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Activation of hepatic stellate cells (HSCs) causes hepatic fibrosis and results in chronic liver diseases. Although activated HSC functions are facilitated by an increase in the cytosolic Ca2+ concentration ([Ca2+]cyt), the pathophysiological roles of ion channels are largely unknown. In the present study, functional analyses of the two-pore domain K+ (K2P) channels, which regulate the resting membrane potential and [Ca2+]cyt, were performed using the human HSC line, LX-2. Expression analyses revealed that TREK1 (also known as KCNK2 and K2P2.1) channels are expressed in LX-2 cells. Whole-cell K+ currents were activated by 10 μM arachidonic acid and the activation was abolished by 100 μM tetrapentylammonium, which are pharmacological characteristics of TREK1 channels. The siRNA knockdown of TREK1 channels caused membrane depolarization and reduced [Ca2+]cyt. In addition, TREK1 knockdown downregulated the gene expression of collage type I and platelet-derived growth factor. Furthermore, TREK1 knockdown inhibited the proliferation of LX-2 cells. In conclusion, the activity of TREK1 channels determines the resting membrane potential and [Ca2+]cyt, which play a role in extracellular matrix production and cell proliferation in HSCs. This study may help elucidate the molecular mechanism underlying hepatic fibrosis in HSCs and provide a potential therapeutic target for hepatic fibrosis.

Articular chondrocytes are exposed to dynamic osmotic environments during normal joint loading, and thus, require effective volume regulatory mechanisms. A regulatory volume decrease (RVD) is one of the mechanisms for protecting chondrocytes from swelling and damage. Swelling-activated Cl− currents (ICl,swell) are responsible for the RVD, but the molecular identity in chondrocytes is largely unknown. In this study, we reveal that in human OUMS-27 chondrocytes, ICl,swell can be elicited by hypoosmotic stimulation (180 mOsm) and be inhibited by classical Cl− channel blockers, 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS) and niflumic acid, and be attenuated by siRNA knockdown of ClC-3. Our molecular analyses revealed that ClC-3A is expressed as a major splice variant in both human articular chondrocytes and OUMS-27 cells. The onset and early phase of RVD following hypoosmotic stress in OUMS-27 cells were affected by DIDS and ClC-3 knockdown. Hypoosmotic stimulation caused Ca2+ influx and subsequent release of prostaglandin E2 (PGE2) in OUMS-27 cells, and both of these responses were reduced by DIDS and ClC-3 knockdown. These results strongly suggest that ClC-3 is responsible for ICl,swell and RVD under the hypoosmotic environments. It is likely that ClC-3 is associated with the pathogenesis of cartilage degenerative diseases including osteoarthritis via PGE2 release.

Pancreatic neuroendocrine tumors (pNETs) occur due to the abnormal growth of pancreatic islet cells and predominantly develop in the duodenal-pancreatic region. Somatostatinoma is one of the pNETs associated with tumors of pancreatic δ cells, which produce and secrete somatostatin. Limited information is currently available on the pathogenic mechanisms of somatostatinoma. The large-conductance Ca2+-activated K+ (BKCa) channel is expressed in several types of cancer cells and regulates cell proliferation, migration, invasion, and metastasis. In the present study, the functional expression of the BKCa channel was examined in a human somatostatinoma QGP-1 cell line. In QGP-1 cells, outward currents were elicited by membrane depolarization at pCa 6.5 (300 nM) in the pipette solution and inhibited by the specific BKCa channel blocker, paxilline. Paxilline-sensitive currents were detected, even at pCa 8.0 (10 nM) in the pipette solution, in QGP-1 cells. In addition to the α and β2-4 subunits of the BKCa channel, the novel regulatory γ1 subunit (BKCaγ1) was co-localized with the α subunit in QGP-1 cells. Paxilline-sensitive currents at pCa 8.0 in the pipette solution were reduced by the siRNA knockdown of BKCaγ1. Store-operated Ca2+ entry was smaller in BKCaγ1 siRNA-treated QGP-1 cells. The proliferation of QGP-1 cells was attenuated by paxilline or the siRNA knockdown of BKCaγ1. These results strongly suggest that BKCaγ1 facilitates the proliferation of human somatostatinoma cells. Therefore, BKCaγ1 may be a novel therapeutic target for somatostatinoma.

