Effectiveness of Columbianadin,a Bioactive Coumarin Derivative,in Perturbing Transient and Persistent INa

Columbianadin (CBN) is a bioactive coumarin-type compound with various biological activities. However, the action of CBN on the ionic mechanism remains largely uncertain, albeit it was reported to inhibit voltage-gated Ca²⁺ current or to modulate TRP-channel activity. In this study, whole-cell patch-clamp current recordings were undertaken to explore the modifications of CBN or other related compounds on ionic currents in excitable cells (e.g., pituitary GH₃ cells and HL-1 atrial cardiomyocytes). GH₃-cell exposure to CBN differentially decreased peak or late component of voltage-gated Na⁺ current (I_Na) with effective IC₅₀ of 14.7 or 2.8 µM, respectively. The inactivation time course of I_Na activated by short depolarization became fastened in the presence of CBN with estimated K_D value of 3.15 µM. The peak I_Na diminished by 10 µM CBN was further suppressed by subsequent addition of either sesamin (10 µM), ranolazine (10 µM), or tetrodotoxin (1 µM), but it was reversed by 10 µM tefluthrin (Tef); however, further application of 10 µM nimodipine failed to alter CBN-mediated inhibition of I_Na. CBN (10 µM) shifted the midpoint of inactivation curve of I_Na to the leftward direction. The CBN-mediated inhibition of peak I_Na exhibited tonic and use-dependent characteristics. Using triangular ramp pulse, the hysteresis of persistent I_Na enhanced by Tef was noticed, and the behavior was attenuated by subsequent addition of CBN. The delayed-rectifier or erg-mediated K⁺ current was mildly inhibited by 10 µM CBN, while it also slightly inhibited the amplitude of hyperpolarization-activated cation current. In HL-1 atrial cardiomyocytes, CBN inhibited peak I_Na and raised the inactivation rate of the current; moreover, further application of 10 µM Tef attenuated CBN-mediated decrease in I_Na. Collectively, this study provides an important yet unidentified finding revealing that CBN modifies I_Na in electrically excitable cells.     

Midazolam’s Effects on Delayed-Rectifier K+ Current and Intermediate-Conductance Ca2+-Activated K+ Channel in Jurkat T-lymphocytes

Midazolam (MDZ) could affect lymphocyte immune functions. However, the influence of MDZ on cell’s K⁺ currents has never been investigated. Thus, in the present study, the effects of MDZ on Jurkat T lymphocytes were studied using the patch-clamp technique. Results showed that MDZ suppressed the amplitude of delayed-rectifier K⁺ current (I_K(DR)) in concentration-, time-, and state-dependent manners. The IC₅₀ for MDZ-mediated reduction of I_K(DR) density was 5.87 μM. Increasing MDZ concentration raised the rate of current-density inactivation and its inhibitory action on I_K(DR) density was estimated with a dissociation constant of 5.14 μM. In addition, the inactivation curve of I_K(DR) associated with MDZ was shifted to a hyperpolarized potential with no change on the slope factor. MDZ-induced inhibition of I_K(DR) was not reversed by flumazenil. In addition, the activity of intermediate-conductance Ca²⁺-activated K⁺ (IK_Ca) channels was suppressed by MDZ. Furthermore, inhibition by MDZ on both I_K(DR) and IK_Ca-channel activity appeared to be independent from GABAA receptors and affected immune-regulating cytokine expression in LPS/PMA-treated human T lymphocytes. In conclusion, MDZ suppressed current density of I_K(DR) in concentration-, time-, and state-dependent manners in Jurkat T-lymphocytes and affected immune-regulating cytokine expression in LPS/PMA-treated human T lymphocytes.     

