Calcium homeostasis modulator 2 (Calhm2) as slowly activating membrane current channel in mouse B cells.

In mouse B lymphocytes, an unidentified slow-activating voltage-dependent current resembling the characteristics of the Calhm family ion channel (ICalhm-L) was investigated. RT-PCR analysis revealed the presence of Calhm2 and 6 transcripts, with subsequent whole-cell patch-clamp studies indicating that the ICalhm-L is augmented by heat, alkaline pH, and low extracellular [Ca2+]. Overexpression of Calhm2, but not Calhm6, in N2A cells recapitulated ICalhm-L. Moreover, Calhm2 knockdown in Bal-17 cells abolished ICalhm-L. We firstly identify the voltage-dependent ion channel function of the Calhm2 in the mouse immune cells. ATP release assays in primary mouse B cells suggested a significant contribution of Calhm2 for purinergic signaling at physiological temperature.

Identification and Characterization of a Novel Large-Conductance Calcium-Activated Potassium Channel Activator, CTIBD, and Its Relaxation Effect on Urinary Bladder Smooth Muscle.

The large-conductance calcium-activated potassium channel (BKCa channel) is expressed on various tissues and is involved in smooth muscle relaxation. The channel is highly expressed on urinary bladder smooth muscle cells and regulates the repolarization phase of the spontaneous action potentials that control muscle contraction. To discover novel chemical activators of the BKCa channel, we screened a chemical library containing 8364 chemical compounds using a cell-based fluorescence assay. A chemical compound containing an isoxazolyl benzene skeleton (compound 1) was identified as a potent activator of the BKCa channel and was structurally optimized through a structure-activity relationship study to obtain 4-(4-(4-chlorophenyl)-3-(trifluoromethyl)isoxazol-5-yl)benzene-1,3-diol (CTIBD). When CTIBD was applied to the treated extracellular side of the channel, the conductance-voltage relationship of the channel shifted toward a negative value, and the maximum conductance increased in a concentration-dependent manner. CTIBD altered the gating kinetics of the channel by dramatically slowing channel closing without effecting channel opening. The effects of CTIBD on bladder muscle relaxation and micturition function were tested in rat tissue and in vivo. CTIBD concentration-dependently reduced acetylcholine-induced contraction of urinary bladder smooth muscle strips. In an acetic acid-induced overactive bladder (OAB) model, intraperitoneal injection of 20 mg/kg CTIBD effectively restored frequent voiding contraction and lowered voiding volume without affecting other bladder function parameters. Thus, our results indicate that CTIBD and its derivatives are novel chemical activators of the bladder BKCa channel and potential candidates for OAB therapeutics. SIGNIFICANCE STATEMENT: The novel BKCa channel activator CTIBD was identified and characterized in this study. CTIBD directly activates the BKCa channel and relaxes urinary bladder smooth muscle of rat, so CTIBD can be a potential candidate for overactive bladder therapeutics.

Targeted epigenetic modulation using a DNA-based histone deacetylase inhibitor enhances cardiomyogenesis in mouse embryonic stem cells.

The epigenome has an essential role in orchestrating transcriptional activation and modulating key developmental processes. Previously, we developed a library of pyrrole-imidazole polyamides (PIPs) conjugated with suberoylanilide hydroxamic acid (SAHA), a histone deacetylase (HDAC) inhibitor, for the purpose of sequence-specific modification of epigenetics. Based on the gene expression profile of SAHA-PIPs and screening studies using the α-myosin heavy chain promoter-driven reporter and SAHA-PIP library, we identified that SAHA-PIP G activates cardiac-related genes. Studies in mouse ES cells showed that SAHA-PIP G could enhance the generation of spontaneous beating cells, which is consistent with upregulation of several cardiac-related genes. Moreover, ChIP-seq results confirmed that the upregulation of cardiac-related genes is highly correlated with epigenetic activation, relevant to the sequence-specific binding of SAHA-PIP G. This proof-of-concept study demonstrating the applicability of SAHA-PIP not only improves our understanding of epigenetic alterations involved in cardiomyogenesis but also provides a novel chemical-based strategy for stem cell differentiation.