Brain capillary endothelial cells (BCECs) form the blood-brain barrier (BBB) and play an essential role in the regulation of its functions. Oxidative stress accumulates excessive reactive oxygen species (ROS) and facilitates the death of BCECs, leading to a dysfunctional BBB. However, the mechanisms underlying the death of BCECs under oxidative stress remain unclear. In the present study, the effects of oxidative stress on cell viability, ROS production, intracellular Ca2+ concentration, and protein expression were examined using a cell line derived from bovine BCECs, t-BBEC117. When t-BBEC117 cells were exposed to oxidative stress induced by hydrogen peroxide (H2O2, 10–100 μM), cell growth was inhibited in a dose-dependent manner. Oxidative stress by 30 μM H2O2 increased the production of ROS and its effects were blocked by the ROS scavenger, 10 mM N-acetyl- l -cysteine (NAC). In addition, oxidative stress reduced store-operated Ca2+ entry (SOCE) and this decrease was recovered by NAC or the Orai channel activator, 5 μM 2-aminoethyl diphenylborinate (2-APB). The siRNA knockdown of Orai1 revealed that Orai1 was mainly responsible for SOCE channels and its activity was decreased by oxidative stress. However, the protein expression of Orai1 and STIM1 was not affected by oxidative stress. Oxidative stress-induced cell death was rescued by 2-APB, NAC, or the STIM-Orai activating region. In conclusion, oxidative stress reduces Orai1-mediated SOCE and, thus, facilitates the death of BCECs.

Functional coupling between large-conductance Ca2+-activated K+ (BKCa) channels in the plasma membrane (PM) and ryanodine receptors (RyRs) in the sarcoplasmic reticulum (SR) is an essential mechanism for regulating mechanical force in most smooth muscle (SM) tissues. Spontaneous Ca2+ release through RyRs (Ca2+ sparks) and subsequent BKCa channel activation occur within the PM-SR junctional sites. We report here that a molecular interaction of caveolin-1 (Cav1), a caveola-forming protein, with junctophilin-2 (JP2), a bridging protein between PM and SR, positions BKCa channels near RyRs in SM cells (SMCs) and thereby contributes to the formation of a molecular complex essential for Ca2+ microdomain function. Approximately half of all Ca2+ sparks occurred within a close distance (

Two-pore domain K+ (K2P) channels are thought to be druggable targets. However, only a few agents specific for K2P channels have been identified, presumably due to the lack of an efficient screening system. To develop a new high-throughput screening (HTS) system targeting these channels, we have established a HEK293-based "test cell" expressing a mutated Na+ channel (Nav1.5) with markedly slowed inactivation, as well as a K+ channel (Kir2.1) that sets the membrane potential quite negative, close to K+ equilibrium potential. We found in this system that Kir2.1 block by 100 μM Ba2+ application consistently elicited a large depolarization like a long-lasting action potential. This maneuver resulted in cell death, presumably due to the sustained Na+ influx. When either the TWIK-related acid-sensitive K+ (TASK)-1 or TASK-3 channel was expressed in the test cells, Ba2+-induced cell death was markedly weakened. Stronger activation of TASK-1 by extracellular acidification further decreased the cell death. In contrast, the presence of K2P channel blockers enhanced cell death. IC50 values for TASK-1 and/or TASK-3 blockers acquired by measurements of relative cell viability were comparable to those obtained using patch-clamp recordings. Both blockers and openers of K2P channels can be accurately assessed with high efficiency and throughput by this novel HTS system.

The mechanisms underlying neuronal cell death induced by the rise of intracellular Na+ concentration ([Na+]i) following abnormal hyperexcitation are not fully understood. Previously, we have established a recombinant cell line derived from HEK293 cells, in which the occurrence of a sustained action potential (AP) induces cell death. Mutated voltage-gated Nav1.5 channel (IFM/QQQ) lacking inactivation, and inward rectifying K+ channel (Kir2.1) were co-expressed in HEK293 cells (IFM/QQQ + Kir2.1 cells). In this cell line, the rise of [Na+]i due to a sustained AP reached maximum within 15 min without concomitant [Ca2+]i rise, and then elicited significant externalization of phosphatidylserine and enhancement of caspase activity. Marked decreases in mitochondrial transmembrane potential and ATP concentration were also detected. The significant cell death occurred at 3 h from the AP onset and reached a steady state at around 12 h. The significant release of lactate dehydrogenase was not detected even after 12 h. These results provide novel findings that Na+ accumulation or/and possibly concomitant K+ loss elicits apoptosis presumably due to the mitochondrial dysfunction, which is attributable to neither the membrane depolarization nor [Ca2+]i change. This apoptotic mechanism may be involved, at least in part, in neuronal cell death under pathophysiological settings with abnormal hyperexcitability.

