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Transporter-mediated clearance is determined by two factors, its single-molecule clearance, and expression level. However, no reliable method has been developed to evaluate them separately. This study aimed to develop a reliable method for evaluating the single-molecule activity of membrane transporters, such as organic anion transporting polypeptide (OATP) 2B1.HEK293 cells that co-expressed large conductance calcium-activated potassium (BK) channel and OATP2B1 were established and used for the following experiments. i) BK channel-mediated whole-cell conductance was measured using patch-clamp technique and divided by its unitary conductance to estimate the number of channels on plasma membrane (QI). ii) Using plasma membrane fraction, quantitative targeted absolute proteomics determined the stoichiometric ratio (ρ) of OATP2B1 to BK channel. iii) The uptake of estrone 3-sulfate was evaluated to calculate the Michaelis constant and uptake clearance (CL) per cell. Single-molecule clearance (CLint) was calculated by dividing CL by QI·ρ.QI and ρ values were estimated to be 916 and 2.16, respectively, yielding CLint of 5.23 fL/min/molecule. We successfully developed a novel method to reliably measure the single-molecule activity of a transporter, which could be used to evaluate the influences of factors such as genetic variations and post-translational modifications on the intrinsic activity of transporters.

Pulmonary vessels play a pivotal role in oxygen circulation. We previously demonstrated that pimaric acid (PiMA) activated large-conductance Ca2+-activated K+ (BKCa) channels and inhibited voltage-dependent Ca2+ channels (VDCCs). In the present study, PiMA attenuated vasoconstriction induced by high K+ or endothelin-1 in rat pulmonary arterial smooth muscles (PASMs). PiMA also reduced high K+-induced cytosolic [Ca2+] increase in PASM cells. PiMA increased BKCa currents and decreased VDCC currents. BKCa channels and VDCCs were formed by the α/β1 and α1C/α1D/β2/β3 subunits, respectively. These results indicate that PiMA induces vasorelaxation through the dual effects of BKCa channel activation and VDCC inhibition in PASMs.

Pulmonary arterial hypertension (PAH) is a progressive and fatal disease that is characterized by vascular remodeling of the pulmonary artery. PAH remodeling is primarily caused by the excessive proliferation of pulmonary arterial smooth muscle cells (PASMCs). Therefore, an inhibitory mechanism is expected as a target for the treatment of PAH. Corosolic acid (CRA) is a pentacyclic triterpenoid extracted from the leaves of Banaba (Lagerstroemia speciosa) that exerts anti-diabetic, anti-inflammatory, and anti-tumor effects. In the present study, the effects of CRA on PAH remodeling were examined using PASMCs from idiopathic pulmonary arterial hypertension (IPAH) patients and monocrotaline (MCT)-induced pulmonary hypertensive (PH) rats. CRA inhibited the excessive proliferation of IPAH-PASMCs in a concentration-dependent manner (IC50 = 14.1 μM). It also reduced the migration of IPAH-PASMCs. The CRA treatment downregulated the expression of signal transducer and activator of transcription 3 (STAT3) in IPAH-PASMCs. In MCT-PH rats, the administration of CRA (1 mg/kg/day) attenuated increases in right ventricular systolic pressure, pulmonary vascular remodeling, and right ventricular hypertrophy. CRA also decreased the expression of STAT3 in pulmonary arterial smooth muscles from MCT-PH rats. In conclusion, the anti-proliferative and anti-migratory effects of CRA in PASMCs ameliorated PAH remodeling by downregulating STAT3 signaling pathways.

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.

Pulmonary arterial hypertension (PAH) is a rare, progressive, and fatal cardiovascular/lung disease. The incidence rate is affected by age. Monocrotaline (MCT, 60 mg/kg)-treated rats are widely used as an experimental PAH model. Here, we found that young rats died at a mean of 23.4 days after MCT injection, whereas adult rats survived for over 42 days. However, young (7-week-old) and adult (20-week-old) MCT-treated rats developed PAH, and had upregulated Ca2+-sensing receptor and transient receptor potential canonical subfamily 6 channel expression in pulmonary arteries. The present study provides novel information for elucidating the mechanism underlying the age difference in PAH patients.

