Id2 Represses Aldosterone-Stimulated Cardiac T-Type Calcium Channels Expression.

Aldosterone excess is a cardiovascular risk factor. Aldosterone can directly stimulate an electrical remodeling of cardiomyocytes leading to cardiac arrhythmia and hypertrophy. L-type and T-type voltage-gated calcium (Ca2+) channels expression are increased by aldosterone in cardiomyocytes. To further understand the regulation of these channels expression, we studied the role of a transcriptional repressor, the inhibitor of differentiation/DNA binding protein 2 (Id2). We found that aldosterone inhibited the expression of Id2 in neonatal rat cardiomyocytes and in the heart of adult mice. When Id2 was overexpressed in cardiomyocytes, we observed a reduction in the spontaneous action potentials rate and an arrest in aldosterone-stimulated rate increase. Accordingly, Id2 siRNA knockdown increased this rate. We also observed that CaV1.2 (L-type Ca2+ channel) or CaV3.1, and CaV3.2 (T-type Ca2+ channels) mRNA expression levels and Ca2+ currents were affected by Id2 presence. These observations were further corroborated in a heart specific Id2- transgenic mice. Taken together, our results suggest that Id2 functions as a transcriptional repressor for L- and T-type Ca2+ channels, particularly CaV3.1, in cardiomyocytes and its expression is controlled by aldosterone. We propose that Id2 might contributes to a protective mechanism in cardiomyocytes preventing the presence of channels associated with a pathological state.

A long-lasting cardiomyogenic gene expression by PEI-based transfection induces endogenous cardiac mRNAs in human adipose-derived stem cells.

Our previous work revealed that a polyethyleneimine (PEI)-based gene delivery causes robust and sustained expression of exogenous genes in human adipose-derived stem cells (hADSCs). Here we use this method to test whether a single introduction of cDNAs for the three cardiomyogenic reprogramming genes (GATA4, MEF2C, and TBX5) might be sufficient to induce transdifferentiation of hADSCs towards the cardiomyogenic lineage. A single transfection results in sustained expression of the introduced genes for more than two weeks. hADSCs exhibit undetectable or very low levels of mRNAs for endogenous GATA4, MEF2C and TBX5. However, mRNAs for these endogenous factors become apparent at ∼2 weeks after transfection and keep increasing until the end of experimental period at the fifth week. Concordant with these cardiomyogenic genes, Nkx2.5 mRNA becomes significant at ∼2 weeks and gradually increases until the end of experimental period. Several other cardiomyogenic mRNAs were also significant at 5 weeks. Thus, a single transfection of cDNAs for the cardiomyogenic reprogramming genes using a PEI-based method induces transdifferentiation of ADSCs.

Effective gene delivery into adipose-derived stem cells: transfection of cells in suspension with the use of a nuclear localization signal peptide-conjugated polyethylenimine.

BACKGROUND AIMS: Adipose-derived stem cells have the ability to turn into several clinically important cell types. However, it is difficult to transfect these cells with the use of conventional cationic lipid-based reagents. Polyethylenimine (PEI) is considered to be an inexpensive and effective tool for delivery of nucleic acids into mammalian cells. METHODS: We used a linear PEI conjugated with the nuclear localization signal (NLS) peptide of Simian vacuolating virus 40 large T antigen (PEI-NLS) for transfection of plasmid DNA into adipose-derived cells. We also tested if transfection of cells in suspension might improve the degree and duration of exogenous gene expression. RESULTS: Transfection of cells in suspension with the use of a PEI conjugated with an NLS peptide resulted in high levels of reporter gene expression for an extended period of time in clonal 3T3-L1 preadipocytes and native human adipose-derived stem cells. The reporter gene expression increased for 3 days after the addition of the PEI-NLS peptide-DNA mixture in cell suspension and remained significant for at least 7 days. Cell density did not influence the level of reporter gene expression. Thus, the suspension method with the use of an NLS peptide-conjugated PEI leads to a robust and sustained expression of exogenous genes in adipose-derived cells. CONCLUSIONS: The devised transfection method may be useful for reprogramming of adipose-derived stem cells and cell-based therapy.