Conversion of intracellular Ca2+ signals to electrical activity results in multiple and differing physiological impacts depending on cell types. In some organs such as gastrointestinal and urinary systems, spontaneous Ca2+ oscillation in pacermaker cells can function essentially as a Ca2+ clock mechanism, which has been originally found in pacemaking in sinoatrial node cell of the heart. The conversion of discrete Ca2+ clock events to spontaneous electrical activity is an essential step for the initiation and propagation of pacemaker activity through the multicellular organs resulting in synchronized physiological functions. Here, a model of intracellular signal transduction from a Ca2+ oscillation to initiation of electrical slow waves and their propagation were reconstituted in HEK293 cells. This was accomplished based on ryanodine receptor (RyR) type 3, Ca2+-activated ion channels, i.e. small conductance Ca2+-activated K+ channel (SK2) or Ca2+-activated Cl− channel (TMEM16A), and connexin43 being heterologously co-expressed. The propagation of electrical waves was abolished or substantially reduced by treatment with selective blockers of the expressed channels and 18β-glycyrrhetinic acid, a gap junction inhibitor, respectively. Thus, we demonstrated that the conversion of Ca2+ oscillation to electrical signals with cell to cell propagation can be reconstituted as a model of Ca2+ clock pacemaker activity by combinational expression of critical elements in heterologous expression system.

Voltage-gated K+ (KV) channels, which control firing and shape of action potentials in excitable cells, are supposed to be potential therapeutic targets in many types of diseases. Pimaric acid (PiMA) is a unique opener of large conductance Ca2+-activated K+ channel. Here, we report that PiMA modulates recombinant rodent KV channel activity. The enhancement was significant at low potentials (

Ca(2+) influx via store-operated Ca(2+) entry (SOCE) plays critical roles in many essential cellular functions. The Ca(2+) release-activated Ca(2+) (CRAC) channel complex, consisting of Orai and STIM, is one of the major components of store-operated Ca(2+) (SOC) channels. Our previous study demonstrated that histamine can cause sustained Ca(2+) entry through SOC channels in OUMS-27 cells derived from human chondrosarcoma. This SOCE was increased by low- and decreased by high-concentrations of 2-aminoethoxydiphenyl borate. Quantitative reverse transcription PCR and Western blot analyses revealed abundant expressions of Orai1, Orai2 and STIM1. Introduction of dominant negative mutant of Orai1, or siOrai1 knockdown significantly attenuated SOCE. Following histamine application, single molecule imaging using total internal reflection fluorescence (TIRF) microscopy demonstrated punctate Orai1-STIM1 complex formation in plasma membrane. In contrast, knockdown or over-expression of Orai2 resulted in an increase or a decrease in SOCE, respectively. Finally, TIRF imaging revealed direct coupling between Orai1 and Orai2, and suggested that Orai2 reduces Orai1 function by formation of a hetero-tetramer. These results provide substantial evidence that Orai1, Orai2 and STIM1 form functional CRAC channels in OUMS-27 cells and that these complexes are responsible for sustained Ca(2+) entry in response to agonist stimulation.

Store-operated Ca(2+) entry (SOCE) via Orai1 and STIM1 complex is supposed to have obligatory roles in the regulation of cellular functions of vascular endothelial cells, while little is known about the contribution of Orai2. Quantitative PCR and Western blot analyses indicated the expression of Orai2 and STIM2, in addition to Orai1 and STIM1 in bovine brain capillary endothelial cell line, t-BBEC117. During the exponential growth of t-BBEC117, the knockdown of Orai1 and STIM1 significantly reduced the SOCE activity, whereas Orai2 and STIM2 siRNAs had no effect. To examine whether endogenous SOCE activity contributes to the regulation of cell cycle progression, t-BBEC117 were synchronized using double thymidine blockage. At the G2/M phase, Ca(2+) influx via SOCE was decreased and Orai2 expression was increased compared to the G0/G1 phase. When Orai2 was knocked down at the G2/M phase, the decrease in SOCE was removed, and cell proliferation was partly attenuated. Taken together, Orai1 significantly contributes to cell proliferation via the functional expression, which is presumably independent of the cell cycle phases. In construct, Orai2 is specifically up-regulated during the G2/M phase, negatively modulates the SOCE activity, and may contribute to the regulation of cell cycle progression in brain capillary endothelial cells.