Nanoscale imaging of rat atrial myocytes by scanning ion conductance microscopy reveals heterogeneity of T-tubule openings and ultrastructure of the cell membrane.

In contrast to ventricular myocytes, the structural and functional importance of atrial transverse tubules (T-tubules) is not fully understood. Therefore, we investigated the ultrastructure of T-tubules of living rat atrial myocytes in comparison with ventricular myocytes. Nanoscale cell surface imaging by scanning ion conductance microscopy (SICM) was accompanied by confocal imaging of intracellular T-tubule network, and the effect of removal of T-tubules on atrial excitation-contraction coupling (EC-coupling) was observed. By SICM imaging, we classified atrial cell surface into 4 subtypes. About 38% of atrial myocytes had smooth cell surface with no clear T-tubule openings and intracellular T-tubules (smooth-type). In 33% of cells, we found a novel membrane nanostructure running in the direction of cell length and named it 'longitudinal fissures' (LFs-type). Interestingly, T-tubule openings were often found inside the LFs. About 17% of atrial cells resembled ventricular myocytes, but they had smaller T-tubule openings and a lower Z-groove ratio than the ventricle (ventricular-type). The remaining 12% of cells showed a mixed structure of each subtype (mixed-type). The LFs-, ventricular-, and mixed-type had an appreciable amount of reticular form of intracellular T-tubules. Formamide-induced detubulation effectively removed atrial T-tubules, which was confirmed by both confocal images and decreased cell capacitance. However, the LFs remained intact after detubulation. Detubulation reduced action potential duration and L-type Ca2+channel (LTCC) density, and prolonged relaxation time of the myocytes. Taken together, we observed heterogeneity of rat atrial T-tubules and membranous ultrastructure, and the alteration of atrial EC-coupling by disruption of T-tubules.

TrpA1 Regulates Defecation of Food-Borne Pathogens under the Control of the Duox Pathway.

Pathogen expulsion from the gut is an important defense strategy against infection, but little is known about how interaction between the intestinal microbiome and host immunity modulates defecation. In Drosophila melanogaster, dual oxidase (Duox) kills pathogenic microbes by generating the microbicidal reactive oxygen species (ROS), hypochlorous acid (HOCl) in response to bacterially excreted uracil. The physiological function of enzymatically generated HOCl in the gut is, however, unknown aside from its anti-microbial activity. Drosophila TRPA1 is an evolutionarily conserved receptor for reactive chemicals like HOCl, but a role for this molecule in mediating responses to gut microbial content has not been described. Here we identify a molecular mechanism through which bacteria-produced uracil facilitates pathogen-clearing defecation. Ingestion of uracil increases defecation frequency, requiring the Duox pathway and TrpA1. The TrpA1(A) transcript spliced with exon10b (TrpA1(A)10b) that is present in a subset of midgut enteroendocrine cells (EECs) is critical for uracil-dependent defecation. TRPA1(A)10b heterologously expressed in Xenopus oocytes is an excellent HOCl receptor characterized with elevated sensitivity and fast activation kinetics of macroscopic HOCl-evoked currents compared to those of the alternative TRPA1(A)10a isoform. Consistent with TrpA1's role in defecation, uracil-excreting Erwinia carotovora showed higher persistence in TrpA1-deficient guts. Taken together, our results propose that the uracil/Duox pathway promotes bacteria expulsion from the gut through the HOCl-sensitive receptor, TRPA1(A)10b, thereby minimizing the chances that bacteria adapt to survive host defense systems.

Deficiency of Anoctamin 5/TMEM16E causes nuclear positioning defect and impairs Ca2+ signaling of differentiated C2C12 myotubes.