Functional Transient Receptor Potential Ankyrin 1 and Vanilloid 1 Ion Channels Are Overexpressed in Human Oral Squamous Cell Carcinoma

Oral squamous cell carcinoma (OSCC) is a common cancer with poor prognosis. Transient Receptor Potential Ankyrin 1 (TRPA1) and Vanilloid 1 (TRPV1) receptors are non-selective cation channels expressed on primary sensory neurons and epithelial and immune cells. TRPV1 mRNA and immunopositivity, as well as TRPA1-like immunoreactivity upregulation, were demonstrated in OSCC, but selectivity problems with the antibodies still raise questions and their functional relevance is unclear. Therefore, here, we investigated TRPA1 and TRPV1 expressions in OSCC and analyzed their functions. TRPA1 and TRPV1 mRNA were determined by RNAscope in situ hybridization and qPCR. Radioactive ₄₅Ca²⁺ uptake and ATP-based luminescence indicating cell viability were measured in PE/CA-PJ41 cells in response to the TRPA1 agonist allyl-isothiocyanate (AITC) and TRPV1 agonist capsaicin to determine receptor function. Both TRPA1 and TRPV1 mRNA are expressed in the squamous epithelium of the human oral mucosa and in PE/CA-PJ41 cells, and their expressions are significantly upregulated in OSCC compared to healthy mucosa. TRPA1 and TRPV1 activation (100 µM AITC, 100 nM capsaicin) induced ₄₅Ca²⁺-influx into PE/CA-PJ41 cells. Both AITC (10 nM–5 µM) and capsaicin (100 nM–45 µM) reduced cell viability, reaching significant decrease at 100 nM AITC and 45 µM capsaicin. We provide the first evidence for the presence of non-neuronal TRPA1 receptor in the OSCC and confirm the expression of TRPV1 channel. These channels are functionally active and might regulate cancer cell viability.     

Ca2+ Signalling Induced by NGF Identifies a Subset of Capsaicin-Excitable Neurons Displaying Enhanced Chemo-Nociception in Dorsal Root Ganglion Explants from Adult pirt-GCaMP3 Mouse

Nociceptors sense hazards via plasmalemmal cation channels, including transient receptor potential vanilloid 1 (TRPV1). Nerve growth factor (NGF) sensitises TRPV1 to capsaicin (CAPS), modulates nociceptor excitability and induces thermal hyperalgesia, but cellular mechanisms remain unclear. Confocal microscopy was used to image changes in intracellular Ca²⁺ concentration ([Ca²⁺]_i) across neuronal populations in dorsal root ganglia (DRG) explants from pirt-GCaMP3 adult mice, which express a fluorescent reporter in their sensory neurons. Raised [Ca²⁺]_i was detected in 84 neurons of three DRG explants exposed to NGF (100 ng/mL) and most (96%) of these were also excited by 1 μM CAPS. NGF elevated [Ca²⁺]_i in about one-third of the neurons stimulated by 1 μM CAPS, whether applied before or after the latter. In neurons excitable by NGF, CAPS-evoked [Ca²⁺]_i signals appeared significantly sooner (e.g., respective lags of 1.0 ± 0.1 and 1.9 ± 0.1 min), were much (>30%) brighter and lasted longer (6.6 ± 0.4 vs. 3.9 ± 0.2 min) relative to those non-responsive to the neurotrophin. CAPS tachyphylaxis lowered signal intensity by ~60% but was largely prevented by NGF. Increasing CAPS from 1 to 10 μM nearly doubled the number of cells activated but only modestly increased the amount co-activated by NGF. In conclusion, a sub-population of the CAPS-sensitive neurons in adult mouse DRG that can be excited by NGF is more sensitive to CAPS, responds with stronger signals and is further sensitised by transient exposure to the neurotrophin.     