Ca 2+ -activated Cl − channel (CaCC) often plays substantial roles in the regulation of membrane excitability in smooth muscle cells (SMCs). TMEM16A, a member of the TMEM16 family, has been suggested as the molecular entity responsible for CaCC in several types of SMCs. In this study, the expression of TMEM16A splicing variants and their contribution to CaCC activity were examined in murine portal vein SMCs (mPVSMCs). Four transcripts of TMEM16A splicing variants, which include four alternatively spliced segments (“ a ” and “ b ” in N-terminus and “ c ” and “ d ” in the first intracellular loop), were identified; the expression ratio of four transcripts of “ abc ”, “ acd ”, “ abcd ” and “ ac ” was 64.5, 25.8, 4.8 and 4.8%, respectively. The immunostaining of isolated mPVSMCs with anti-TMEM16A antibody indicates the abundant expression of TMEM16A on the cell membrane. CaCC currents recorded in mPVSMCs were markedly reduced by T16A inh -A01, a specific TMEM16A inhibitor. When the two major TMEM16A splicing variants, abc and acd isoforms, were expressed separately in HEK293 cells, the CaCC currents, which possess similar electrophysiological characteristics to those in mPVSMCs were observed. The single-molecule photobleaching analyses using total internal reflection fluorescence (TIRF) microscope indicated that the distribution of stepwise photobleaching events was fit well with a binomial distribution for homodimer. Additionally, the heterodimer formation was suggested by fluorescence resonance energy transfer (FRET) analyses in HEK293 cells co-expressing CFP- or YFP-tagged variants. In conclusion, alternatively spliced variants of TMEM16A abc and acd in mPVSMCs are two major molecular entities of CaCC and may form hetero-/homo-dimers to be functional as CaCC in the regulation of membrane excitability and contractility in mPVSMCs.

Caveolin family is supposed to be essential molecules for the formation of not only caveola structure on cell membrane but also functional molecular complexes in them with direct and/or indirect interaction with other membrane and/or submembrane associated proteins. The direct coupling of caveolin-1 (cav1) with large conductance Ca(2+)-activated K(+) channel, KCa1.1 has been established in several types of cells and in expression system as well. The possible interaction of caveolin-3 (cav3), which shows expression in some differential tissues from cav1, with KCa1.1 remains to be determined. In the present study, the density of KCa1.1 current expressed in HEK293 cells was significantly reduced by the co-expression of cav3, as well as cav1. The co-localization and direct interaction of GFP- or CFP-labeled cav3 (GFP/CFP-cav3) with YFP- or mCherry-labeled KCa1.1 (KCa1.1-YFP/mCherry) were clearly demonstrated by single molecular image analyses using total internal reflection fluorescence (TIRF) microscopy and fluorescence resonance energy transfer (FRET) analyses with acceptor photobleaching method. The deletion of suggested cav1-binding motif in C terminus region of KCa1.1 (KCa1.1ΔCB-YFP) resulted in the marked decrease in cell surface expression, co-localization and FRET efficiency with CFP-cav3 and CFP-cav1. The FLAG-KCa1.1 co-immunoprecipitation with GFP-cav3 or GFP-cav1 also supported their direct molecular interaction. These results strongly suggest that cav3 possesses direct interaction with KCa1.1, presumably at the same domain for cav1 binding. This interaction regulates KCa1.1 expression to cell surface and the formation of functional molecular complex in caveolae in living cells.

