Slow inactivation of the NaV1.4 sodium channel in mammalian cells is impeded by co-expression of the β1 subunit

In response to sustained depolarization or prolonged bursts of activity in spiking cells, sodium channels enter long-lived non-conducting states from which recovery at hyperpolarized potentials occurs over hundreds of milliseconds to seconds. The molecular basis for this slow inactivation remains unknown, although many functional domains of the channel have been implicated. Expression studies in Xenopus oocytes and mammalian cell lines have suggested a role for the accessory β1 subunit in slow inactivation, but the effects have been variable. We examined the effects of the β1 subunit on slow inactivation of skeletal muscle (NaV1.4) sodium channels expressed in HEK cells. Co-expression of the β1 subunit impeded slow inactivation elicited by a 30-s depolarization, such that the voltage dependence was right shifted (depolarized) and recovery was hastened. Mutational studies showed this effect was dependent upon the extracellular Ig-like domain, but was independent of the intracellular C-terminal tail. Furthermore, the β1 effect on slow inactivation was shown to be independent of the negative coupling between fast and slow inactivation.     

Regulation of Nav1.6 and Nav1.8 peripheral nerve Na+ channels by auxiliary β-subunits

Voltage-gated Na(+) (Na(v)) channels are composed of a pore-forming α-subunit and one or more auxiliary β-subunits. The present study investigated the regulation by the β-subunit of two Na(+) channels (Na(v)1.6 and Na(v)1.8) expressed in dorsal root ganglion (DRG) neurons. Single cell RT-PCR was used to show that Na(v)1.8, Na(v)1.6, and β(1)-β(3) subunits were widely expressed in individually harvested small-diameter DRG neurons. Coexpression experiments were used to assess the regulation of Na(v)1.6 and Na(v)1.8 by β-subunits. The β(1)-subunit induced a 2.3-fold increase in Na(+) current density and hyperpolarizing shifts in the activation (-4 mV) and steady-state inactivation (-4.7 mV) of heterologously expressed Na(v)1.8 channels. The β(4)-subunit caused more pronounced shifts in activation (-16.7 mV) and inactivation (-9.3 mV) but did not alter the current density of cells expressing Na(v)1.8 channels. The β(3)-subunit did not alter Na(v)1.8 gating but significantly reduced the current density by 31%. This contrasted with Na(v)1.6, where the β-subunits were relatively weak regulators of channel function. One notable exception was the β(4)-subunit, which induced a hyperpolarizing shift in activation (-7.6 mV) but no change in the inactivation or current density of Na(v)1.6. The β-subunits differentially regulated the expression and gating of Na(v)1.8 and Na(v)1.6. To further investigate the underlying regulatory mechanism, β-subunit chimeras containing portions of the strongly regulating β(1)-subunit and the weakly regulating β(2)-subunit were generated. Chimeras retaining the COOH-terminal domain of the β(1)-subunit produced hyperpolarizing shifts in gating and increased the current density of Na(v)1.8, similar to that observed for wild-type β(1)-subunits. The intracellular COOH-terminal domain of the β(1)-subunit appeared to play an essential role in the regulation of Na(v)1.8 expression and gating.     

Reversal of diabetes by βTC3 cells encapsulated in alginate beads generated by emulsion and internal gelation

Encapsulation of insulin-producing cells in alginate beads could improve the treatment of type 1 diabetes by reducing or eliminating the need for immunosuppression. We have recently adapted an emulsion and internal gelation process to β-cell encapsulation. This process has the advantages of being well suited for m(3)/h production rates and allowing the use of increased alginate concentrations. Compared with 1.5% alginate beads generated by a standard extrusion process, 5% alginate emulsion-generated beads demonstrated greater in vitro stability and greater volumetric exclusion of antibody-sized pullulan. When βTC3 cells were transplanted into streptozotocin-induced allogeneic diabetic mice, a significant decrease in the blood glucose levels was seen within 2 days with the 5% emulsion-generated beads but not until >16 days with the 1.5% extrusion-generated beads. This was correlated with higher cell survival and lower graft-specific plasma immunoglobulin levels. These results suggest that higher-concentration alginate beads generated by emulsion and internal gelation have improved graft immunoprotection. The emulsion process is a promising and scalable technology for cellular therapies requiring immune isolation.     