Differential contribution of Kv4-containing channels to A-type, voltage-gated potassium currents in somatic and visceral dorsal root ganglion neurons.

Little is known about electrophysiological differences of A-type transient K(+) (KA) currents in nociceptive afferent neurons that innervate somatic and visceral tissues. Staining with isolectin B4 (IB4)-FITC classifies L6-S1 dorsal root ganglion (DRG) neurons into three populations with distinct staining intensities: negative to weak, moderate, and intense fluorescence signals. All IB4 intensely stained cells are negative for a fluorescent dye, Fast Blue (FB), injected into the bladder wall, whereas a fraction of somatic neurons labeled by FB, injected to the external urethral dermis, is intensely stained with IB4. In whole-cell, patch-clamp recordings, phrixotoxin 2 (PaTx2), a voltage-gated K(+) (Kv)4 channel blocker, exhibits voltage-independent inhibition of the KA current in IB4 intensely stained cells but not the one in bladder-innervating cells. The toxin also shows voltage-independent inhibition of heterologously expressed Kv4.1 current, whereas its inhibition of Kv4.2 and Kv4.3 currents is voltage dependent. The swapping of four amino acids at the carboxyl portion of the S3 region between Kv4.1 and Kv4.2 transfers this characteristic. RT-PCRs detected Kv4.1 and the long isoform of Kv4.3 mRNAs without significant Kv4.2 mRNA in L6-S1 DRGs. Kv4.1 and Kv4.3 mRNA levels were higher in laser-captured, IB4-stained neurons than in bladder afferent neurons. These results indicate that PaTx2 acts differently on channels in the Kv4 family and that Kv4.1 and possibly Kv4.3 subunits functionally participate in the formation of KA channels in a subpopulation of somatic C-fiber neurons but not in visceral C-fiber neurons innervating the bladder.

Enigma homolog 1 promotes myogenic gene expression and differentiation of C2C12 cells.

The Enigma homolog (ENH) gene generates several splicing variants. The initially identified ENH1 possesses one PDZ and three LIM domains, whereas ENH2~4 lack the latter domains. The splicing switch from ENH1 to LIM-less ENHs occurs during development/maturation of skeletal and heart muscles. We examined for the roles of ENH splicing variants in muscle differentiation using C2C12 cells. Cells stably expressing ENH1 exhibited significantly higher MyoD and myogenin mRNA levels before differentiation and after 5 days in low serum-differentiating medium than mock-transfected cells. ENH1-stable transformants also retained the ability to exhibit elongated morphology with well-extended actin fibers following differentiation. In contrast, cells stably expressing ENH3 or ENH4 did not show myotube-like morphology or reorganization of actin fibers following culture in the differentiating medium. Transient overexpression of ENH1 using adenovirus supported the increased expression of muscle marker mRNAs and the formation of well-organized stress fibers, whereas ENH4 overexpression prevented these morphological changes. Furthermore, specific suppression of ENH1 expression by RNAi caused a significant reduction in MyoD mRNA level and blocked the morphological changes. These results suggest that ENH1 with multiple protein-protein interaction modules is essential for differentiation of striated muscles, whereas ectopic expression of LIM-less ENH disrupts normal muscle differentiation.

Cellular basis of burn-induced cardiac dysfunction and prevention by mesenteric lymph duct ligation.

BACKGROUND: Myocardial contractile depression develops 4 to 24 h after major burn injury. We have reported previously that in a rat burn injury model (≈40% of total body surface area burn), mesenteric lymph duct ligation (LDL) prior to burn prevented myocardial dysfunction. However, the underlying cellular and molecular mechanisms are not well understood. MATERIALS AND METHODS: Left ventricular myocytes were isolated from sham burn (control), sham burn with LDL (sham + LDL), burn, and burn with LDL (burn + LDL) rats at 4 and 24 h after burn or sham burn. Electrophysiological techniques were used to study myocyte size, contractility and L-type Ca2+ channel current (ICa). Further studies examined changes in the messenger RNA expression levels of pore-forming subunit of the L-type Ca(2+) channel, α1C, and its auxiliary subunits, ß1, ß2, ß3, and α2δ1, which modulate the abundance of the ICa in post-burn hearts. RESULTS: Depressed myocyte contractility (≈20%) developed during 4 to 24 h post-burn compared with control, sham + LDL, or burn + LDL groups, a pattern of changes consistent with whole heart studies. There was no significant alteration in myocyte size. The ICa density was significantly decreased (≈30%) at 24 h post-burn, whereas the messenger RNA expression levels of Ca(2+) channel gene were not significantly altered at 4 and 24 h after burn injury. CONCLUSIONS: These results suggest that the post-burn contractile phenotype in vivo was also present in isolated myocytes in vitro, but cellular remodeling was not a major factor. The results also suggest that changes in ICa regulation, but not from Ca(2+) channel gene modification, may be a key element involved in post-burn contractile depression and the beneficial effects of LDL.