In smooth muscles (SMs), cytosolic Ca 2+ ([Ca 2+ ] cyt ) dynamics during an action potential are triggered by Ca 2+ influx through voltage-dependent Ca 2+ channels (VDCCs) in the plasma membrane. The physiological significance of Ca 2+ amplification by subsequent Ca 2+ release through ryanodine receptors (RyRs) from the sarcoplasmic reticulum (SR) is still a matter of topics in SMs. In the present study, depolarization-evoked local Ca 2+ dynamics in Ca 2+ microdomain were imaged using total internal reflection fluorescence (TIRF) microscopy in mouse urinary bladder SM cells (UBSMCs). Upon depolarization under whole-cell voltage-clamp, the rapid and local elevation of [Ca 2+ ] cyt was followed by larger [Ca 2+ ] cyt increase with propagation occurred in a limited TIRF zone within ∼200 nm from cell surface. The depolarization-evoked [Ca 2+ ] cyt increase in a TIRF zone was abolished or greatly reduced by the pretreatment with Cd 2+ or ryanodine, respectively. The initial local [Ca 2+ ] cyt increases were mediated by Ca 2+ influx through single or clustered VDCCs as Ca 2+ sparklets, and the following step was elicited by Ca 2+ -induced Ca 2+ release (CICR) through RyR from SR. The depolarization-induced outward currents, mainly due to large-conductance Ca 2+ -activated K + channel activation, were also markedly reduced by Cd 2+ and ryanodine. In addition, TIRF analyses showed that the fluorescent signals of individual or clustered VDCC distributed in relatively uniform fashion and that a subset of RyRs in the subplasmalemmal SR also located in TIRF zone. In conclusion, fast TIRF imaging successfully demonstrated two step Ca 2+ events upon depolarization in Ca 2+ microdomain of UBSMCs; the initial Ca 2+ influx as Ca 2+ sparklets through discrete VDCC or their clusters and the following CICR via the activation of loosely coupled RyRs in SR located in the Ca 2+ microdomains.

To provide a high-throughput screening method for human ether-a-go-go-gene-related gene (hERG) K(+) channel inhibition, a new recombinant cell line, in which single action potential (AP)-induced cell death was produced by gene transfection. Mutated human cardiac Na(+) channel Nav1.5 (IFM/Q3), which shows extremely slow inactivation, and wild-type inward rectifier K(+) channel, Kir2.1, were stably co-expressed in HEK293 cells (IFM/Q3+Kir2.1). In IFM/Q3+Kir2.1, application of single electrical stimulation (ES) elicited a long AP lasting more than 30 s and led cells to die by more than 70%, whereas HEK293 co-transfected with wild-type Nav1.5 and Kir2.1 fully survived. The additional expression of hERG K(+) channels in IFM/Q3+Kir2.1 shortened the duration of evoked AP and thereby markedly reduced the cell death. The treatment of the cells with hERG channel inhibitors such as nifekalant, E-4031, cisapride, terfenadine, and verapamil, recovered the prolonged AP and dose-dependently facilitated cell death upon ES. The EC(50) values to induce the cell death were 3 µM, 19 nM, 17 nM, 74 nM, and 3 µM, respectively, whereas 10 µM nifedipine did not induce cell death. Results indicate the high utility of this cell system for hERG K(+) channel safety assay.

Brain capillary endothelial cells (BCECs) form blood brain barrier (BBB) to maintain brain homeostasis. Cell turnover of BCECs by the balance of cell proliferation and cell death is critical for maintaining the integrity of BBB. Here we found that stimuli with tunicamycin, endoplasmic reticulum (ER) stress inducer, up-regulated inward rectifier K(+) channel (K(ir)2.1) and facilitated cell death in t-BBEC117, a cell line derived from bovine BCECs. The activation of K(ir) channels contributed to the establishment of deeply negative resting membrane potential in t-BBEC117. The deep resting membrane potential increased the resting intracellular Ca(2+) concentration due to Ca(2+) influx through non-selective cation channels and thereby partly but significantly regulated cell death in t-BBEC117. The present results suggest that the up-regulation of K(ir)2.1 is, at least in part, responsible for cell death/cell turnover of BCECs induced by a variety of cellular stresses, particularly ER stress, under pathological conditions.

