Recently, we demonstrated that a large conductance Ca2+-activated K+ channel (BK(Ca)) in human umbilical cord vein-derived mesenchymal cells (hUC-MSCs). In this study, we studied effects of quercetin, the flavonoid used for the modulator of cell cycle and the treatment of anti-tumor, on BK(Ca) in hUC-MSCs using single channel and/or whole cell configuration. Single channel conductance was 277.8+/-36.2 pS in hUC-MSCs and the value is similar to that of typical BK(Ca) channel reported in other cells. The single channel current was activated by increasing of intracellular Ca2+. Quercetin (30~100 micrometer) increased BK(Ca) in whole cell patch configuration whereas EGCG was not affected on the channel. The activation effect of quercetin on BK(Ca) channel was also observed in inside-out configuration. The quercetin-induced BK(Ca) was increased a concentration-dependent manner with an IC50 value of 19.9 micrometer. Taken together, BK(Ca) in hUC-MSCs can be an important target for the action of quercetin and the channels are partly may contribute to maintaining homeostasis by regulation of K ion flux in hUC-MSCs.
Cell Cycle ; Homeostasis ; Humans ; Inhibitory Concentration 50 ; Mesenchymal Stromal Cells ; Quercetin ; Umbilical Cord

Bladder cancer is a common cancer in smoking men and may correlate with mechanosensitive potassium channels because the urinary bladder is a stretch sensing organ. Two-pore K+ channels (K2P), such as TASK3 and TREK1, have recently been shown to play a critical role in both cell apoptosis and tumorigenesis. Of the channels, TREK1 can be activated by many physiological stimuli, including polyunsaturated fatty acids, and intracellular pH, hypoxia, and neurotransmitters. Here we attempted to determine whether TREK1 is functionally expressed in bladder cancer 253J cells. K2P channels, including TREK1, TREK2, TASK1, TASK3, and TWIK1, were quantified in cultured human bladder cancer 253J cells using real time quantitative RT-PCR (qRT-PCR) analysis. Among them, TREK1-like channel was recorded at a single channel level using the patch-clamp technique. The TREKl-like channel, with single-channel conductance of ~90 pS at -80 mV, was recorded in symmetrical 150 mM KCl using an excised inside-out patch configuration. The current-voltage relationships were linear and were insensitive to tetraethylammonium. The channel was activated by membrane stretch, free fatty acids, and intracellular acidosis. These results with electrophysiological properties resemble to those of K2P channel, for instance, TREK1. Therefore, we conclude that TREK1 channel is functionally present in bladder cancer 253J cells.
Acidosis ; Anoxia ; Apoptosis ; Cell Transformation, Neoplastic ; Fatty Acids, Nonesterified ; Fatty Acids, Unsaturated ; Humans* ; Hydrogen-Ion Concentration ; Male ; Membranes ; Neurotransmitter Agents ; Patch-Clamp Techniques ; Potassium Channels* ; Potassium* ; Smoke ; Smoking ; Tetraethylammonium ; Urinary Bladder Neoplasms ; Urinary Bladder*

BACKGROUND AND OBJECTIVES: Marfan syndrome is an autosomal dominant heritable disease of connective tissue which is characterized by cardinal features mainly in the cardiovascular, ocular and skeletal systems. Aneurysms or dissections of the aorta are the major cardiovascular complications of the disorder causing early mortality. Mutations in the fibrillin-1 (FBN1) gene on chromosome 15q21.1 have been found to be major causes of Marfan syndrome. The purpose of this study was to characterize the molecular defect in Korean Marfan patients, thus contributing to the effort of correlating the genotype with the phenotype. SUBJECTS AND METHODS: We screened all 65 exons of the FBN1 gene in 14 subjects diagnosed as Marfan syndrome by the method of single strand conformation polymorphism-heteroduplex analysis. RESULTS: We found mutations in only 10 among 14 patients. This study identified 8 novel mutations and 2 previously reported mutations in 14 Korean Marfan patients. Two cases were nonsense mutations and 8 were missense mutations, including 3 frameshift. Seven cases of the mutations occurred in one of the 43 calcium binding epidermal growth factor-like domains within an FBN1 gene. Mutations in Marfan patients occurred variably over the whole field of this FBN1 gene. CONCLUSION: Our results will contribute to the establishment of a database of Korean Marfan patients. Extending this study and using the database will help early detection of the disease and prevention of complications.
Aneurysm ; Aorta ; Calcium ; Codon, Nonsense ; Connective Tissue ; Exons ; Genotype ; Humans ; Marfan Syndrome* ; Mortality ; Mutation, Missense ; Phenotype

