Charade of the SR K-Channel: Two Ion-Channels, TRIC-A and TRIC-B, Masquerade as a Single K-Channel

The presence of a sarcoplasmic reticulum (SR) K⁺-selective ion-channel has been known for >30 years yet the molecular identity of this channel has remained a mystery. Recently, an SR trimeric intracellular cation channel (TRIC-A) was identified but it did not exhibit all expected characteristics of the SR K⁺-channel. We show that a related SR protein, TRIC-B, also behaves as a cation-selective ion-channel. Comparison of the single-channel properties of purified TRIC-A and TRIC-B in symmetrical 210 mM K⁺ solutions, show that TRIC-B has a single-channel conductance of 138 pS with subconductance levels of 59 and 35 pS, whereas TRIC-A exhibits full- and subconductance open states of 192 and 129 pS respectively. We suggest that the K⁺-current fluctuations observed after incorporating cardiac or skeletal SR into bilayers, can be explained by the gating of both TRIC-A and TRIC-B channels suggesting that the SR K⁺-channel is not a single, distinct entity. Importantly, TRIC-A is regulated strongly by trans-membrane voltage whereas TRIC-B is activated primarily by micromolar cytosolic Ca²⁺ and inhibited by luminal Ca²⁺. Thus, TRIC-A and TRIC-B channels are regulated by different mechanisms, thereby providing maximum flexibility and scope for facilitating monovalent cation flux across the SR membrane.     

Melting Curve Analysis after T Allele Enrichment (MelcaTle) as a Highly Sensitive and Reliable Method for Detecting the JAK2V617F Mutation

Detection of the JAK2V617F mutation is essential for diagnosing patients with classical myeloproliferative neoplasms (MPNs). However, detection of the low-frequency JAK2V617F mutation is a challenging task due to the necessity of discriminating between true-positive and false-positive results. Here, we have developed a highly sensitive and accurate assay for the detection of JAK2V617F and named it melting curve analysis after T allele enrichment (MelcaTle). MelcaTle comprises three steps: 1) two cycles of JAK2V617F allele enrichment by PCR amplification followed by BsaXI digestion, 2) selective amplification of the JAK2V617F allele in the presence of a bridged nucleic acid (BNA) probe, and 3) a melting curve assay using a BODIPY-FL-labeled oligonucleotide. Using this assay, we successfully detected nearly a single copy of the JAK2V617F allele, without false-positive signals, using 10 ng of genomic DNA standard. Furthermore, MelcaTle showed no positive signals in 90 assays screening healthy individuals for JAK2V617F. When applying MelcaTle to 27 patients who were initially classified as JAK2V617F-positive on the basis of allele-specific PCR analysis and were thus suspected as having MPNs, we found that two of the patients were actually JAK2V617F-negative. A more careful clinical data analysis revealed that these two patients had developed transient erythrocytosis of unknown etiology but not polycythemia vera, a subtype of MPNs. These findings indicate that the newly developed MelcaTle assay should markedly improve the diagnosis of JAK2V617F-positive MPNs.     

Monocarboxylate transporter-1 is required for cell death in mouse chondrocytic ATDC5 cells exposed to interleukin-1beta via late phase activation of nuclear factor kappaB and expression of phagocyte-type NADPH oxidase

Interleukin-1β (IL-1β) induces cell death in chondrocytes in a nitric oxide (NO)- and reactive oxygen species (ROS)-dependent manner. In this study, increased production of lactate was observed in IL-1β-treated mouse chondrocytic ATDC5 cells prior to the onset of their death. IL-1β-induced cell death in ATDC5 cells was suppressed by introducing an siRNA for monocarboxylate transporter-1 (MCT-1), a lactate transporter distributed in plasma and mitochondrial inner membranes. Mct-1 knockdown also prevented IL-1β-induced expression of phagocyte-type NADPH oxidase (NOX-2), an enzyme specialized for production of ROS, whereas it did not have an effect on inducible NO synthase. Suppression of IL-1β-induced cell death by Nox-2 siRNA indicated that NOX-2 is involved in cell death. Phosphorylation and degradation of inhibitor of κBα (IκBα) from 5 to 20 min after the addition of IL-1β was not affected by Mct-1 siRNA. In addition, IκBα was slightly decreased after 12 h of incubation with IL-1β, and the decrease was prominent after 36 h, whereas activation of p65/RelA was observed from 12 to 48 h after exposure to IL-1β. These changes were not seen in Mct-1-silenced cells. Forced expression of IκBα super repressor as well as treatment with the IκB kinase inhibitor BAY 11-7082 suppressed NOX-2 expression. Furthermore, Mct-1 siRNA lowered the level of ROS generated after 15-h exposure to IL-1β, whereas a ROS scavenger, N-acetylcysteine, suppressed both late phase degradation of IκBα and Nox-2 expression. These results suggest that MCT-1 contributes to NOX-2 expression via late phase activation of NF-κB in a ROS-dependent manner in ATDC5 cells exposed to IL-1β.     