The intermediate conductance Ca(2+)-activated K(+) channel (IK(Ca) channel) encoded by K(Ca)3.1 is responsible for the control of proliferation and differentiation in various types of cells. We identified novel spliced variants of K(Ca)3.1 (human (h) K(Ca)3.1b) from the human thymus, which were lacking the N-terminal domains of the original hK(Ca)3.1a as a result of alternative splicing events. hK(Ca)3.1b was significantly expressed in human lymphoid tissues. Western blot analysis showed that hK(Ca)3.1a proteins were mainly expressed in the plasma membrane fraction, whereas hK(Ca)3.1b was in the cytoplasmic fraction. We also identified a similar N terminus lacking K(Ca)3.1 variants from mice and rat lymphoid tissues (mK(Ca)3.1b and rK(Ca)3.1b). In the HEK293 heterologous expression system, the cellular distribution of cyan fluorescent protein-tagged hK(Ca)3.1a and/or YFP-tagged hK(Ca)3.1b isoforms showed that hK(Ca)3.1b suppressed the localization of hK(Ca)3.1a to the plasma membrane. In the Xenopus oocyte translation system, co-expression of hK(Ca)3.1b with hK(Ca)3.1a suppressed IK(Ca) channel activity of hK(Ca)3.1a in a dominant-negative manner. In addition, this study indicated that up-regulation of mK(Ca)3.1b in mouse thymocytes differentiated CD4(+)CD8(+) phenotype thymocytes into CD4(-)CD8(-) ones and suppressed concanavalin-A-stimulated thymocyte growth by down-regulation of mIL-2 transcripts. Anti-proliferative effects and down-regulation of mIL-2 transcripts were also observed in mK(Ca)3.1b-overexpressing mouse thymocytes. These suggest that the N-terminal domain of K(Ca)3.1 is critical for channel trafficking to the plasma membrane and that the fine-tuning of IK(Ca) channel activity modulated through alternative splicing events may be related to the control in physiological and pathophysiological conditions in T-lymphocytes.

The expression of large-conductance Ca(2+)-activated K(+) (BK) channel protein in amygdala complex was higher in adult (8-10 weeks old) male rats than in female. Castration at 4-6 weeks old significantly reduced BK channel expression in amygdala to the level similar to that in female. Immunocytochemical analyses of pyramidal-like neurons isolated from amygdala revealed that somas with relatively large size were highly immunoreactive to both anti-androgen receptor (AR) and anti-BK channel antibodies, while those with smaller size were not. The double-immunopositive neurons were dominant (60%) among pyramidal-like neurons isolated from amygdala of male rats but rare among those from female. The membrane current sensitive to penitrem A, a BK channel blocker, was the major K(+) current component in large neurons and showed higher current-density than that in smaller ones. These results suggest the gender-dependent cell population expressing BK channels in amygdala complex and its up-regulation by AR stimulation.

Malignant melanoma is the most deadly form of skin cancer and its incidence is steadily increasing worldwide. The plasma membrane in melanoma cells possesses a variety of ion channels, so its profile is thought to lead to a novel target for medical treatment for malignant melanoma. Here we showed that human melanoma G-361 cells expressed the epithelial Na(+) channel delta subunit (ENaC delta), which is largely unknown in physiological and pathological functions in non-neuronal tissues. Expression analyses at the level of mRNA clearly revealed that ENaC delta transcript was strongly expressed in human melanoma cells using reverse transcription-polymerase chain reaction and cell-based in situ hybridization techniques. Other ENaC subunits (alpha, beta, and gamma) were also distributed in human melanoma cells. In addition, human melanoma cells possessed an abundant expression of ENaC delta protein by immunocytochemistry. These results provide an attractive target for drug development of malignant melanoma.

The ubiquitin E3 protein ligase Nedd4-2 is a physiological regulator of the epithelial sodium channel ENaC, which is essential for transepithelial Na+ transport and is linked to Liddle's syndrome, an autosomal dominant disorder of human salt-sensitive hypertension. Nedd4-2 function is negatively regulated by phosphorylation via a serum- and glucocorticoid-inducible protein kinase (Sgk1), which serves as a mechanism to inhibit the ubiquitination-dependent degradation of ENaC. We report here that 14-3-3 proteins participate in this regulatory process through a direct interaction with a phosphorylated form of human Nedd4-2 (a human gene product of KIAA0439, termed hNedd4-2). The interaction is dependent on Sgk1-catalyzed phosphorylation of hNedd4-2 at Ser-468. We found that this interaction preserved the activity of the Sgk1-stimulated ENaC-dependent Na+ current while disrupting the interaction decreased ENaC density on the Xenopus laevis oocytes surface possibly by enhancing Nedd4-2-mediated ubiquitination that leads to ENaC degradation. Our findings suggest that 14-3-3 proteins modulate the cell surface density of ENaC cooperatively with Sgk1 kinase by maintaining hNedd4-2 in an inactive phosphorylated state.