The blood–brain barrier (BBB) is mainly formed by brain capillary endothelial cells (BCECs) and is exposed to hypoxic environments under pathological conditions. The effects of hypoxia on the expression and activity of Ca2+-activated Cl− (ClCa) channels, TMEM16A, were examined in bovine brain endothelial t-BBEC117 cells and mouse BCECs. The expression of TMEM16A was upregulated by hypoxia. Whole-cell ClCa currents increased under hypoxia. Hypoxia also increased cell proliferation and trans-endothelial permeability, which were attenuated by ClCa channel blockers or TMEM16A siRNA. These findings are useful for elucidating the pathological role of TMEM16A ClCa channels in the BBB during cerebral ischemia.

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 (

Ion channels play pivotal roles in a wide variety of cellular functions; therefore, their physiological characteristics, pharmacological responses, and molecular structures have been extensively investigated. However, the mobility of an ion channel itself in the cell membrane has not been examined in as much detail. A total internal reflection fluorescence (TIRF) microscope allows fluorophores to be imaged in a restricted region within an evanescent field of less than 200 nm from the interface of the coverslip and plasma membrane in living cells. Thus the TIRF microscope is useful for selectively visualizing the plasmalemmal surface and subplasmalemmal zone. In this review, we focused on a single-molecule analysis of the dynamic movement of ion channels in the plasma membrane using TIRF microscopy. We also described two single-molecule imaging techniques under TIRF microscopy: fluorescence resonance energy transfer (FRET) for the identification of molecules that interact with ion channels, and subunit counting for the determination of subunit stoichiometry in a functional channel. TIRF imaging can also be used to analyze spatiotemporal Ca2+ events in the subplasmalemma. Single-molecule analyses of ion channels and localized Ca2+ signals based on TIRF imaging provide beneficial pharmacological and physiological information concerning the functions of ion channels.

Abstract.: TMEM16A is a major component of Ca2+-activated Cl− channel (CaCC) conductance in murine portal vein smooth muscle cells (mPVSMCs). Here, the regulation of CaCC activity by the actin cytoskeleton was examined in mPVSMCs. Actin disruption by cytochalasin D did not affect the current density, but increased the deactivation time constant in mPVSMCs. The elongated deactivation was recovered by jasplakinolide. When murine TMEM16A was transfected into HEK293 cells that have a poorly developed actin cytoskeleton, electrophysiological properties of CaCC currents were not changed by cytochalasin D. In conclusion, the CaCC activity in mPVSMCs is modified by the interaction of TMEM16A with abundant actin cytoskeleton. Keywords:: TMEM16A, actin cytoskeleton, portal vein

Abstract.: Upregulation of the Kir2.1 channel during endoplasmic reticulum (ER) stress in t-BBEC117, an immortalized bovine brain endothelial cell line, caused a sustained increase in intracellular Ca2+ concentration ([Ca2+]i) and a facilitation of cell death. Expressions of Ca2+ influx channels (TRPC, Orai1, STIM1) were unchanged by ER stress. The ER stress–induced [Ca2+]i increase was mainly attributed to the deeper resting membrane potential due to Kir2.1 upregulation. ER stress arrested at the G2/M phase and it was attenuated by an inhibitor of Kir2.1. These results indicate that Kir2.1 upregulation by ER stress facilitates cell death via regulation of cell cycle progression in t-BBEC117. Keywords:: brain capillary endothelial cell, store operated calcium entry, endoplasmic reticulum stress