Anoctamin 5 (ANO5)/TMEM16E belongs to a member of the ANO/TMEM16 family member of anion channels. However, it is a matter of debate whether ANO5 functions as a genuine plasma membrane chloride channel. It has been recognized that mutations in the ANO5 gene cause many skeletal muscle diseases such as limb girdle muscular dystrophy type 2L (LGMD2L) and Miyoshi muscular dystrophy type 3 (MMD3) in human. However, the molecular mechanisms of the skeletal myopathies caused by ANO5 defects are poorly understood. To understand the role of ANO5 in skeletal muscle development and function, we silenced the ANO5 gene in C2C12 myoblasts and evaluated whether it impairs myogenesis and myotube function. ANO5 knockdown (ANO5-KD) by shRNA resulted in clustered or aggregated nuclei at the body of myotubes without affecting differentiation or myotube formation. Nuclear positioning defect of ANO5-KD myotubes was accompanied with reduced expression of Kif5b protein, a kinesin-related motor protein that controls nuclear transport during myogenesis. ANO5-KD impaired depolarization-induced [Ca2+]i transient and reduced sarcoplasmic reticulum (SR) Ca2+ storage. ANO5-KD resulted in reduced protein expression of the dihydropyridine receptor (DHPR) and SR Ca2+-ATPase subtype 1. In addition, ANO5-KD compromised co-localization between DHPR and ryanodine receptor subtype 1. It is concluded that ANO5-KD causes nuclear positioning defect by reduction of Kif5b expression, and compromises Ca2+ signaling by downregulating the expression of DHPR and SERCA proteins.

Positive feedback control between STIM1 and NFATc3 is required for C2C12 myoblast differentiation.

Up-regulation of STIM1-mediated store-operated Ca(2+) entry (SOCE) and Ca(2+)-dependent NFAT signaling is important for myogenic differentiation. However, the molecular mechanisms for differentiation specific up-regulation of STIM1/SOCE-mediated signaling are poorly understood. This study explored whether functional crosstalk between STIM1 and a member of NFAT transcription factor is important for C2C12 myoblast differentiation. Transient increase of NFATc3 expression was observed in the initial phase of differentiation, and the increased activity of NFATc3 isoform was correlated with up-regulation of STIM1 expression. Overexpression of NFATc3 increased STIM1 expression, SOCE activity, and myotube formation, whereas NFATc3 knockdown showed the opposite effects. Overexpression of STIM1 increased the activity and expression level of NFATc3, and enhanced myotube formation, whereas STIM1 knockdown resulted in the opposite effects. Taken together, our findings suggest that a positive feedback control between STIM1/SOCE and NFATc3 is required for efficient induction and progression of myoblast differentiation.

Mechanism of Relaxation Via TASK-2 Channels in Uterine Circular Muscle of Mouse.

Plasma pH can be altered during pregnancy and at labor. Membrane excitability of smooth muscle including uterine muscle is suppressed by the activation of K(+) channels. Because contractility of uterine muscle is regulated by extracellular pH and humoral factors, K(+) conductance could be connected to factors regulating uterine contractility during pregnancy. Here, we showed that TASK-2 inhibitors such as quinidine, lidocaine, and extracellular acidosis produced contraction in uterine circular muscle of mouse. Furthermore, contractility was significantly increased in pregnant uterine circular muscle than that of non-pregnant muscle. These patterns were not changed even in the presence of tetraetylammonium (TEA) and 4-aminopyridine (4-AP). Finally, TASK-2 inhibitors induced strong myometrial contraction even in the presence of L-methionine, a known inhibitor of stretchactivated channels in myometrium. When compared to non-pregnant myometrium, pregnant myometrium showed increased immunohistochemical expression of TASK-2. Therefore, TASK-2, seems to play a key role during regulation of myometrial contractility in the pregnancy and provides new insight into preventing preterm delivery.

Differential pathways for calcium influx activated by concanavalin A and CD3 stimulation in Jurkat T cells.