Characterization of a bicistronic knock-in reporter mouse model for investigating the role of CABLES2 in vivo

Two members of the CDK5 and ABL enzyme substrate (CABLES) family, CABLES1 and CABLES2, share a highly homologous C-terminus. They interact and associate with cyclin-dependent kinase 3 (CDK3), CDK5, and c-ABL. CABLES1 mediates tumor suppression, regulates cell proliferation, and prevents protein degradation. Although Cables2 is ubiquitously expressed in adult mouse tissues at RNA level, the role of CABLES2 in vivo remains unknown. Here, we generated bicistronic Cables2 knock-in reporter mice that expressed CABLES2 tagged with 3×FLAG and 2A-mediated fluorescent reporter tdTomato. Cables2-3×FLAG-2A-tdTomato (Cables2^Tom) mice confirmed the expression of Cables2 in various mouse tissues. Interestingly, high intensity of tdTomato fluorescence was observed in the brain, testis and ovary, especially in the corpus luteum. Furthermore, immunoprecipitation analysis using the brain and testis in Cables2^Tom/Tom revealed interaction of CABLES2 with CDK5. Collectively, our new Cables2 knock-in reporter model will enable the comprehensive analysis of in vivo CABLES2 function.     

The Yeast RER2 Gene, Identified by Endoplasmic Reticulum Protein Localization Mutations, Encodes cis-Prenyltransferase, a Key Enzyme in Dolichol Synthesis

As an approach to understand the molecular mechanisms of endoplasmic reticulum (ER) protein sorting, we have isolated yeast rer mutants that mislocalize a Sec12-Mfα1p fusion protein from the ER to later compartments of the secretory pathway (S. Nishikawa and A. Nakano, Proc. Natl. Acad. Sci. USA 90:8179–8183, 1993). The temperature-sensitive rer2 mutant mislocalizes different types of ER membrane proteins, suggesting that RER2 is involved in correct localization of ER proteins in general. The rer2 mutant shows several other characteristic phenotypes: slow growth, defects in N and O glycosylation, sensitivity to hygromycin B, and abnormal accumulation of membranes, including the ER and the Golgi membranes. RER2 and SRT1, a gene whose overexpression suppresses rer2, encode novel proteins similar to each other, and their double disruption is lethal. RER2 homologues are found not only in eukaryotes but also in many prokaryote species and thus constitute a large gene family which has been well conserved during evolution. Taking a hint from the phenotype of newly established mutants of the Rer2p homologue of Escherichia coli, we discovered that the rer2 mutant is deficient in the activity of cis-prenyltransferase, a key enzyme of dolichol synthesis. This and other lines of evidence let us conclude that members of the RER2 family of genes encode cis-prenyltransferase itself. The difference in phenotypes between the rer2 mutant and previously obtained glycosylation mutants suggests a novel, as-yet-unknown role of dolichol.     