The Evidence for Effective Inhibition of INa Produced by Mirogabalin ((1R,5S,6S)-6-(aminomethyl)-3-ethyl-bicyclo [3.2.0] hept-3-ene-6-acetic acid), a Known Blocker of CaV Channels

Mirogabalin (MGB, Tarlige^®), an inhibitor of the α₂δ-1 subunit of voltage-gated Ca²⁺ (Ca_V) channels, is used as a way to alleviate peripheral neuropathic pain and diabetic neuropathy. However, to what extent MGB modifies the magnitude, gating, and/or hysteresis of various types of plasmalemmal ionic currents remains largely unexplored. In pituitary tumor (GH₃) cells, we found that MGB was effective at suppressing the peak (transient, I_Na(T)) and sustained (late, I_Na(L)) components of the voltage-gated Na⁺ current (I_Na) in a concentration-dependent manner, with an effective IC₅₀ of 19.5 and 7.3 μM, respectively, while the K_D value calculated on the basis of minimum reaction scheme was 8.2 μM. The recovery of I_Na(T) inactivation slowed in the presence of MGB, although the overall current–voltage relation of I_Na(T) was unaltered; however, there was a leftward shift in the inactivation curve of the current. The magnitude of the window (I_Na(W)) or resurgent I_Na (I_Na(R)) evoked by the respective ascending or descending ramp pulse (V_ramp) was reduced during cell exposure to MGB. MGB-induced attenuation in I_Na(W) or I_Na(R) was reversed by the further addition of tefluthrin, a pyrethroid insecticide known to stimulate I_Na. MGB also effectively lessened the strength of voltage-dependent hysteresis of persistent I_Na in response to the isosceles triangular V_ramp. The cumulative inhibition of I_Na(T), evoked by pulse train stimulation, was enhanced in its presence. Taken together, in addition to the inhibition of Ca_V channels, the Na_V channel attenuation produced by MGB might have an impact in its analgesic effects occurring in vivo.     

Evidence for Inhibitory Perturbations on the Amplitude,Gating, and Hysteresis of A-Type Potassium Current,Produced by Lacosamide,a Functionalized Amino Acid with Anticonvulsant Properties

Lacosamide (Vimpat^®, LCS) is widely known as a functionalized amino acid with promising anti-convulsant properties; however, adverse events during its use have gradually appeared. Despite its inhibitory effect on voltage-gated Na⁺ current (I_Na), the modifications on varying types of ionic currents caused by this drug remain largely unexplored. In pituitary tumor (GH₃) cells, we found that the presence of LCS concentration-dependently decreased the amplitude of A-type K⁺ current (I_K(A)) elicited in response to membrane depolarization. The I_K(A) amplitude in these cells was sensitive to attenuation by the application of 4-aminopyridine, 4-aminopyridine-3-methanol, or capsaicin but not by that of tetraethylammonium chloride. The effective IC₅₀ value required for its reduction in peak or sustained I_K(A) was calculated to be 102 or 42 µM, respectively, while the value of the dissociation constant (K_D) estimated from the slow component in I_K(A) inactivation at varying LCS concentrations was 52 µM. By use of two-step voltage protocol, the presence of this drug resulted in a rightward shift in the steady-state inactivation curve of I_K(A) as well as in a slowing in the recovery time course of the current block; however, no change in the gating charge of the inactivation curve was detected in its presence. Moreover, the LCS addition led to an attenuation in the degree of voltage-dependent hysteresis for I_K(A) elicitation by long-duration triangular ramp voltage commands. Likewise, the I_K(A) identified in mouse mHippoE-14 neurons was also sensitive to block by LCS, coincident with an elevation in the current inactivation rate. Collectively, apart from its canonical action on I_Na inhibition, LCS was effective at altering the amplitude, gating, and hysteresis of I_K(A) in excitable cells. The modulatory actions on I_K(A), caused by LCS, could interfere with the functional activities of electrically excitable cells (e.g., pituitary tumor cells or hippocampal neurons).     