Splicing transitions of the anchoring protein ENH during striated muscle development.

The ENH (PDLIM5) protein acts as a scaffold to tether various functional proteins at subcellular sites via PDZ and three LIM domains. Splicing of the ENH primary transcript generates various products with different repertories of protein interaction modules. Three LIM-containing ENH predominates in neonatal cardiac tissue, whereas LIM-less ENHs are abundant in adult hearts, as well as skeletal muscles. Here we examine the timing of splicing transitions of ENH gene products during postnatal heart development and C2C12 myoblast differentiation. Real-time PCR analysis shows that LIM-containing ENH1 mRNA is gradually decreased during postnatal heart development, whereas transcripts with the short exon 5 appear in the late postnatal period and continues to increase until at least one month after birth. The splicing transition from LIM-containing ENH1 to LIM-less ENHs is also observed during the early period of C2C12 differentiation. This transition correlates with the emergence of ENH transcripts with the short exon 5, as well as the expression of myogenin mRNA. In contrast, the shift from the short exon 5 to the exon 7 occurs in the late differentiation period. The timing of this late event corresponds to the appearance of mRNA for the skeletal myosin heavy chain MYH4. Thus, coordinated and stepwise splicing transitions result in the production of specific ENH transcripts in mature striated muscles.

Localization of Kv1.3 channels in the immunological synapse modulates the calcium response to antigen stimulation in T lymphocytes.

The immunological synapse (IS), a highly organized structure that forms at the point of contact between a T cell and an APC, is essential for the proper development of signaling events, including the Ca(2+) response. Kv1.3 channels control Ca(2+) homeostasis in human T cells and move into the IS upon Ag presentation. However, the process involved in channel accumulation in the IS and the functional implications of this localization are not yet known. Here we define the movement of Kv1.3 into the IS and study whether Kv1.3 localization into the IS influences Ca(2+) signaling in Jurkat T cells. Crosslinking of the channel protein with an extracellular Ab limits Kv1.3 mobility and accumulation at the IS. Moreover, Kv1.3 recruitment to the IS does not involve the transport of newly synthesized channels and it does not occur through recycling of membrane channels. Kv1.3 localization in the IS modulates the Ca(2+) response. Blockade of Kv1.3 movement into the IS by crosslinking significantly increases the amplitude of the Ca(2+) response triggered by anti-CD3/anti-CD28-coated beads, which induce the formation of the IS. On the contrary, the Ca(2+) response induced by TCR stimulation without the formation of the IS with soluble anti-CD3/anti-CD28 Abs is unaltered. The results presented herein indicate that, upon Ag presentation, membrane-incorporated Kv1.3 channels move along the plasma membrane to localize in the IS. This localization is important to control the amplitude of the Ca(2+) response, and disruption of this process can account for alterations of downstream Ca(2+)-dependent signaling events.

Immature adipocyte-derived exosomes inhibit expression of muscle differentiation markers.