To develop a simple screening system for blockers of voltage-gated Kv1.3 and Kv1.5 channels, new cell lines co-expressing mutated Nav1.5 (IFM/Q3), Kir2.1 (Kir), and Kv1.3 or Kv1.5 were introduced as IFM/Q3+Kir+Kv1.3 and IFM/Q3+Kir+Kv1.5, respectively. Electrical stimulation (ES) of a cell line, IFM/Q3+Kir, induced prolonged action potentials due to the slow inactivation of IFM/Q3 and subsequent cell death. Additional co-expression of Kv1.3 or Kv1.5 to IFM/Q3+Kir shortened the evoked action potentials and prevented cell death. In the presence of margatoxin, a selective Kv1.3-blocker, ES induced cell death in IFM/Q3+Kir+Kv1.3, but not in IFM/Q3+Kir+Kv1.5. In the presence of 4-aminopyridine, a non-selective Kv-channel blocker, ES application elicited cell death in both cell lines. The IC50s of acacetin, a Kv1.5-blocker, was 10.2 μM in IFM/Q3+Kir+Kv1.3 and almost identical to that in IFM/Q3+Kir+Kv1.5 (7.6 μM). The IC50s of citalopram, a 5-HT uptake-inhibitor, were 1.8 μM in IFM/Q3+Kir+Kv1.3 and 1.5 μM in IFM/Q3+Kir+Kv1.5, respectively. These IC50s were comparable to those determined electrophysiologically. In conclusion, acacetin and citalopram block both Kv1.3 and Kv1.5 without selectivity. The Kv1.3 or Kv1.5 channel inhibition assay using these new cell lines may be applicable to high–throughput screening because of its simplicity, accuracy, and high cost-performance. Keywords:: Kv1.3, Kv1.5, high-throughput screening, acacetin, citalopram

The rise of Ca2+ concentration ([Ca2+] i ) by reducing external Na+ in urinary bladder smooth muscle cells (UBSMCs) from transgenic mice overexpressing Na+/Ca2+ exchanger type-1.3 (NCX1.3tg/tg) was about 4 times as large as that in the wild-type (WT). NCX1 protein expression in UB increased about 4-fold in NCX1.3tg/tg. The Ca2+ release by caffeine in UBSMCs was comparable between NCX1.3tg/tg and WT, but [Ca2+]i decay was faster in NCX1.3tg/tg. Contractions induced by acetylcholine, 60 mM K+, or electrical stimulation were significantly smaller in UB segments of NCX1.3tg/tg. NCX worked in Ca2+-extrusion mode during these contractions in UBSMCs of both WT and NCX1.3tg/tg. Keywords:: Na+-Ca2+ exchanger, urinary bladder smooth muscle, transgenic mouse

Abstract.: The contribution of Cl− conductance relative to that of K+ in the regulation of membrane potential was examined using OUMS-27 cells, a model cell-line of human chondrocytes. Application of 100 μM niflumic acid (NFA) and other anion-channel blockers induced significant membrane hyperpolarization. The NFA-sensitive membrane current under voltage-clamp was predominantly Cl− current. Application of NFA induced small but significant increase in intracellular Ca2+ concentration ([Ca2+]i) and markedly enhanced the late component of [Ca2+]i rise induced by 1 μM histamine. In conclusion, Cl− conductance substantially contributes to the regulation of resting membrane potential and [Ca2+]i in OUMS-27 cells. Keywords:: chondrocyte, chloride channel, niflumic acid

Large-conductance Ca2+-activated K+ (BK) channel regulates action potential firing in pancreatic β-cells. We cloned novel spliced variants of the BK-channel β2-subunit (BKβ2b), which consisted of 36 amino acids including the N-terminal in the original human BKβ2 (BKβ2a), from human and rodent pancreas. Real-time PCR analysis showed the abundant expression of BKβ2b transcripts in human and rodent pancreas and also in the RINm5f insulinoma cell line. In addition, up-regulation of both BK-channel α-subunit (BKα) and BKβ2b transcripts was observed in pancreas tissues from diabetes mellitus patients. In HEK293 cells co-expressing BKα and BKβ2b, the inactivation of BK-channel currents, which is typical for BKα + BKβ2a, was not observed, and electrophysiological and pharmacological properties of BKα + BKβ2b were almost identical to those of BKα alone. In HEK293 cells stably expressing BKα, the transient co-expression of yellow fluorescence protein (YFP)-tagged BKβ2a proteins resulted in their distribution along the cell membrane. In contrast, the co-expression of YFP-tagged BKβ2b with BKα showed diffusely distributed fluorescence signals throughout the cell body. Taken together, the predominant splicing of BKβ2b versus that of BKβ2a presumably enhances the contribution of BK channels to membrane potential and may possibly be a factor modulating insulin secretion in a suppressive manner in pancreatic β-cells. Keywords:: Ca2+-activated K+ channel, β2-subunit, spliced variant, pancreatic islet β-cell, diabetes mellitus