Comprehensive Simulation of Ca2+ Transients in the Continuum of Mouse Skeletal Muscle Fiber Types

Mag-Fluo-4 has revealed differences in the kinetics of the Ca²⁺ transients of mammalian fiber types (I, IIA, IIX, and IIB). We simulated the changes in [Ca²⁺] through the sarcomere of these four fiber types, considering classical (troponin –Tn–, parvalbumin –Pv–, adenosine triphosphate –ATP–, sarcoplasmic reticulum Ca²⁺ pump –SERCA–, and dye) and new (mitochondria –MITO–, Na⁺/Ca²⁺ exchanger –NCX–, and store-operated calcium entry –SOCE–) Ca²⁺ binding sites, during single and tetanic stimulation. We found that during a single twitch, the sarcoplasmic peak [Ca²⁺] for fibers type IIB and IIX was around 16 µM, and for fibers type I and IIA reached 10–13 µM. The release rate in fibers type I, IIA, IIX, and IIB was 64.8, 153.6, 238.8, and 244.5 µM ms⁻¹, respectively. Both the pattern of change and the peak concentrations of the Ca²⁺-bound species in the sarcoplasm (Tn, PV, ATP, and dye), the sarcolemma (NCX, SOCE), and the SR (SERCA) showed the order IIB ≥ IIX > IIA > I. The capacity of the NCX was 2.5, 1.3, 0.9, and 0.8% of the capacity of SERCA, for fibers type I, IIA, IIX, and IIB, respectively. MITO peak [Ca²⁺] ranged from 0.93 to 0.23 µM, in fibers type I and IIB, respectively, while intermediate values were obtained in fibers IIA and IIX. The latter numbers doubled during tetanic stimulation. In conclusion, we presented a comprehensive mathematical model of the excitation–contraction coupling that integrated most classical and novel Ca²⁺ handling mechanisms, overcoming the limitations of the fast- vs. slow-fibers dichotomy and the use of slow dyes.     

Thermoelectric properties of Ca0.8Dy0.2MnO3 synthesized by solution combustion process

High-quality Ca_0.8Dy_0.2MnO₃ nano-powders were synthesized by the solution combustion process. The size of the synthesized Ca_0.8Dy_0.2MnO₃ powders was approximately 23 nm. The green pellets were sintered at 1150-1300°C at a step size of 50°C. Sintered Ca_0.8Dy_0.2MnO₃ bodies crystallized in the perovskite structure with an orthorhombic symmetry. The sintering temperature did not affect the Seebeck coefficient, but significantly affected the electrical conductivity. The electrical conductivity of Ca_0.8Dy_0.2MnO₃ increased with increasing temperature, indicating a semiconducting behavior. The absolute value of the Seebeck coefficient gradually increased with an increase in temperature. The highest power factor (3.7 × 10^-5 Wm^-1 K^-2 at 800°C) was obtained for Ca_0.8Dy_0.2MnO₃ sintered at 1,250°C. In this study, we investigated the microstructure and thermoelectric properties of Ca_0.8Dy_0.2MnO₃, depending on sintering temperature.     

Snapin Specifically Up-Regulates Cav1.3 Ca2+ Channel Variant with a Long Carboxyl Terminus

Ca²⁺ entry through Ca_v1.3 Ca²⁺ channels plays essential roles in diverse physiological events. We employed yeast-two-hybrid (Y2H) assays to mine novel proteins interacting with Ca_v1.3 and found Snapin2, a synaptic protein, as a partner interacting with the long carboxyl terminus (CT_L) of rat Ca_v1.3_L variant. Co-expression of Snapin with Ca_v1.3_L/Ca_vβ₃/α₂δ₂ subunits increased the peak current density or amplitude by about 2-fold in HEK-293 cells and Xenopus oocytes, without affecting voltage-dependent gating properties and calcium-dependent inactivation. However, the Snapin up-regulation effect was not found for rat Ca_v1.3_S containing a short CT (CT_S) in which a Snapin interaction site in the CT_L was deficient. Luminometry and electrophysiology studies uncovered that Snapin co-expression did not alter the membrane expression of HA tagged Ca_v1.3_L but increased the slope of tail current amplitudes plotted against ON-gating currents, indicating that Snapin increases the opening probability of Ca_v1.3_L. Taken together, our results strongly suggest that Snapin directly interacts with the CT_L of Ca_v1.3_L, leading to up-regulation of Ca_v1.3_L channel activity via facilitating channel opening probability.     