TREK (TWIK-RElated K+ channels) and TRAAK (TWIK-Related Arachidonic acid Activated K+ channels) were expressed in COS-7 cells, and the channel activities were recorded from inside-out membrane patches using holding potential of -40 mV in symmetrical 150 mM K+ solution. Intracellular application of an oxidizing agent, 5,5'-dithio-bis (2-nitrobenzoic acid) (DTNB), markedly decreased the activity of the TREK2, and the activity was partially reversed by the reducing agent, dithiothreitol (DTT). In order to examine the possibility that the target sites for the oxidizing agents might be located in the C-terminus of TREK2, two chimeras were constructed: TREK2 (1-383)/TASK3C and TREK2 (1-353)/TASK3C. The channel activity in the TREK2 (1-383)/TASK3C chimera was still inhibited by DTNB, but not in the TREK2 (1-353)/TASK3C chimera. These results indicate that TREK2 is inhibited by oxidation, and that the target site for oxidation is located between the amino acid residues 353 and 383 in the C-terminus of the TREK2 protein.
Animals ; Arachidonic Acid ; Chimera ; COS Cells ; Dithionitrobenzoic Acid ; Dithiothreitol ; Membranes ; Oxidants

No abstract available.
Asymptomatic Diseases ; Carotid Artery, Internal ; Carotid Stenosis ; Fingers

Human bone marrow mesenchymal stem cells (hBM-MSCs) represent a potentially valuable cell type for clinical therapeutic applications. The present study was designed to evaluate the effect of long-term culturing (up to 10th passages) of hBM-MSCs from eight individual amyotrophic lateral sclerosis (ALS) patients, focusing on functional ion channels. All hBM-MSCs contain several MSCs markers with no significant differences, whereas the distribution of functional ion channels was shown to be different between cells. Four types of K+ currents, including noise-like Ca+2-activated K+ current (IKCa), a transient outward K+ current (Ito), a delayed rectifier K+ current (IKDR), and an inward-rectifier K+ current (Kir) were heterogeneously present in these cells, and a TTX-sensitive Na+ current (INa,TTX) was also recorded. In the RT-PCR analysis, Kv1.1, heag1, Kv4.2, Kir2.1, MaxiK, and hNE-Na were detected. In particular, INa,TTX showed a significant passage-dependent increase. This is the first report showing that functional ion channel profiling depend on the cellular passage of hBM-MSCs
Amyotrophic Lateral Sclerosis ; Bone Marrow ; Humans ; Ion Channels ; Mesenchymal Stromal Cells ; Stem Cells

Bladder cancer is the seventh most common cancer in men that smoke, and the incidence of disease increases with age. The mechanism of occurrence has not yet been established. Potassium channels have been linked with cell proliferation. Some two-pore domain K+ channels (K2P), such as TASK3 and TREK1, have recently been shown to be overexpressed in cancer cells. Here we focused on the relationship between cell growth and the mechanosensitive K2P channel, TREK2, in the human bladder cancer cell line, 253J. We confirmed that TREK2 was expressed in bladder cancer cell lines by Western blot and quantitative real-time PCR. Using the patch-clamp technique, the mechanosensitive TREK2 channel was recorded in the presence of symmetrical 150 mM KCl solutions. In 253J cells, the TREK2 channel was activated by polyunsaturated fatty acids, intracellular acidosis at -60 mV and mechanical stretch at -40 mV or 40 mV. Furthermore, small interfering RNA (siRNA)-mediated TREK2 knockdown resulted in a slight depolarization from -19.9 mV+/-0.8 (n=116) to -8.5 mV+/-1.4 (n=74) and decreased proliferation of 253J cells, compared to negative control siRNA. 253J cells treated with TREK2 siRNA showed a significant increase in the expression of cell cycle boundary proteins p21 and p53 and also a remarkable decrease in protein expression of cyclins D1 and D3. Taken together, the TREK2 channel is present in bladder cancer cell lines and may, at least in part, contribute to cell cycle-dependent growth.
Acidosis ; Blotting, Western ; Cell Cycle ; Cell Line ; Cell Proliferation ; Cyclins ; Fatty Acids, Unsaturated ; Humans* ; Incidence ; Male ; Patch-Clamp Techniques ; Potassium Channels ; Real-Time Polymerase Chain Reaction ; RNA, Small Interfering ; Smoke ; Urinary Bladder Neoplasms ; Urinary Bladder*