Sustained increase in [Ca(2+)](c) (Δ[Ca(2+)](c)) is a critical early signal from T-cell receptor (TCR/CD3). In general, Ca(2+)-release activated Ca(2+) channels (CRAC) are responsible for the Ca(2+) influx and Δ[Ca(2+)](c) after TCR/CD3 stimulation. However, T cells also express Ca(2+)-permeable nonselective cation channels such as TRPM2 and TRPC. Gd(3+) is a relatively selective blocker for CRAC at micromolar concentrations. Here, Jurkat T cells were used to investigate the Gd(3+)-resistant Ca(2+) influx (Δ[Ca(2+)](c,Gd)) induced by concanavalin A (ConA, 1 µg/ml), a widely used mitogenic agent for T cells, or by anti-CD3 Ab (αCD3). αCD3-induced Δ[Ca(2+)](c) was partly (~60%) inhibited by 1 µM Gd(3+) while thapsigargin-induced Δ[Ca(2+)] was almost completely abolished. ConA-induced Δ[Ca(2+)] was mostly inhibited by 1 µM Gd(3+) during the early phase (<30 s of ConA application) and became resistant during the late phase (>2 min). Induction of Δ[Ca(2+)](c,Gd) by αCD3 and ConA was inhibited by 2-aminoethoxydiphenyl borate (2-APB) and by N-(p-amylcinnamoyl) anthranilic acid, indicating that TRPM2 and TRPC are involved in this process. Treatment with Pyr-3, a TRPC3-specific inhibitor, potently suppressed Δ[Ca(2+)](c,Gd) by αCD3 (IC(50), 0.16 µM). Patch clamp experiments demonstrated that the TRPM2 channels were activated by ConA, and the TRPC-like channels were activated by αCD3. Our present study suggests that TRPM2 and TRPC3 are activated by ConA and TCR/CD3, respectively, in Jurkat T cells and are responsible for the induction of Δ[Ca(2+)](c,Gd).

Kir3.1 channel is functionally involved in TLR4-mediated signaling.

We aimed to study the involvement of Kir3.1 channel in TLR4-mediated signaling. LPS stimulation induced the recruitment of TLR4 and Kir3.1 into the lipid raft in THP-1 cells. Treatment with Tertiapin-Q, an inhibitor of Kir3.1, markedly abolished the recruitment of TLR4 into the lipid raft and inhibited the LPS-induced NF-κB activation, resulting in decreased production of TNF-α, IL-1ß, and IL-6. To verify the specific role of the Kir3.1 channel, we generated Kir3.1-knockdown THP-1 cells. The Kir3.1(KD) THP-1 cells exhibited inhibition of NF-κB activation and production of these pro-inflammatory cytokines in response to TLR4 stimulation. Taken together, our results demonstrate that the Kir3.1 channel is involved in the TLR4-mediated signal at an early event by facilitating the recruitment of TLR4 into lipid raft.

The three muscle layers in the pyloric sphincter and their possible function during antropyloroduodenal motility.

This study was conducted to determine the muscular arrangement of the human pyloric sphincter using a comprehensive approach that involved microdissection, histology, and microcomputed tomography (micro-CT). The stomachs of 80 embalmed Korean adult cadavers were obtained. In all specimens, loose muscular tissue of the innermost aspect of the sphincter wall ran aborally, forming the newly found inner longitudinal muscle bundles, entered the duodenum, and connected with the nearby circular bundles. In all specimens, approximately one-third of the outer longitudinal layer of the sphincter entered its inner circular layer, divided the circular layer into several parts, and finally connected with the circular bundles. Anatomical findings around the sphincter were confirmed in micro-CT images. The sphincter wall comprised three layers: an inner layer of longitudinal bundles, a middle layer of major circular and minor longitudinal bundles, and an outer layer of longitudinal bundles. The stomach outer longitudinal bundles were connected to the sphincter circular bundles. The inner longitudinal bundles of the sphincter were connected to the adjacent circular bundles of the duodenum.

Biomimetic cell-actuated artificial muscle with nanofibrous bundles.

Biohybrid artificial muscle produced by integrating living muscle cells and their scaffolds with free movement in vivo is promising for advanced biomedical applications, including cell-based microrobotic systems and therapeutic drug delivery systems. Herein, we provide a biohybrid artificial muscle constructed by integrating living muscle cells and their scaffolds, inspired by bundled myofilaments in skeletal muscle. First, a bundled biohybrid artificial muscle was fabricated by the integration of skeletal muscle cells and hydrophilic polyurethane (HPU)/carbon nanotube (CNT) nanofibers into a fiber shape similar to that of natural skeletal muscle. The HPU/CNT nanofibers provided a stretchable basic backbone of the 3-dimensional fiber structure, which is similar to actin-myosin scaffolds. The incorporated skeletal muscle fibers contribute to the actuation of biohybrid artificial muscle. In fact, electrical field stimulation reversibly leads to the contraction of biohybrid artificial muscle. Therefore, the current development of cell-actuated artificial muscle provides great potential for energy delivery systems as actuators for implantable medibot movement and drug delivery systems. Moreover, the innervation of the biohybrid artificial muscle with motor neurons is of great interest for human-machine interfaces.

