to the membrane regions of cell-cell contact and insertion I2 targeting the protein to the nucleus.     

Neurite outgrowth promotion by the alternatively spliced region of tenascin-C is influenced by cell-type specific binding.

We have investigated the impact of cellular environment on the neurite outgrowth promoting properties of the alternatively spliced fibronectin type-III region (fnA-D) of tenascin-C. FnA-D promoted neurite outgrowth in vitro when bound to the surface of BHK cells or cerebral cortical astrocytes, but the absolute increase was greater on astrocytes. In addition, different neurite outgrowth promoting sites were revealed within fnA-D bound to the two cellular substrates. FnA-D also promoted neurite outgrowth as a soluble ligand; however, the actions of soluble fnA-D were not affected by cell type. Therefore, we hypothesized that different mechanisms of cellular binding can alter the growth promoting actions of bound fnA-D. We found that fnA-D utilizes two distinct sequences to bind to the BHK cell surface as opposed to the BHK extracellular matrix. In contrast, only one of these sequences is utilized to bind to the astrocyte matrix as opposed to the astrocyte surface. Furthermore, Scatchard analysis indicated two types of receptors for fnA-D on BHK cells and only one type on astrocytes. These results suggest that active sites for neurite outgrowth within fnA-D are differentially revealed depending on cell-specific fnA-D binding sites. Therefore, the function of tenascin-C and its various domains must be considered in terms of cellular context.     

Mutagenesis of the DI/DIII linker in dengue virus envelope protein impairs viral particle assembly

The dengue virus (DV) envelope (E) protein is important in mediating viral entry and assembly of progeny virus during cellular infection. Domains I and III (DI and DIII, respectively) of the DV E protein are connected by a highly conserved but poorly ordered region, the DI/DIII linker. Although the flexibility of the DI/DIII linker is thought to be important for accommodating the structural rearrangements undergone by the E protein during viral entry, the function of the linker in the DV infectious cycle is not well understood. In this study, we performed site-directed mutagenesis on conserved residues in the DI/DIII linker of the DV2 E protein and showed that the resulting mutations had little or no effect on the entry process but greatly affected virus assembly. Biochemical fractionation and immunofluorescence microscopy experiments performed on infectious virus as well as in a virus-like particle (VLP) system indicate that the DI/DIII linker mutants express the DV structural proteins at the sites of particle assembly near the ER but fail to form infectious particles. This defect is not due to disruption of E's interaction with prM and pr in immature and mature virions, respectively. Serial passaging of the DV2 mutant E-Y299F led to the identification of a mutation in the membrane-proximal stem region of E that fully compensates for the assembly defect of this DI/DIII linker mutant. Together, our results suggest a critical and previously unidentified role for the E protein DI/DIII linker region during the DV2 assembly process.     

Actin cytoskeleton remodeling by the alternatively spliced isoform of PDLIM4/RIL protein

RIL (product of PDLIM4 gene) is an actin-associated protein that has previously been shown to stimulate actin bundling by interacting with actin-cross-linking protein α-actinin-1 and increasing its affinity to filamentous actin. Here, we report that the alternatively spliced isoform of RIL, denoted here as RILaltCterm, functions as a dominant-negative modulator of RIL-mediated actin reorganization. RILaltCterm is regulated at the level of protein stability, and this protein isoform accumulates particularly in response to oxidative stress. We show that the alternative C-terminal segment of RILaltCterm has a disordered structure that directs the protein to rapid degradation in the core 20 S proteasomes. Such degradation is ubiquitin-independent and can be blocked by binding to NAD(P)H quinone oxidoreductase NQO1, a detoxifying enzyme induced by prolonged exposure to oxidative stress. We show that either overexpression of RILaltCterm or its stabilization by stresses counteracts the effects produced by full-length RIL on organization of actin cytoskeleton and cell motility. Taken together, the data suggest a mechanism for fine-tuning actin cytoskeleton rearrangement in response to stresses.     

Genetic polymorphism and sequence evolution of an alternatively spliced exon of the glial fibrillary acidic protein gene,GFAP

Isoform GFAPepsilon of the human cytoskeletal protein GFAP carries, as the result of alternative splicing of exon 7a of GFAP, a novel 42-amino-acid-long C-terminal region with binding capacity for the presenilin proteins. Here we show that exon 7a is present in a variety of mammals but absent from GFAP of chicken and fish. Comparison of the mouse and human GFAP exons showed an increased rate of nonsynonymous nucleotide substitutions in exon 7a compared to the other exons. This resulted in 10 nonconservative and 2 conservative amino acid substitutions and suggests that exon 7a has evolved under different functional constraints. Exons 7a of humans and higher primates are 100% identical apart from alanine codon 426, which is conserved in only 9% of the human alleles, while 21 and 70% of the alleles, respectively, have a valine or a threonine codon at that position. Threonine represents a potential phosphorylation site, and positive selection of that effect could explain the high allele frequency.     

A sequence compilation and comparison of exons that are alternatively spliced in neurons.