Aging decreases the contribution of MaxiK channel in regulating vascular tone in mesenteric artery by unparallel downregulation of α- and β1-subunit expression

Vascular disease increases in incidence with age and is the commonest cause of morbidity and mortality among elderly people. Large-conductance Ca²⁺-activated K⁺(MaxiK) channel, with pore-forming α-subunit and modulatory β1-subunit, is a key regulator of vascular tone. This study explored functional and molecular evidence of MaxiK alteration with aging in the mesenteric artery(MA). Young, Middle-aged, and Old male Wistar rats were used. Selective MaxiK channel blocker (Iberiotoxin) induced a significant increase of vascular tension in MA in all three age groups. However, these effects were greatly decreased in Old animals. The amplitude and frequency of spontaneous transient outward currents were significantly decreased with aging. Single channel recording revealed that aging induced a decrease of the open probability and the mean open time, but an increase of the mean closed time of MaxiK channel. The Ca²⁺/voltage sensitivity of MaxiK was also decreased. Western blotting showed that the protein expression of MaxiK β1- and α-subunit was significantly reduced with aging, and the suppression of β1 subunits was larger than that of α subunits. These data suggest that aging decreases capability of MaxiK channel in regulating vascular tone in the MA, which may be partially mediated by unparallel downregulation of α- and β1-subunit expression.     

Modulation of the function of dendritic cells in adolescents with chronic HBV infection by IFN-λ1

The exact immunology pathogenesis of hepatitis B virus (HBV) infection remains unclear currently. The dendritic cells (DCs) dysfunction is evident in adolescents with chronic HBV infection in the immune tolerant phase. DCs, as the most efficient professional antigen-presenting cells (APCs), possess the strongest antigen presenting the effect in the body and can stimulate the initial T cell activation and proliferation, depending on their stage of maturation. The recently classified type III interferon group, interferon-λ1 (IL-29), interferon-λ2 (IL-28A), and interferon-λ3 (IL-28B) displays immunomodulatory and antiviral activity. In the current study, we describe a way to stimulate the DCs maturation. As a result, IFN-λ1 combined with recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF) and recombinant human interleukin-4 (rhIL-4) can induce the DCs maturation and promote the costimulatory molecules such as CD80, CD83, CD86 and human leucocyte antigen DR (HLA-DR) expression in the immune tolerance and the clearance phases. This study demonstrates that the DCs function is remarkably impaired both in the immune tolerant phase and the immune clearance phase in adolescents with chronic HBV infection compared with healthy youth control. At the same time, this study has developed a theoretical basis for the application of IFN-λ1 breaking immune tolerance and improving the body’s immune system to clear HBV.     

β1-subunit modulates the Nav1.4 sodium channel by changing the surface charge

Voltage-gated sodium channels, comprised of a pore-forming α-subunit and additional regulatory (β) subunits, play a critical role in regulation of neuronal excitability. Mechanisms of regulation of β-subunits remain elusive. We have tested the functional effects of β1 sodium channel subunit on surface charges as a mechanism for channel modulation. HEK-293 cell lines permanently transfected with the sole rat skeletal muscle sodium channel α-subunit (Nav1.4), or co-expressing the sodium channel α-subunit and β1-subunit were studied with the whole-cell mode of the patch–clamp technique. At physiological extracellular Ca²⁺ concentration (2 mM), expression of β1-subunit did not produce any significant effect on the voltage-dependent properties of sodium currents. However, a shift of half-activation potentials of sodium channel by changing the extracellular Ca²⁺ was potentiated when β1 was co-expressed with α-subunit. In contrast, the expression of β1-subunit did not affect the Ca²⁺ binding to the open or to the closed sodium channel pore, difference of the effect provoked by extracellular Ca²⁺ could therefore be attributed to an increased in negative surface charge determined by the presence of β1-subunit. These data are in agreement with the hypothesis of a modulation of the sodium current by the expression of the highly sialylated β1-subunit, which would alter the channel gating by increasing the density of surface negative charges in the vicinity of the sodium channel voltage sensing machinery.     