Molecular pathogenesis of a novel mutation, G108D, in short-chain acyl-CoA dehydrogenase identified in subjects with short-chain acyl-CoA dehydrogenase deficiency

Short-chain acyl-CoA dehydrogenase (SCAD) is a mitochondrial enzyme involved in the β-oxidation of fatty acids. Genetic defect of SCAD was documented to cause clinical symptoms such as progressive psychomotor retardation, muscle hypotonia, and myopathy in early reports. However, clinical significance of SCAD deficiency (SCADD) has been getting ambiguous, for some variants in the ACADS gene, which encodes the SCAD protein, has turned out to be widely prevailed among general populations. Accordingly, the pathophysiology of SCADD has not been clarified thus far. The present report focuses on two suspected cases of SCADD detected through the screening of newborns by tandem mass spectrometry. In both subjects, compound heterozygous mutations in ACADS were detected. The mutated genes were expressed in a transient gene expression system, and the enzymatic activities of the obtained mutant SCAD proteins were measured. The activities of the mutant SCAD proteins were significantly lower than that of the wild-type enzyme, confirming the mechanism underlying the diagnosis of SCADD in both subjects. Moreover, the mutant SCAD proteins gave rise to mitochondrial fragmentation and autophagy, both of which were proportional to the decrease in SCAD activities. The association of autophagy with programed cell death suggests that the mutant SCAD proteins are toxic to mitochondria and to the cells in which they are expressed. The expression of recombinant ACADS-encoded mutant proteins offers a technique to evaluate both the nature of the defective SCAD proteins and their toxicity. Moreover, our results provide insight into possible molecular pathophysiology of SCADD.     

Surface-expressed TLR6 participates in the recognition of diacylated lipopeptide and peptidoglycan in human cells

Recognition of microbial components by TLR2 requires cooperation with other TLRs. TLR6 has been shown to be required for the recognition of diacylated lipoproteins and lipopeptides derived from mycoplasma and to activate the NF-kappaB signaling cascade in conjunction with TLR2. Human TLR2 is expressed on the cell surface in a variety of cells, including monocytes, neutrophils, and monocyte-derived, immature dendritic cells (iDCs), whereas the expression profile of TLR6 in human cells remains obscure. In this study we produced a function-blocking mAb against human TLR6 and analyzed TLR6 expression in human blood cells and cell lines and its participation in ligand recognition. TLR6 was expressed, although at a lower level than TLR2, on the cell surface in monocytes, monocyte-derived iDCs, and neutrophils, but not on B, T, or NK cells. Confocal microscopic analysis revealed that TLR6 was colocalized with TLR2 at the plasma membrane of monocytes. Importantly, TLR2/6 signaling did not require endosomal maturation, and anti-TLR6 mAb inhibited cytokine production in monocytes and iDCs stimulated with synthetic macrophage-activating lipopeptide-2 or peptidoglycan, indicating that TLR6 recognized diacylated lipopeptide and peptidoglycan at the cell surface. In addition, TLR2 mutants C30S and C36S (Cys(30) and Cys(36) in TLR2 were substituted with Ser), which were expressed intracellularly in HEK293 cells, failed to induce NF-kappaB activation upon macrophage-activating lipopeptide-2 stimulation even in the presence of TLR6. Thus, coexpression of TLR2 and TLR6 at the cell surface is crucial for recognition of diacylated lipopeptide and peptidoglycan and subsequent cellular activation in human cells.     

Activation of protein kinase C epsilon inhibits the two-pore domain K+ channel, TASK-1, inducing repolarization abnormalities in cardiac ventricular myocytes

Activation of the platelet-activating factor (PAF) receptor leads to a decrease in outward current in murine ventricular myocytes by inhibiting the TASK-1 channel. TASK-1 carries a background or "leak" current and is a member of the two-pore domain potassium channel family. Its inhibition is sufficient to delay repolarization, causing prolongation of the action potential duration, and in some cases, early after depolarizations. We set out to determine the cellular mechanisms that control regulation of TASK-1 by PAF. Inhibition of TASK-1 via activation of the PAF receptor is protein kinase C (PKC)-dependent. Using isoform-specific PKC inhibitor or activator peptides in patch clamp experiments, we now demonstrate that activation of PKCepsilon is both necessary and sufficient to regulate murine TASK-1 current in a heterologous expression system and to induce repolarization abnormalities in isolated myocytes. Furthermore, site-directed mutagenesis studies have identified threonine 381, in the C-terminal tail of murine TASK-1, as a critical residue in this regulation.