Delayed Ventricular Repolarization and Sodium Channel Current Modification in a Mouse Model of Rett Syndrome

Rett syndrome (RTT) is a severe developmental disorder that is strongly linked to mutations in the MECP2 gene. RTT has been associated with sudden unexplained death and ECG QT interval prolongation. There are mixed reports regarding QT prolongation in mouse models of RTT, with some evidence that loss of Mecp2 function enhances cardiac late Na current, I_Na,Late. The present study was undertaken in order to investigate both ECG and ventricular AP characteristics in the Mecp2^Null/Y male murine RTT model and to interrogate both fast I_Na and I_Na,Late in myocytes from the model. ECG recordings from 8–10-week-old Mecp2^Null/Y male mice revealed prolongation of the QT and rate corrected QT (QTc) intervals and QRS widening compared to wild-type (WT) controls. Action potentials (APs) from Mecp2^Null/Y myocytes exhibited longer APD₇₅ and APD₉₀ values, increased triangulation and instability. I_Na,Late was also significantly larger in Mecp2^Null/Y than WT myocytes and was insensitive to the Nav1.8 inhibitor A-803467. Selective recordings of fast I_Na revealed a decrease in peak current amplitude without significant voltage shifts in activation or inactivation V_0.5. Fast I_Na ‘window current’ was reduced in RTT myocytes; small but significant alterations of inactivation and reactivation time-courses were detected. Effects of two I_Na,Late inhibitors, ranolazine and GS-6615 (eleclazine), were investigated. Treatment with 30 µM ranolazine produced similar levels of inhibition of I_Na,Late in WT and Mecp2^Null/Y myocytes, but produced ventricular AP prolongation not abbreviation. In contrast, 10 µM GS-6615 both inhibited I_Na,Late and shortened ventricular AP duration. The observed changes in I_Na and I_Na,Late can account for the corresponding ECG changes in this RTT model. GS-6615 merits further investigation as a potential treatment for QT prolongation in RTT.     

Elongation and Contraction of Scallop Sarcoplasmic Reticulum (SR): ATP Stabilizes Ca2+-ATPase Crystalline Array Elongation of SR Vesicles

The Ca²⁺-ATPase is an integral transmembrane Ca²⁺ pump of the sarcoplasmic reticulum (SR). Crystallization of the cytoplasmic surface ATPase molecules of isolated scallop SR vesicles was studied at various calcium concentrations by negative stain electron microscopy. In the absence of ATP, round SR vesicles displaying an assembly of small crystalline patches of ATPase molecules were observed at 18 µM [Ca²⁺]. These partly transformed into tightly elongated vesicles containing ATPase crystalline arrays at low [Ca²⁺] (≤1.3 µM). The arrays were classified as ‘’tetramer’’, “two-rail” (like a railroad) and ‘’monomer’’. Their crystallinity was low, and they were unstable. In the presence of ATP (5 mM) at a low [Ca²⁺] of ~0.002 µM, “two-rail” arrays of high crystallinity appeared more frequently in the tightly elongated vesicles and the distinct tetramer arrays disappeared. During prolonged (~2.5 h) incubation, ATP was consumed and tetramer arrays reappeared. A specific ATPase inhibitor, thapsigargin, prevented both crystal formation and vesicle elongation in the presence of ATP. Together with the second part of this study, these data suggest that the ATPase forms tetramer units and longer tetramer crystalline arrays to elongate SR vesicles, and that the arrays transform into more stable “two-rail” forms in the presence of ATP at low [Ca²⁺].     