Late Na+ current produced by human cardiac Na+ channel isoform Nav1.5 is modulated by its β1 subunit

Experimental data accumulated over the past decade show the emerging importance of the late sodium current (I _NaL) for the function of both normal and, especially, failing myocardium, in which I _NaL is reportedly increased. While recent molecular studies identified the cardiac Na⁺ channel (NaCh) α subunit isoform (Na_v1.5) as a major contributor to I _NaL, the molecular mechanisms underlying alterations of I _NaL in heart failure (HF) are still unknown. Here we tested the hypothesis that I _NaL is modulated by the NaCh auxiliary β subunits. tsA201 cells were transfected simultaneously with human Na_v1.5 (former hH1a) and cardiac β₁ or β₂ subunits, and whole-cell patch-clamp experiments were performed. We found that I _NaL decay kinetics were significantly slower in cells expressing α + β₁ (time constant τ = 0.73 ± 0.16 s, n = 14, mean ± SEM, P < 0.05) but remained unchanged in cells expressing α + β₂ (τ = 0.52 ± 0.09 s, n = 5), compared with cells expressing Na_v1.5 alone (τ = 0.54 ± 0.09 s, n = 20). Also, β₁, but not β₂, dramatically increased I _NaL relative to the maximum peak current, I _NaT (2.3 ± 0.48%, n = 14 vs. 0.48 ± 0.07%, n = 6, P < 0.05, respectively) and produced a rightward shift of the steady-state availability curve. We conclude that the auxiliary β₁ subunit modulates I _NaL, produced by the human cardiac Na⁺ channel Na_v1.5 by slowing its decay and increasing I _NaL amplitude relative to I _NaT. Because expression of Na_v1.5 reportedly decreases but β₁ remains unchanged in chronic HF, the relatively higher expression of β₁ may contribute to the known I _NaL increase in HF via the modulation mechanism found in this study. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Modulation of ATP-sensitive potassium channel activity by flash-photolysis of ‘caged-ATP’ in rat heart cells

We have used caged-ATP to investigate the kinetic behavior of K_ATP channels in ventricular cells from rat heart. In whole cells, loaded with caged-ATP, an increase of intracellular [ATP] following a UV light flash produced a decrease of K_ATP channel current that was too slow ( 300 ms) to be explained by the expected timecourse of ATP release ( 3 ms) and the time-course of channel blockade by ATP ( 20 ms). In isolated membrane patches, caged-ATP itself caused partial blockade of K_ATP channels. Under these conditions, photorelease of ATP caused channel activity to decline further. The results suggest that caged-ATP can bind to the K_ATP channel but, on binding, decreases the open probability to a lesser extent than does ATP. Additionally, the observations indicate that for photolytically-generated ATP to bind to the channel, caged-ATP must first unbind (slowly) from the channel. We conclude that caged-ATP is not fully caged with respect to its allosteric action on the K_ATP channel.     

Induction of β-catenin by the suppression of signal regulatory protein α1 in K562 cells

The signal regulatory protein (SIRP) α1 is a cell surface receptor expressed predominantly in monocytes, granulocytes, dendritic cells, as well as hematopoietic stem cells. In contrast, SIRPα1 expression is significantly reduced in the majority of myeloid malignancies. SIRPα1 is a negative regulator of signaling and its reduced expression is considered to play a role in the pathogenesis of these diseases through aberrant signaling. To identify SIRPα1 downstream target genes, we established SIRP α1-knockdown chronic myeloid leukemia K562 (K562SIRPα1KD) cells expressing reduced levels of SIRPα1 by stably transfecting SIRPα1 siRNAs. Microarray analysis demonstrated that several genes, including β-catenin, were significantly induced in K562SIRPα1KD cells. Real-time PCR and Western blot analyses, confirmed the induction of this gene. Phosphorylation of Ser9 of glycogen synthesis kinase (GSK) -3β, results in the inactivation of GSK-3β, leading to the induction of β-catenin. We found significant phosphorylation of extracellular signal-regulated kinase (ERK), Akt, as well as of GSK-3β-Ser9, which may play a role in the up-regulation of β-catenin in K562SIRPα1KD cells. To our knowledge, this is a first report demonstrating the relationships between SIRPα1 and β-catenin in leukemia cells.     