Abstract.: Ca2+ release from intracellular store sites via the ryanodine receptor (RyR) and hormonal regulation by flutamide, an androgen-receptor (AR) antagonist, on it were examined in vas deferens (VD) smooth muscle cells (SMCs). VD and VDSMCs were obtained from two groups of male rats that were treated p.o. with 100 mg/kg flutamide (Flu) or vehicle (Vehicle). Both spontaneous and caffeine-induced Ca2+ releases were markedly smaller in single VDSMCs from Flu than in those from Vehicle. Interestingly, [Ca2+]i rise by 100 μM norepinephrine in VDSMCs from Flu was larger than that in those from Vehicle. The contractions induced by direct electrical stimulation in tissue preparations from Flu showed lower susceptibility to 30 μM ryanodine than those from Vehicle. Real-time PCR analyses revealed that the transcripts of ryanodine receptor (RyR) type 2 and type 3 (RyR2 and RyR3) were expressed in VD and markedly reduced in Flu. The protein expression of total RyR was significantly reduced by flutamide treatment, but that of inositol 1,4,5-trisphosphate receptor (IP3R) was not affected. It can be strongly suggested that long term block of AR by flutamide reduced the expression of RyR and its contribution to the contraction, but not those of IP3R in VDSMCs. Keywords:: ryanodine receptor, Ca2+ release, flutamide, vas deferens, smooth muscle

In this study, we provide evidence of critical changes in the expression of non-selective cation currents (NSCC) during culture in rat aortic myocytes. A selective TRPV4 agonist, 4α-phorbol 12,13-didecanoate (4αPDD), had little effect on membrane currents and intracellular Ca2+ (Ca2+i) in freshly isolated cells from the aorta. In contrast, in cultured aortic myocytes with and without serum, 4αPDD at a concentration range between 0.3 and 3 μM effectively elevated Ca2+i, which was abolished in the absence of external Ca2+. Application of 4αPDD to cultured aortic myocytes also activated NSCC, which had a reversal potential of +3 mV. Both of these signals were blocked by ruthenium red (RuR), an effective blocker of TRPVs. Although the expression of TRPV4 mRNA transcript was found in cultured as well as non-cultured aortic myocytes, significant immunoreactivity to TRPV4 protein was only detected in cultured rat aortic myocytes. Moreover, cultured human pulmonary arterial smooth muscle cells (hPASM) had a substantial response to 4αPDD, which was susceptible to the removal of external Ca2+ and application of RuR. These results provide a strong basis for our proposal that endogenous TRPV4 functions as an important regulator of Ca2+i in vascular myocytes under some physiological and pathophysiological conditions. Keywords:: TRPV4, 4α-phorbol 12,13-didecanoate, cell-culture

We examined the effects of methyl-β-cyclodextrin (MβCD) on Ca2+-induced Ca2+ release (CICR) in smooth muscle cells (SMCs) of mouse urinary bladder (UB). Short depolarization of UBSMCs under voltage-clamp elicited several local Ca2+ transients (Ca2+ hot spots) via CICR within 20 ms in discrete sub-sarcolemmal areas. Then, the Ca2+ wave spread to whole areas. The pretreatment with 10 mM MβCD significantly attenuated Ca2+ hot spots in UBSMCs and reduced contraction by single direct electrical pulse stimulation in UBSM strips. MβCD may prevent CICR by attenuating the coupling between voltage-dependent Ca2+ channels and ryanodine receptors in Ca2+ hot spot areas. Keywords:: methyl-β-cyclodextrin, Ca2+-induced Ca2+ release, smooth muscle

Abstract.: We examined effects of small and intermediate conductance Ca2+-activated K+ (SK and IK) channel openers, DCEBIO (5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazol-2-one) and NS309 (3-oxime-6,7-dichloro-1H-indole-2,3-dione), on L-type Ca2+ channel current (ICa) that was measured in smooth muscle cells isolated from mouse urinary bladder under whole cell voltage-clamp. The ICa was concentration-dependently inhibited by DCEBIO and NS309; half inhibition was obtained at 71.6 and 10.6 µM, respectively. The specificity of NS309 to the IK channel over the Ca2+ channel appears to be high and higher than that of DCEBIO. DCEBIO and even NS309 may, however, substantially block Ca2+ channels when used as SK channel openers. Keywords:: voltage-dependent Ca2+ channel, DCEBIO, NS309