Nitric Oxide Does Not Inhibit but Is Metabolized by the Cytochrome bcc-aa3 Supercomplex

Nitric oxide (NO) is a well-known active site ligand and inhibitor of respiratory terminal oxidases. Here, we investigated the interaction of NO with a purified chimeric bcc-aa₃ supercomplex composed of Mycobacterium tuberculosis cytochrome bcc and Mycobacterium smegmatis aa₃-type terminal oxidase. Strikingly, we found that the enzyme in turnover with O₂ and reductants is resistant to inhibition by the ligand, being able to metabolize NO at 25 °C with an apparent turnover number as high as ≈303 mol NO (mol enzyme)⁻¹ min⁻¹ at 30 µM NO. The rate of NO consumption proved to be proportional to that of O₂ consumption, with 2.65 ± 0.19 molecules of NO being consumed per O₂ molecule by the mycobacterial bcc-aa₃. The enzyme was found to metabolize the ligand even under anaerobic reducing conditions with a turnover number of 2.8 ± 0.5 mol NO (mol enzyme)⁻¹ min⁻¹ at 25 °C and 8.4 µM NO. These results suggest a protective role of mycobacterial bcc-aa₃ supercomplexes against NO stress.     

The Effectiveness in Activating M-Type K+ Current Produced by Solifenacin ([(3R)-1-azabicyclo[2.2.2]octan-3-yl] (1S)-1-phenyl-3,4-dihydro-1H-isoquinoline-2-carboxylate): Independent of Its Antimuscarinic Action

Solifenacin (Vesicare^®, SOL), known to be a member of isoquinolines, is a muscarinic antagonist that has anticholinergic effect, and it has been beneficial in treating urinary incontinence and neurogenic detrusor overactivity. However, the information regarding the effects of SOL on membrane ionic currents is largely uncertain, despite its clinically wide use in patients with those disorders. In this study, the whole-cell current recordings revealed that upon membrane depolarization in pituitary GH₃ cells, the exposure to SOL concentration-dependently increased the amplitude of M-type K⁺ current (I_K(M)) with effective EC₅₀ value of 0.34 μM. The activation time constant of I_K(M) was concurrently shortened in the SOL presence, hence yielding the K_D value of 0.55 μM based on minimal reaction scheme. As cells were exposed to SOL, the steady-state activation curve of I_K(M) was shifted along the voltage axis to the left with no change in the gating charge of the current. Upon an isosceles-triangular ramp pulse, the hysteretic area of I_K(M) was increased by adding SOL. As cells were continually exposed to SOL, further application of acetylcholine (1 μM) failed to modify SOL-stimulated I_K(M); however, subsequent addition of thyrotropin releasing hormone (TRH, 1 μM) was able to counteract SOL-induced increase in I_K(M) amplitude. In cell-attached single-channel current recordings, bath addition of SOL led to an increase in the activity of M-type K⁺ (K_M) channels with no change in the single channel conductance; the mean open time of the channel became lengthened. In whole-cell current-clamp recordings, the SOL application reduced the firing of action potentials (APs) in GH₃ cells; however, either subsequent addition of TRH or linopirdine was able to reverse SOL-mediated decrease in AP firing. In hippocampal mHippoE-14 neurons, the I_K(M) was also stimulated by adding SOL. Altogether, findings from this study disclosed for the first time the effectiveness of SOL in interacting with K_M channels and hence in stimulating I_K(M) in electrically excitable cells, and this noticeable action appears to be independent of its antagonistic activity on the canonical binding to muscarinic receptors expressed in GH₃ or mHippoE-14 cells.     