Exosomes are released from a variety of cells to communicate with recipient cells. Exosomes contain microRNAs (miRNAs), which are noncoding RNAs that suppress target genes. Our previous proteomic study (FEBS Open Bio 2016, 6, 816-826) demonstrated that 3T3-L1 adipocytes secrete exosome components as well as growth factors, inspiring us to investigate what type of miRNA is involved in adipocyte-secreted exosomes and what functions they carry out in recipient cells. Here, we profiled miRNAs in 3T3-L1 adipocyte-secreted exosomes and revealed suppression of muscle differentiation by adipocyte-derived exosomes. Through our microarray analysis, we detected over 300 exosomal miRNAs during adipocyte differentiation. Exosomal miRNAs present during adipocyte differentiation included not only pro-adipogenic miRNAs but also miRNAs associated with muscular dystrophy. Gene ontology analysis predicted that the target genes of miRNAs are associated primarily with transcriptional regulation. To further investigate whether adipocyte-secreted exosomes regulate the expression levels of genes involved in muscle differentiation, we treated cultured myoblasts with adipocyte-derived exosome fractions. Intriguingly, the expression levels of myogenic regulatory factors, Myog and Myf6, and other muscle differentiation markers, myosin heavy-chain 3 and insulin-like growth factor 2, were significantly downregulated in myoblasts treated with adipocyte-derived exosomes. Immature adipocyte-derived exosomes exhibited a stronger suppressive effect than mature adipocyte-derived exosomes. Our results suggest that adipocytes suppress the expression levels of muscle differentiation-associated genes in myoblasts via adipocyte-secreted exosomes containing miRNAs.

Effects of bladder outlet obstruction on properties of Ca2+-activated K+ channels in rat bladder.

In this study, we investigated the effects of bladder outlet obstruction (BOO) on the expression and function of large conductance (BK) and small conductance (SK) Ca(2+)-activated K(+) channels in detrusor smooth muscle. The bladder from adult female Sprague-Dawley rats with 6-wk BOO were used. The mRNA expression of the BK channel alpha-subunit, beta1-, beta2-, and beta4-subunits and SK1, SK2, and SK3 channels were investigated using real-time RT-PCR. All subunits except for the BK-beta2, SK2, and SK3 channels were predominantly expressed in the detrusor smooth muscle rather than in the mucosa. The mRNA expression of the BK channel alpha-subunit was not significantly changed in obstructed bladders. However, the expression of the BK channel beta1-subunit and the SK3 channel was remarkably increased in obstructed bladders. On the other hand, the expression of the BK channel beta4-subunit was decreased as the severity of BOO-induced bladder overactivity progressed. In detrusor smooth muscle strips from obstructed bladders, blockade of BK channels by iberiotoxin (IbTx) or charybdotoxin (CTx) and blockade of SK channels by apamin increased the amplitude of spontaneous contractions. These blockers also increased the contractility and affinity of these strips for carbachol during cumulative applications. The facilitatory effects elicited by these K(+) channel blockers were larger in the strips from obstructed bladders compared with control bladders. These results suggest that long-term exposure to BOO for 6 wk enhances the function of both BK and SK types of Ca(2+)-activated K(+) channels in the detrusor smooth muscle to induce an inhibition of bladder contractility, which might be a compensatory mechanism to reduce BOO-induced bladder overactivity.

Fermented Garlic Extract Increases Oxygen Consumption and UCP-1 mRNA Expression in Human Adipose-Derived Stem Cells.

Fermented garlic, often called black garlic, is a traditional food ingredient used in Asian cuisine and possesses various health benefits including anti-obesity activity. The anti-obesity effects of fermented garlic might, in part, might be mediated through direct actions of its components on adipocytes. To test this hypothesis, we examined whether fermented garlic extract might stimulate the metabolic activity of human adipose-derived stem cells (ADSCs) in culture. Cell viability measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay exhibited a complex doseresponse relationship. The lowest concentration (0.4 mg/ml) reduced cell viability (P<0.05 compared to no extract, Bonferroni's multiple comparison), whereas higher concentrations (0.8 and 1.0 mg/ml) resulted in higher cell viability (P<0.05 as compared to 0.4 mg/ml). However, the extract at concentrations >2 mg/ml markedly decreased cell viability. Higher cell viability observed following treatment with 0.8~1.0 mg/ml might be associated with raised oxygen consumption. Fluorescent dye-based measurement revealed that the garlic extract at 1.0 mg/ml significantly increased oxygen consumption. We also detected a significant increase in mRNA expression levels of uncoupling protein-1 (UCP- 1). These findings suggest that fermented garlic stimulates the basal metabolic activity of human ADSCs.

Mitogen-activated protein kinases control cardiac KChIP2 gene expression.