The amiloride-sensitive epithelial Na+ channel (ENaC) regulates Na+ homeostasis into cells and across epithelia. So far, four homologous subunits of mammalian ENaC have been isolated and are denoted as alpha, beta, gamma, and delta. The chemical agents acting on ENaC are, however, largely unknown, except for amiloride and benzamil as ENaC inhibitors. In particular, there are no agonists currently known that are selective for ENaCdelta, which is mainly expressed in the brain. Here we demonstrate that capsazepine, a competitive antagonist for transient receptor potential vanilloid subfamily 1, potentiates the activity of human ENaCdeltabetagamma (hENaCdeltabetagamma) heteromultimer expressed in Xenopus oocytes. The inward currents at a holding potential of -60 mV in hENaCdeltabetagamma-expressing oocytes were markedly enhanced by the application of capsazepine (or =1 microM), and the capsazepine-induced current was mostly abolished by the addition of 100 microM amiloride. The stimulatory effects of capsazepine on the inward current were concentration-dependent with an EC50 value of 8 microM. Neither the application of other vanilloid compounds (capsaicin, resiniferatoxin, and olvanil) nor a structurally related compound (dopamine) modulated the inward current. Although hENaCdelta homomer was also significantly activated by capsazepine, unexpectedly, capsazepine had no effect on hENaCalpha and caused a slight decrease on the hENaCalphabetagamma current. In conclusion, capsazepine acts on ENaCdelta and acts together with protons. Other vanilloids tested do not have any effect. These findings identify capsazepine as the first known chemical activator of ENaCdelta.

The amiloride-sensitive epithelial Na(+) channel (ENaC) controls Na(+) transport into cells and across epithelia. So far, four homologous subunits of mammalian ENaC have been isolated and are denoted as alpha, beta, gamma, and delta. ENaCdelta can associate with beta and gamma subunits and generate a constitutive current that is 2 orders of magnitude larger than that of homomeric ENaCdelta. However, the distribution pattern of ENaCdelta is not consistent with that of the beta and gamma subunits. ENaCdelta is expressed mainly in the brain in contrast to beta and gamma subunits, which are expressed in non-neuronal tissues. To explain this discrepancy, we searched for novel functional properties of homomeric ENaCdelta and investigated the detailed tissue distribution in humans. When human ENaCdelta was expressed in Xenopus oocytes and Chinese hamster ovary cells, a reduction of extracellular pH activated this channel (half-maximal pH for an activation of 5.0), and the acid-induced current was abolished by amiloride. The most striking finding was that the desensitization of the acid-evoked current was much slower (by approximately 10% 120 s later), dissociating from the kinetics of acid-sensing ion channels in the degenerin/epithelial Na(+) channel family, which were rapidly desensitized during acidification. RNA dot-blot analyses showed that ENaCdelta mRNA was widely distributed throughout the brain and was also expressed in the heart, kidney, and pancreas in humans. Northern blotting confirmed that ENaCdelta was expressed in the cerebellum and the hippocampus. In conclusion, human ENaCdelta activity is regulated by protons, indicating that it may contribute to the pH sensation and/or pH regulation in the human brain.

The beta subunits of voltage-dependent calcium channels are known to modify calcium channel currents through pore-forming alpha1 subunits. Of the four beta subunits reported to date, the beta3 subunit is highly expressed in smooth muscle cells and is thought to consist of L-type calcium channels. To determine the role of the beta3 subunit in the voltage-dependent calcium channels of the cardiovascular system in situ, we performed a series of experiments in beta3-null mice. Western blot analysis indicated a significant reduction in expression of the alpha1 subunit in the plasma membrane of beta3-null mice. Dihydropyridine binding experiments also revealed a significant decrease in the calcium channel population in the aorta. Electrophysiological analyses indicated a 30% reduction in Ca2+ channel current density, a slower inactivation rate, and a decreased dihydropyridine-sensitive current in beta3-null mice. The reductions in the peak current density and inactivation rate were reproduced in vitro by co-expression of the calcium channel subunits in Chinese hamster ovary cells. Despite the reduced channel population, beta3-null mice showed normal blood pressure, whereas a significant reduction in dihydropyridine responsiveness was observed. A high salt diet significantly elevated blood pressure only in the beta3-null mice and resulted in hypertrophic changes in the aortic smooth muscle layer and cardiac enlargement. In conclusion, this study demonstrates the involvement and importance of the beta3 subunit of voltage-dependent calcium channels in the cardiovascular system and in regulating channel populations and channel properties in vascular smooth muscle cells.