Production and expression of RANTES (CCL5) by human disc cells and modulation by IL-1-β and TNF-α in 3D culture

Chemokines act as important secondary inflammatory mediators which are released by cells in response to a variety of stimuli. Chemokines bind to cell surface receptors and act as second-order cytokines with specialized functions in inflammation. The role of RANTES (Regulated upon Activation, Normal T-cell Expressed, and Secreted) (also called CCL5 (chemokine (C–C motif) ligand 5)) has received little attention to date in disc tissue. Microarray analyses of lumbar disc annulus tissue revealed that RANTES expression was significantly upregulated in more degenerated Thompson grades IV and V discs compared to expression levels in grades I, II and III discs (p = 0.032). Immunolocalization confirmed the presence of RANTES in the annulus and nucleus of the disc, and localized the RANTES receptors CCR1, CCR3 and CCR5 to cells in the disc. In vitro studies with IL-1-β and TNF-α challenges, both proinflammatory cytokines resulted in elevated levels of RANTES in conditioned media (p < 0.01); TNF-α exposure, however, produced significantly greater levels than did IL-1alpha (p < 0.0001), suggesting a differential regulation by TNF-α. Local production of RANTES in vivo by annulus and nucleus cells, and in vitro induction of RANTES by proinflammatory cytokines suggest that disc cells are primary effector cells as well as target cells, and thus can mediate physiological immune-related processes during disc degeneration by both autocrine and paracrine signaling.     

Arachidonic acid activation of BKCa (Slo1) channels associated to the β1-subunit in human vascular smooth muscle cells

Arachidonic acid (AA) is a polyunsaturated fatty acid involved in a complex network of cell signaling. It is well known that this fatty acid can directly modulate several cellular target structures, among them, ion channels. We explored the effects of AA on high conductance Ca²⁺- and voltage-dependent K⁺ channel (BK_Ca) in vascular smooth muscle cells (VSMCs) where the presence of β₁-subunit was functionally demonstrated by lithocholic acid activation. Using patch-clamp technique, we show at the single channel level that 10 μM AA increases the open probability (Po) of BK_Ca channels tenfold, mainly by a reduction of closed dwell times. AA also induces a left-shift in Po versus voltage curves without modifying their steepness. Furthermore, AA accelerates the kinetics of the voltage channel activation by a fourfold reduction in latencies to first channel opening. When AA was tested on BK_Ca channel expressed in HEK cells with or without the β₁-subunit, activation only occurs in presence of the modulatory subunit. These results contribute to highlight the molecular mechanism of AA-dependent BK_Ca activation. We conclude that AA itself selectively activates the β₁-associated BK_Ca channel, destabilizing its closed state probably by interacting with the β₁-subunit, without modifying the channel voltage sensitivity. Since BK_Ca channels physiologically contribute to regulation of VSMCs contractility and blood pressure, we used the whole-cell configuration to show that AA is able to activate these channels, inducing significant cell hyperpolarization that can lead to VSMCs relaxation.     

Structural and functional analysis of rare missense mutations in human chorionic gonadotrophin β-subunit

Heterodimeric hCG is one of the key hormones determining early pregnancy success. We have previously identified rare missense mutations in hCGβ genes with potential pathophysiological importance. The present study assessed the impact of these mutations on the structure and function of hCG by applying a combination of in silico (sequence and structure analysis, molecular dynamics) and in vitro (co-immunoprecipitation, immuno- and bioassays) approaches. The carrier status of each mutation was determined for 1086 North-Europeans [655 patients with recurrent miscarriage (RM)/431 healthy controls from Estonia, Finland and Denmark] using PCR-restriction fragment length polymorphism. The mutation CGB5 p.Val56Leu (rs72556325) was identified in a single heterozygous RM patient and caused a structural hindrance in the formation of the hCGα/β dimer. Although the amount of the mutant hCGβ assembled into secreted intact hCG was only 10% compared with the wild-type, a stronger signaling response was triggered upon binding to its receptor, thus compensating the effect of poor dimerization. The mutation CGB8 p.Pro73Arg (rs72556345) was found in five heterozygotes (three RM cases and two control individuals) and was inherited by two of seven studied live born children. The mutation caused ~50% of secreted β-subunits to acquire an alternative conformation, but did not affect its biological activity. For the CGB8 p.Arg8Trp (rs72556341) substitution, the applied in vitro methods revealed no alterations in the assembly of intact hCG as also supported by an in silico analysis. In summary, the accumulated data indicate that only mutations with neutral or mild functional consequences might be tolerated in the major hCGβ genes CGB5 and CGB8.     