Alternative splicing is an important regulatory mechanism to create protein diversity. In order to elucidate possible regulatory elements common to neuron specific exons, we created and statistically analysed a database of exons that are alternatively spliced in neurons. The splice site comparison of alternatively and constitutively spliced exons reveals that some, but not all alternatively spliced exons have splice sites deviating from the consensus sequence, implying diverse patterns of regulation. The deviation from the consensus is most evident at the -3 position of the 3' splice site and the +4 and -3 position of the 5' splice site. The nucleotide composition of alternatively and constitutively spliced exons is different, with alternatively spliced exons being more AU rich. We performed overlapping k-tuple analysis to identify common motifs. We found that alternatively and constitutively spliced exons differ in the frequency of several trinucleotides that cannot be explained by the amino acid composition and may be important for splicing regulation.     

Identification of a new co-factor, MOG1, required for the full function of cardiac sodium channel Nav 1.5

The cardiac sodium channel Nav 1.5 is essential for the physiological function of the heart and contributes to lethal cardiac arrhythmias and sudden death when mutated. Here, we report that MOG1, a small protein that is highly conserved from yeast to humans, is a central component of the channel complex and modulates the physiological function of Nav 1.5. The yeast two-hybrid screen identified MOG1 as a new protein that interacts with the cytoplasmic loop II (between transmembrane domains DII and DIII) of Nav 1.5. The interaction was further demonstrated by both in vitro glutathione S-transferase pull-down and in vivo co-immunoprecipitation assays in both HEK293 cells with co-expression of MOG1 and Nav1.5 and native cardiac cells. Co-expression of MOG1 with Nav1.5 in HEK293 cells increased sodium current densities. In neonatal myocytes, overexpression of MOG1 increased current densities nearly 2-fold. Western blot analysis revealed that MOG1 increased cell surface expression of Nav1.5, which may be the underlying mechanism by which MOG1 increased sodium current densities. Immunostaining revealed that in the heart, MOG1 was expressed in both atrial and ventricular tissues with predominant localization at the intercalated discs. In cardiomyocytes, MOG1 is mostly localized in the cell membrane and co-localized with Nav1.5. These results indicate that MOG1 is a critical regulator of sodium channel function in the heart and reveal a new cellular function for MOG1. This study further demonstrates the functional diversity of Nav1.5-binding proteins, which serve important functions for Nav1.5 under different cellular conditions.     

Modulation of Kv1.5 potassium channel gating by extracellular zinc.

Zinc ions are known to induce a variable depolarizing shift of the ionic current half-activation potential and substantially slow the activation kinetics of most K(+) channels. In Kv1.5, Zn(2+) also reduces ionic current, and this is relieved by increasing the external K(+) or Cs(+) concentration. Here we have investigated the actions of Zn(2+) on the gating currents of Kv1.5 channels expressed in HEK cells. Zn(2+) shifted the midpoint of the charge-voltage (Q-V) curve substantially more (approximately 2 times) than it shifted the V(1/2) of the g-V curve, and this amounted to +60 mV at 1 mM Zn(2+). Both Q1 and Q2 activation charge components were similarly affected by Zn(2+), which indicated free access of Zn(2+) to channel closed states. The maximal charge movement was also reduced by 1 mM Zn(2+) by approximately 15%, from 1.6 +/- 0.5 to 1.4 +/- 0.47 pC (n = 4). Addition of external K(+) or Cs(+), which relieved the Zn(2+)-induced ionic current reduction, decreased the extent of the Zn(2+)-induced Q-V shift. In 135 mM extracellular Cs(+), 200 microM Zn(2+) reduced ionic current by only 8 +/- 1%, compared with 71% reduction in 0 mM extracellular Cs(+), and caused a comparable shift in both the g-V and Q-V relations (17.9 +/- 0.6 mV vs. 20.8 +/- 2.1 mV, n = 6). Our results confirm the presence of two independent binding sites involved in the Zn(2+) actions. Whereas binding to one site accounts for reduction of current and binding to the other site accounts for the gating shift in ionic current recordings, both sites contribute to the Zn(2+)-induced Q-V shift.     

Modulation of calcium channel function by drugs

Calcium channel blocking drugs, or "calcium antagonists", have been increasingly used in the last decade, both as valuable cardiovascular drugs, and as tools to investigate the pharmacology of the calcium channels which play a vital role in the excitation-activation coupling of many excitable cells. Three important developments, "patch clamping" to investigate single calcium channels, ligand binding studies to investigate the calcium antagonist "receptor sites", and the introduction of novel calcium channel activators, or "calcium agonists", have recently led to greater understanding of the mechanism of action of drugs on the calcium channel. We show here how the calcium channel modulators interact with the binding sites to increase or decrease calcium flux, and hence to modulate the activity of many excitable tissues. We predict that these new developments will soon result in the isolation of purified calcium channels, and investigation of their subtypes and drug sensitivities. This information could lead to the introduction of novel, more selective calcium antagonists for a variety of indications such as atherosclerosis or neurological disorders. Of particular interest is the potential of tissue-selective calcium agonistic drugs to combat cardiac failure or endocrinological disorders.