Intermediate-conductance Ca2+-activated K+ channel (IKCa channel) encoded by KCa3.1 is responsible for the control of proliferation and differentiation in various types of cells. We identified novel spliced variants of KCa3.1 (hKCa3.1b) from the human thymus, which were lacking the N-terminal domains of the original hKCa3.1a as a result of alternative splicing. hKCa3.1b was significantly expressed in human lymphoid tissues. Western blot analysis showed that hKCa3.1a proteins were mainly expressed in plasma membrane fraction, whereas hKCa3.1b was in the cytoplasmic fraction. We also identified a similar N-terminus lacking KCa3.1 spliced variants from mice and rat lymphoid tissues (mKCa3.1b, rKCa3.1b). In the HEK293 heterologous expression system, the cellular distribution of CFP-tagged hKCa3.1a and/or YFP-tagged hKCa3.1b isoforms showed that hKCa3.1b suppressed the localization of hKCa3.1a to the plasma membrane. In the Xenopus oocyte translation system, co-expression of hKCa3.1b with hKCa3.1a suppressed IKCa channel activity of hKCa3.1a in a dominant-negative manner. In addition, the present study indicated that up-regulation of mKCa3.1b in mice thymocytes differentiated CD4+CD8+ phenotype thymocytes into CD4-CD8- ones, and suppressed concanavalin-A-stimulated thymocyte growth by down-regulation of mIL-2 transcripts. Anti-proliferative effects and down-regulation of mIL-2 transcripts were also observed in mKCa3.1b over-expressing mice thymocytes. These suggest that the N-terminal domain of KCa3.1 is critical for channel trafficking to the plasma membrane, and that the fine tuning of IKCa channel activity modulated through alternative splicing may be related to the control in physiological and pathophysiological conditions in T-lymphocytes.

Trimeric intracellular cation (TRIC) channel subtypes, namely TRIC-A and TRIC-B, function as intracellular channels conducting monovalent cations throughout tissues and probably mediate counter-ion movements coupled with Ca2+ release from the endo/sarcoplasmic reticulum. Knockout mice lacking both TRIC-A and TRIC-B channels suffer embryonic cardiac failure, and the mutant cardiomyocytes display severe dysfunction in SR Ca2+ handling (Yazawa et al., Nature, 2007). In knockout mice lacking TRIC-B channels with neonatal lethality due to respiratory failure, alveolar epithelial cells exhibit compromised Ca2+ release and thus insufficiently produce and secret surfactant phospholipids (Yamazaki et al., Development, 2009). The observations indicate that TRIC channels act as counter-ion channels functionally coupled with Ca2+ release in various cell types.Here we report the direct linkage of TRIC channels with hypertension. Knockout mice lacking TRIC-A channels showed significant hypertension and bradycardia. Ca2+ channel antagonists, but not blockers for vasoactive agents, exerted antihypertensive effects in the mutant mice. Moreover, despite retaining normal passive diameters in a Ca2+-free bathing solution, the mutant arteries showed narrow diameter results from higher resting Ca2+ level maintained by Ca2+ influx via L-type Ca2+ channel. In vascular smooth muscle cells (VSMCs), spontaneous opening of ryanodine receptor channels generates local Ca2+ release called Ca2+ sparks, activates cell-surface Ca2+-dependent K+ channels and reduces membrane potential toward relaxation. Our physiological and pharmacological studies suggested that the loss of TRIC-A channels compromises Ca2+ spark and spontaneous transient outward currents (STOCs) generation. In membrane potential monitoring using the voltage-dependent dye, the mutant VSMCs exhibited elevated resting membrane potential and prolonged repolarizing phases after high KCl-induced depolarization. These results suggested that the loss of TRIC-A in VSMCs enhanced the basal tonus induced by higher resting Ca2+ level results from decrease of Ca2+ sparks, STOCs frequency and depolarization of membrane potential.