Hypertrophied myocardium is associated with reductions in the transient outward K(+) current (Ito) and expression of pore-forming Kv4.2/4.3 and auxiliary KChIP2 subunits. Here we show that KChIP2 mRNA and protein levels are dramatically decreased to 10% to 30% of control levels in the left ventricle of aorta-constricted rats in vivo and phenylephrine (PE)-treated myocytes in vitro. PE also markedly decreases Ito density. Inhibition of protein kinase Cs (PKCs) does not affect the PE-induced reduction in KChIP2 mRNA level, whereas activation of PKC with phorbol ester (phorbol myristate [PMA]) causes a marked reduction in KChIP2 mRNA level. Pharmacological inhibition of MEKs or overexpression of a dominant-negative MEK1 increases the basal KChIP2 mRNA expression and blocks the PMA-induced decrease in auxiliary subunit mRNA level. In addition, a constitutively active MEK1 decreases the basal KChIP2 mRNA level, and PMA causes no further reduction in auxiliary subunit mRNA level in active MEK1-expressing cells. Furthermore, pharmacological inhibition of JNKs or overexpression of a dominant-negative JNK1 prevents the PE-induced, but not PMA-induced, reduction in KChIP2 mRNA expression. These results suggest that downregulation of KChIP2 expression significantly contributes to the hypertrophy-associated reduction in Ito density. They also indicate that the expression of KChIP2 mRNA is controlled by the 2 branches of mitogen-activated protein kinase pathways: JNKs play a predominant role in mediating the PE-induced reduction, whereas the MEK-ERK pathway influences the basal expression and mediates the PKC-mediated downregulation.

A new system for detecting mutations in arabidopsis thaliana and the mutational spectra resulting from ethylmethanesulfonate treatment.

A system was developed for the detection and analysis of mutations occurring on chromosomal DNA in plants. The plasmid pML4, carrying the Escherichia coli rpsL gene, a target gene for mutagenesis, was inserted into a shuttle vector, pCGN5138, to construct a plasmid which could be used for the transformation of plants. pML4 sequences were introduced into Arabidopsis thaliana mediated by Agrobacterium. The pML4 DNA was rescued from transgenic Arabidopsis plants exposed to mutagens, and the plasmids were introduced into Escherichia coli DH10B to isolate mutant clones. In this system, any form of inactivation mutation in the rpsL gene can be positively selected since it makes the E. coli cells resistant to streptomycin. Here we report that the system could detect the mutagenic effect of ethylmethanesulfonate (EMS). Further characterization of the mutants revealed that G:C to A:T transitions predominated among the EMS-induced mutations. This assay system is useful for the detection and analysis of mutations arising on chromosomal DNA in plants, and should be useful for evaluating analysis of the effects of environmental mutagens.

Fly DPP10 acts as a channel ancillary subunit and possesses peptidase activity.

Mammalian DPP6 (DPPX) and DPP10 (DPPY) belong to a family of dipeptidyl peptidases, but lack enzyme activity. Instead, these proteins form complexes with voltage-gated K(+) channels in Kv4 family to control their gating and other properties. Here, we find that the fly DPP10 ortholog acts as an ancillary subunit of Kv4 channels and digests peptides. Similarly to mammalian DPP10, the fly ortholog tightly binds to rat Kv4.3 protein. The association causes negative shifts in voltage dependence of channel activation and steady state inactivation. It also results in faster inactivation and recovery from inactivation. In addition to its channel regulatory role, fly DPP10 exhibits significant dipeptidyl peptidase activity with Gly-Pro-MCA (glycyl-L-proline 4-methylcoumaryl-7-amide) as a substrate. Heterologously expressed Flag-tagged fly DPP10 and human DPP4 show similar Km values towards this substrate. However, fly DPP10 exhibits approximately a 6-times-lower relative kcat value normalized with anti-Flag immunoreactivity than human DPP4. These results demonstrate that fly DPP10 is a dual functional protein, controlling Kv4 channel gating and removing bioactive peptides.

CPD photolyase gene from Spinacia oleracea: repair of UV-damaged DNA and expression in plant organs.