Modulation of hematopoietic colony formation of stem cells in peripheral blood by anti-TGF-β in patients with severe immunosuppression

Summary The influence of transforming growth factor- (TGF-) on hematopoiesis has been evaluated by adding blocking antibodies against TGF- to colony forming assays (CFU-c). When optimum concentrations of recombinant growth factors, granulocyte-macrophage colony stimulating factor (GM-CSF), and interleukin-3 (IL-3) were added to stem cells from the peripheral blood of healthy individuals and certain patients with tumors or HIV infection, the anti-TGF- capable of blocking 5 ng/ml of active TGF- had no significant influence on erythroid or myeloid colony formation. However, in certain immunosuppressed individuals, anti-TGF- resulted in a significant decrease of erythroid colony formation and slight suppression of myeloid colony formation. The significant inhibition of hematopoiesis by plasma of HIV patients could be due to the presence of active forms of TGF-. The results of the blocking experiments are consistent with the concept that TGF- in low concentrations is essential for erythropoiesis and myelopoiesis but that higher levels of TGF- primarily inhibit erythropoiesis in vitro. TGF- serves as a coordinating factor when efficient recruitment of granulocytes and monocytes is more essential than erythropoiesis and stem cell growth.Abbreviations BFU-E burst forming unit-erythroid - CFC colony forming cells - CFU-GEMM colony forming unit-granulocyte/erythroid/macrophage/megacaryocyte - CFU-GM colony forming unit-granulocyte/macrophage - EPO erythropoietin - GM-CSF granulocyte/macrophage-colony stimulating factor - HIV human immunodeficiency virus - IL-1 interleukin-1 - IL-3 interleukin-3 - IMDM Iscove's Modified Dulbecco's medium - PBS phosphate buffered saline - TGF- transforming growth factor- - TNF- tumor necrosis factor-     

Expression of the human β-defensin-1 induced by dehydroandrographolide in human pulmonary gland epithelial cells

In this study we observed the enhanced mRNA and protein expression of human β-defensin-1 (hBD-1) induced by dehydroandrographolide (DA) in human gland epithelial cells and explored the mechanism of DA on infection treatment. Human pulmonary gland epithelial cells were incubated with DA, then were harvested for hBD-1 expression detection. The mRNA expression of hBD-1 was detected by RT-PCR while the protein expression was detected by Western blot. It was found that DA can up-regulate mRNA and protein expression of hBD-1, the optimal concentration was 80 µM, the maximal expression of hBD-1 mRNA and protein occurred after 8 h. The DA can up-regulate mRNA and protein expression of hBD-1 in human gland epithelial cells. It suggests that β-defensin-1 may play an important role in the treatment of infective diseases with DA.     

Synoviocytes in chronic synovitis in situ and cytokine stimulated synovial cells in vitro neo-express α1, α3 and α5 chains of β1 integrins

The expression of the 1 integrins was examined immunohistochemically in synoviocytes from normal synovial membrane and from chronic synovitis of different aetiology and intensity. Normal synoviocytes were 61-positive but lacked 1 through 5. In mild inflammation type A synoviocytes neo-expressed 1, 3, and 5 chains. In severe inflammation both type A and B synoviocytes expressed 3, 4, 5, and 6 chains. The effects of inflammatory cytokines, as single agents or in combination, on the 1 integrin expression in cultured normal synoviocytes was determined by immunocytochemistry and flow cytometry. The 1 chain, while absent in unstimulated synoviocytes, was induced by interleukin-1 (IL-1), tumour necrosis factor- (TNF-), and interferon- (INF-). This effect was enhanced by combining IL-1 and TNF-. Expression of the 3 chain was up-regulated by IL-1 and, more intensely, by IFN-. Transforming growth factor (TGF-) inhibited the up-regulating effect of IL-1 and antagonized the effect of IFN- on 3 chain expression. Expression of the 5 chain was up-regulated significantly by co-stimulation through IL-1 together with TGF- or TNF-. Thus, the 1 integrin profile of cytokine activated synoviocytes in vitro resembled that of synoviocytes in synovitis in situ. These data suggest that IL-1, TNF-, IFN-, and TGF- are likely to be among the effectors regulating 1 integrin expression in synoviocytes in vivo.     