Novel KCNQ4 variants in different functional domains confer genotype- and mechanism-based therapeutics in patients with nonsyndromic hearing loss.

Loss-of-function variant in the gene encoding the KCNQ4 potassium channel causes autosomal dominant nonsyndromic hearing loss (DFNA2), and no effective pharmacotherapeutics have been developed to reverse channel activity impairment. Phosphatidylinositol 4,5-bisphosphate (PIP2), an obligatory phospholipid for maintaining KCNQ channel activity, confers differential pharmacological sensitivity of channels to KCNQ openers. Through whole-exome sequencing of DFNA2 families, we identified three novel KCNQ4 variants related to diverse auditory phenotypes in the proximal C-terminus (p.Arg331Gln), the C-terminus of the S6 segment (p.Gly319Asp), and the pore region (p.Ala271_Asp272del). Potassium currents in HEK293T cells expressing each KCNQ4 variant were recorded by patch-clamp, and functional recovery by PIP2 expression or KCNQ openers was examined. In the homomeric expression setting, the three novel KCNQ4 mutant proteins lost conductance and were unresponsive to KCNQ openers or PIP2 expression. Loss of p.Arg331Gln conductance was slightly restored by a tandem concatemer channel (WT-p.R331Q), and increased PIP2 expression further increased the concatemer current to the level of the WT channel. Strikingly, an impaired homomeric p.Gly319Asp channel exhibited hyperactivity when a concatemer (WT-p.G319D), with a negative shift in the voltage dependence of activation. Correspondingly, a KCNQ inhibitor and chelation of PIP2 effectively downregulated the hyperactive WT-p.G319D concatemer channel. Conversely, the pore-region variant (p.Ala271_Asp272del) was nonrescuable under any condition. Collectively, these novel KCNQ4 variants may constitute therapeutic targets that can be manipulated by the PIP2 level and KCNQ-regulating drugs under the physiological context of heterozygous expression. Our research contributes to the establishment of a genotype/mechanism-based therapeutic portfolio for DFNA2.

TNF-alpha inhibits the CD3-mediated upregulation of voltage-gated K+ channel (Kv1.3) in human T cells.

A long term treatment of T cells with tumor necrosis factor alpha (TNF-alpha) paradoxically inhibits the immunologic responses to TCR/CD3 stimulation. The voltage-gated K(+) channels (K(v)) of T cells attracted attention as a pharmacological target for the treatment of autoimmune diseases. Here, the authors investigated the effects of TNF-alpha on the K(v) current (I(Kv)) and its upregulation by CD3 in human T cells. Acute treatment with TNF-alpha (10 min) temporarily decreased I(Kv) in Jurkat-T cells (cells subsequently recovered after treatment >12h), whereas CD3 stimulation for 24h increased I(Kv) amplitude more than two-fold. Furthermore, chronic pretreatment with TNF-alpha almost completely blocked the I(Kv) increase induced by CD3 stimulation. An immunoblot study confirmed an increase in the protein level of K(v) induced by CD3 stimulation, and its inhibition by TNF-alpha pretreatment. In addition, the facilitation of I(Kv) by CD3 stimulation and its inhibition by pretreatment with TNF-alpha were confirmed in freshly isolated human peripheral CD4(+) T cells, in which the voltage-dependence of I(Kv) was unaffected by TNF-alpha and/or CD3 stimulation. We conclude that the inhibition of CD3-induced K(v) upregulation by TNF-alpha might be associated with the paradoxical suppression of T cell function by TNF-alpha under conditions of chronic inflammation.

Multiple transport modes of the cardiac Na+/Ca2+ exchanger.