The UV-B radiation contained in solar radiation has deleterious effects on plant growth, development and physiology. Specific damage to DNA caused by UV radiation involves the cyclobutyl pyrimidine dimers (CPD) and the pyrimidine (6-4) pyrimidone photoproducts. CPDs are repaired by CPD photolyase via a UV-A/blue light-dependent mechanism. The gene for the class II CPD photolyase has been cloned from higher plants such as Arabidopsis, cucumbers and rice. We isolated and characterized the cDNA and a genomic clone encoding the spinach class II CPD photolyase. The gene consisted of 3777 bases and 9 exons. The sequence of amino acids predicted from the nucleotide sequence of the cDNA of the gene was highly homologous to that of the higher plants listed above. When a photolyase-deficient Escherichia coli strain was transformed with the cDNA, photoreactivation activity was partially restored, by the illumination with photoreactivating light, resulting in an increased survival and decreased content of CPDs in the Escherichia coli genome. In both the male and female plants, the gene was highly expressed in leaves and flowers under the condition of 14-h light and 10-h dark cycle. The expression in the roots was quite low compared with the other organs.

GATA and FOG2 transcription factors differentially regulate the promoter for Kv4.2 K(+) channel gene in cardiac myocytes and PC12 cells.

OBJECTIVE: Kv4.2 subunits are major components of transient outward K(+) channels in cardiac myocytes and somatodendritic A-type channels in neurons. To identify molecular mechanisms underlying transcriptional regulation of Kv4.2 gene, we have isolated and characterized the promoter for the rat Kv4.2 gene. METHODS: PCR-based amplification of cDNA end (5'RACE) and RNase protection assays were used to determine transcription start sites. Transient transfection of Kv4.2 promoter-luciferase reporter constructs into neonatal cardiac myocytes and PC12 cells was employed to measure activity of the Kv4.2 promoter. Cotransfection of expression vectors for the transcription factors, GATA and/or FOG2, was performed to determine the effects of these transcription factors on the Kv4.2 promoter. RESULTS: Transcription of the gene initiates at 552 bp upstream from the translation initiation site in the brain and heart. Deletion analysis revealed that the approximately 200-bp fragment encompassing this start site drives significant transcription in neonatal cardiac myocytes and PC12 cells. The transcription factors GATA4 and 6 differentially enhance activity of the minimum promoter in the two cell types: GATA4 produces a larger increase than GATA6 in cardiac myocytes, whereas the latter results in a more substantial enhancement in PC12 cells. Furthermore, the coregulator of GATA, FOG2, markedly suppresses the GATA-induced increase in myocytes, but enhances it in PC12 cells. The use of GATA mutants that are incapable of forming complexes with FOG2 indicates that the formation of GATA-FOG complexes is required for the FOG2-induced suppression in myocytes, but not for the FOG2-mediated enhancement in PC12 cells. CONCLUSION: These results indicate that GATA and FOG2 transcription factors use distinct mechanisms to control the expression of Kv4.2 gene in cardiac myocytes and PC12 cells. The lack of a GATA-binding consensus sequence in the Kv4.2 minimum promoter suggests that these transcription factors indirectly influence the channel gene transcription.

A sustained increase in the intracellular Ca²âº concentration induces proteolytic cleavage of EAG2 channel.

Voltage-gated EAG2 channel is abundant in the brain and enhances cancer cell growth by controlling cell volume. The channel contains a cyclic nucleotide-binding homology (CNBH) domain and multiple calmodulin-binding motifs. Here we show that a raised intracellular Ca(2+) concentration causes proteolytic digestion of heterologously expressed and native EAG2 channels. A treatment of EAG2-expressing cells with the Ca(2+) ionophore A23187 for 1h reduces the full-length protein by ∼80% with a concomitant appearance of 30-35-kDa peptides. Similarly, a treatment with the Ca(2+)-ATPase inhibitor thapsigargin for 3h removes 30-35-kDa peptides from ∼1/3 of the channel protein. Moreover, an incubation of the isolated rat brain membrane with CaCl2 leads to the generation of fragments with similar sizes. This Ca(2+)-induced digestion is not seen with EAG1. Mutations in a C-terminal calmodulin-binding motif alter the degrees and positions of the cleavage. Truncated channels that mimic the digested proteins exhibit a reduced current density and altered channel gating. In particular, these shorter channels lack a rapid activation typical in EAG channels with more than 20-mV positive shifts in voltage dependence of activation. The truncation also eliminates the ability of EAG2 channel to reduce cell volume. These results suggest that a sustained increase in the intracellular Ca(2+) concentration leads to proteolytic cleavage at the C-terminal cytosolic region following the CNBH domain by altering its interaction with calmodulin. The observed Ca(2+)-induced proteolytic cleavage of EAG2 channel may act as an adaptive response under physiological and/or pathological conditions.