In situ expression of β1, β3 and β4 integrin subunits in non-neoplastic endothelium and vascular tumours

Endothelial cells play an important role in adhesive interactions between circulating cells and extracellular matrix proteins. In vitro studies have shown that many of these processes are mediated by a superfamily of heterodimeric transmembrane glycoproteins called integrins. The distribution patterns of 1, 3 and 4 integrin subunits in endothelial cells (EC) in situ were examined immunohistochemically on serial forzen sections of a wide range of non-neoplastic tissues and of vascular tumours, both benign and malignant. Expression of the 1 subunit was a constitutive feature of EC. Among the 1-associated subunits, 5 and 6 were broadly distributed in EC, irrespective of vessel size and microenvironment. The 3 subunit displayed intermediate levels of expression with a slight preference for small vessel EC. Presence of 1 was confined to EC of capillaries and venules/small veins. Expression of 2 in EC was inconsistent. With rare exceptions, the 4 chain was absent in EC. The 3 and v subunits were expressed in most EC, though not always concomitantly. In contrast to the 1 chain, however, these integrin subunits were absent in EC of glomerular capillaries and were expressed variably in sinusoidal EC. The 4 chain was evenly present in the great majority of EC, except for those of large vessels. In vascular tumours, the patterns of 1 and 1 to 6 subunit expression generally corresponded to those found in their non-neoplastic counterparts. Expression of 3, v and 4 chains, however, decreased in neoplasia, especially in angiosarcomas. These data show that EC dispose of broad and at the same time differential repertoires of integrin subunits that presumably reflect vessel-type associated functional differences among these cells. In vascular tumours, the orthologous distribution patterns of 1 and 1 to 6 chains are conserved in most instances while the amounts of 3, v and 4 subunits expressed in EC tend to decrease in the course of malignant transformation.Dedicated to Prof. Dr. med. Dres. h.c. Wilhelm Doerr on the occasion of his 80th birthday     

Modulation of Na+channel inactivation by the β1 subunit: A deletion analysis

Na⁺ currents recorded from Xenopus oocytes expressing the Na⁺ channel subunit alone inactivate with two exponential components. The slow component predominates in monomeric channels, while coexpression with the ₁ subunit favors the fast component. Macropatch recordings show that the relative rates of these components are much greater than previously estimated from two-electrode measurements (30-fold vs 5-fold). A re-assessment of steady-state inactivation, h (V), shows that there is no depolarized shift of the slow component, provided a sufficiently long prepulse duration and repetition interval are used to achieve steady-state entry and recovery from inactivation, respectively. Deletion mutagenesis of the ₁ subunit was used to define which regions of the subunit are required to modulate inactivation kinetics. The carboxy tail, comprising the entire predicted intracellular domain, can be deleted without a loss of activity; whereas small deletions in the extracellular amino domain or the signal peptide totally disrupt function.     

Absence of regulation of the T-type calcium current by Cav1.1, β1a and γ1 dihydropyridine receptor subunits in skeletal muscle cells

The subunit structure of low voltage activated T-type Ca²⁺ channels is still unknown. Co-expression of dihydropyridine receptor (DHPR) auxiliary subunits with T-type α₁ subunits in heterologous systems has produced conflicting results. In developing foetal skeletal muscle fibres which abundantly express DHPR subunits, Ca_v3.2 (α_1H) subunits are believed to underlie T-type calcium currents which disappear 2 to 3 weeks after birth. Therefore, a possible regulation of foetal skeletal muscle T-type Ca²⁺ channels by DHPR subunits was investigated in freshly isolated foetal skeletal muscle using knockout mice, which provide a powerful tool to address this question. The possible involvement of α_1S (Ca_v1.1), β₁ and γ₁ DHPR subunits was tested using dysgenic (α_1S-null), β_1a and γ₁ knockout mice. The results show that the absence of α_1S, β₁ or γ₁ DHPR subunits does not significantly affect the electrophysiological properties of T-type Ca²⁺ currents in skeletal muscle, suggesting that (1) native Ca_v3.2 is not regulated by β₁ or γ₁ DHPR subunits; (2) T-type and L-type currents have distinct and not interchangeable roles.