The cardiac Na+/Ca2+ exchanger (NCX1; ref. 2) is a bi-directional Ca2+ transporter that contributes to the electrical activity of the heart. When, and if, Ca2+ is exported or imported depends on the Na+/Ca2+ exchange ratio. Whereas a ratio of 3:1 (Na+:Ca2+) has been indicated by Ca2+ flux equilibrium studies, a ratio closer to 4:1 has been indicated by exchange current reversal potentials. Here we show, using an ion-selective electrode technique to quantify ion fluxes in giant patches, that ion flux ratios are approximately 3.2 for maximal transport in either direction. With Na+ and Ca2+ on both sides of the membrane, net current and Ca2+ flux can reverse at different membrane potentials, and inward current can be generated in the absence of cytoplasmic Ca2+, but not Na+. We propose that NCX1 can transport not only 1 Ca2+ or 3 Na+ ions, but also 1 Ca2+ with 1 Na+ ion at a low rate. Therefore, in addition to the major 3:1 transport mode, import of 1 Na+ with 1 Ca2+ defines a Na+-conducting mode that exports 1 Ca2+, and an electroneutral Ca2+ influx mode that exports 3 Na+. The two minor transport modes can potentially determine resting free Ca2+ and background inward current in heart.

Two-Ply Carbon Nanotube Fiber-Typed Enzymatic Biofuel Cell Implanted in Mice.

Implantable devices have emerged as a promising industry. It is inevitable that these devices will require a power source to operate in vivo. Thus, to power implantable medical devices, biofuel cells (BFCs) that generate electricity using glucose without an external power supply have been considered. Although implantable BFCs have been developed for application in vivo, they are limited by their bulky electrodes and low power density. In the present study, we attempted to apply to living mice an implantable enzymatic BFC (EBFC) that was previously reported to be a high-power EBFC comprising carbon nanotube yarn electrodes. To improve their mechanical properties and for convenient implantation, the electrodes were coated with Nafion and twisted into a micro-sized, two-ply, one-body system. When the two-ply EBFC system was implanted in the abdominal cavity of mice, it provided a high-power density of 0.3 mW/cm2. The two-ply EBFC system was injected through a needle using a syringe without surgery and the inflammatory response in vivo initially induced by the injection of the EBFC system was attenuated after 7 days, indicating the biocompatibility of the system in vivo.

Switchable redox activity by proton fuelled DNA nano-machines.

The switching electrochemical property of an SWNT/DNA hybrid can be produced through reversible conformational changes between the closed and open state originating from the pH-responding i-motif DNA which significantly improves its molecular switching and stability by hydrophobic interactions with SWNTs.

Tough supersoft sponge fibers with tunable stiffness from a DNA self-assembly technique.

Tough and soft: Highly porous, spongelike materials self-assemble by calcium ion condensation of DNA-wrapped carbon nanotubes (SWNTs-DNA; see picture, IL = ionic liquid). The toughness, modulus, and swellability of the electrically conductive sponges can be tuned by controlling the density and strength of interfiber junctions. The sponges have compliances similar to the softest natural tissue, while robust interfiber junctions give high toughness.

Discovery of Natural Compounds Promoting Cardiomyocyte Differentiation.

The commitment of pluripotent stem cells to the cardiac lineage has enormous potential in regenerative medicine interventions for several cardiac diseases. Thus, it is necessary to understand and regulate this differentiation process for potential clinical application. In this study, we developed defined conditions with chemical inducers for effective cardiac lineage commitment and elucidated the mechanism for high-efficiency differentiation. First, we designed a robust reporter-based platform to screen chemical inducers of cardiac differentiation in the mouse P19 teratocarcinoma cell line. Using this system, we identified two natural alkaloids, lupinine and ursinoic acid, which enhanced cardiomyocyte differentiation of P19 cells in terms of beating colony numbers with respect to oxytocin, and confirmed their activity in mouse embryonic stem cells. By analyzing the expression of key markers, we found that this enhancement can be attributed to the early and rapid induction of the Wnt signaling pathway. We also found that these natural compounds could not only supersede the action of the Wnt3a ligand but also had a very quick response time, allowing them to act as efficient cardiac mesoderm inducers that subsequently promoted cardiomyocyte differentiation. Thus, this study offers a way to develop chemical-based differentiation strategy for high-efficiency cardiac lineage commitment, which has an advantage over currently available methods with complex medium composition and parameters. Furthermore, it also provides an opportunity to pinpoint the key molecular mechanisms pivotal to the cardiac differentiation process, which are necessary to design an efficient strategy for cardiomyocyte differentiation.