The C-terminus SH3-binding domain of Kv1.3 is required for the actin-mediated immobilization of the channel via cortactin.

Kv1.3 channels play a pivotal role in the activation and migration of T-lymphocytes. These functions are accompanied by the channels' polarization, which is essential for associated downstream events. However, the mechanisms that govern the membrane movement of Kv1.3 channels remain unclear. F-actin polymerization occurs concomitantly to channel polarization, implicating the actin cytoskeleton in this process. Here we show that cortactin, a factor initiating the actin network, controls the membrane mobilization of Kv1.3 channels. FRAP with EGFP-tagged Kv1.3 channels demonstrates that knocking down cortactin decreases the actin-based immobilization of the channels. Using various deletion and mutation constructs, we show that the SH3 motif of Kv1.3 mediates the channel immobilization. Proximity ligation assays indicate that deletion or mutation of the SH3 motif also disrupts interaction of the channel with cortactin. In T-lymphocytes, the interaction between HS1 (the cortactin homologue) and Kv1.3 occurs at the immune synapse and requires the channel's C-terminal domain. These results show that actin dynamics regulates the membrane motility of Kv1.3 channels. They also provide evidence that the SH3 motif of the channel and cortactin plays key roles in this process.

SIRT1 suppresses the senescence-associated secretory phenotype through epigenetic gene regulation.

Senescent cells develop a pro-inflammatory response termed the senescence-associated secretory phenotype (SASP). As many SASP components affect surrounding cells and alter their microenvironment, SASP may be a key phenomenon in linking cellular senesence with individual aging and age-related diseases. We herein demonstrated that the expression of Sirtuin1 (SIRT1) was decreased and the expression of SASP components was reciprocally increased during cellular senescence. The mRNAs and proteins of SASP components, such as IL-6 and IL-8, quickly accumulated in SIRT1-depleted cells, and the levels of these factors were also higher than those in control cells, indicating that SIRT1 negatively regulated the expression of SASP factors at the transcriptional level. SIRT1 bound to the promoter regions of IL-8 and IL-6, but dissociated from them during cellular senescence. The acetylation of Histone H3 (K9) and H4 (K16) of the IL-8 and IL-6 promoter regions gradually increased during cellular senescence. In SIRT1-depleted cells, the acetylation levels of these regions were already higher than those in control cells in the pre-senescent stage. Moreover, these acetylation levels in SIRT1-depleted cells were significantly higher than those in control cells during cellular senescence. These results suggest that SIRT1 repressed the expression of SASP factors through the deacetylation of histones in their promoter regions.

Differential association of the auxiliary subunit Kvbeta2 with Kv1.4 and Kv4.3 K+ channels.

Kvbeta2 subunits associate with Kv1 and Kv4 K+ channels, but the basis of preferential association is not understood. For example, detergent resistance suggests stronger auxiliary subunit association with Kv4.2 than with Kv1.2, but Kvbeta2 preferentially localizes with the latter channels in brain. Here we examine the interaction of Kvbeta2 with two native binding partners in brain: Kv4.3 and Kv1.4. We show that the auxiliary subunit binds more efficiently to Kv1.4 than to Kv4.3 in mammalian cells. However, preexisting Kvbeta2 complexes with Kv1.4 and Kv4.3 have similar detergent sensitivity. Thus, preferential steady state binding may reflect a difference in initial association rather than stability. We also find that that the cytoplasmic C-terminus of Kv4.3 inhibits Kvbeta2 association. Apparently, a region proximal to the Kv4.3 pore contributes to the inefficient auxiliary subunit interaction that produces preferential binding of Kvbeta2 to Kv1 channels.