Interferon Beta Activity Is Modulated via Binding of Specific S100 Proteins

Interferon-β (IFN-β) is a pleiotropic cytokine used for therapy of multiple sclerosis, which is also effective in suppression of viral and bacterial infections and cancer. Recently, we reported a highly specific interaction between IFN-β and S100P lowering IFN-β cytotoxicity to cancer cells (Int J Biol Macromol. 2020; 143: 633–639). S100P is a member of large family of multifunctional Ca²⁺-binding proteins with cytokine-like activities. To probe selectivity of IFN-β—S100 interaction with respect to S100 proteins, we used surface plasmon resonance spectroscopy, chemical crosslinking, and crystal violet assay. Among the thirteen S100 proteins studied S100A1, S100A4, and S100A6 proteins exhibit strictly Ca²⁺-dependent binding to IFN-β with equilibrium dissociation constants, K_d, of 0.04–1.5 µM for their Ca²⁺-bound homodimeric forms. Calcium depletion abolishes the S100—IFN-β interactions. Monomerization of S100A1/A4/A6 decreases K_d values down to 0.11–1.0 nM. Interferon-α is unable of binding to the S100 proteins studied. S100A1/A4 proteins inhibit IFN-β-induced suppression of MCF-7 cells viability. The revealed direct influence of specific S100 proteins on IFN-β activity uncovers a novel regulatory role of particular S100 proteins, and opens up novel approaches to enhancement of therapeutic efficacy of IFN-β.     

Involvement of Ca2+ Signaling in the Synergistic Effects between Muscarinic Receptor Antagonists and β2-Adrenoceptor Agonists in Airway Smooth Muscle

Long-acting muscarinic antagonists (LAMAs) and short-acting β₂-adrenoceptor agonists (SABAs) play important roles in remedy for COPD. To propel a translational research for development of bronchodilator therapy, synergistic effects between SABAs with LAMAs were examined focused on Ca²⁺ signaling using simultaneous records of isometric tension and F340/F380 in fura-2-loaded tracheal smooth muscle. Glycopyrronium (3 nM), a LAMA, modestly reduced methacholine (1 μM)-induced contraction. When procaterol, salbutamol and SABAs were applied in the presence of glycopyrronium, relaxant effects of these SABAs are markedly enhanced, and percent inhibition of tension was much greater than the sum of those for each agent and those expected from the BI theory. In contrast, percent inhibition of F340/F380 was not greater than those values. Bisindolylmaleimide, an inhibitor of protein kinase C (PKC), significantly increased the relaxant effect of LAMA without reducing F340/F380. Iberiotoxin, an inhibitor of large-conductance Ca²⁺-activated K⁺ (K_Ca) channels, significantly suppressed the effects of these combined agents with reducing F340/F380. In conclusion, combination of SABAs with LAMAs synergistically enhances inhibition of muscarinic contraction via decreasing both Ca²⁺ sensitization mediated by PKC and Ca²⁺ dynamics mediated by K_Ca channels. PKC and K_Ca channels may be molecular targets for cross talk between β₂-adrenoceptors and muscarinic receptors.     

Enhanced thermoelectric properties induced by chemical pressure in Ca3Co4O9

We report the investigation of boron substitution on structural, electrical, thermal, and thermoelectric properties of Ca_3−xB_xCo₄O₉ (x=0, 0.5, 0.75, and 1) in the temperature range between 300 K and 5 K. X-ray diffraction studies show that the Ca₃Co₄O₉ phase is successfully preserved as the majority phase in the x=0.5 sample despite the small size of boron ions. Electrical transport measurements confirm that B³⁺ substitution for Ca²⁺ causes an increase in resistivity due to the decrease in carrier concentration. x=0.5 sample is found to have a Seebeck coefficient of 181 μV/K at room temperature which is ~1.5 times higher than that of the pure Ca₃Co₄O₉. Our results indicate that the chemical pressure due to the large ionic radii difference between B³⁺ (0.27 Å) and Ca²⁺ (1 Å) enhances the thermoelectric properties as long as the unique crystal structure of Ca₃Co₄O₉ is preserved.     

Differential Regulation of Ca2+-Activated Cl− Channel TMEM16A Splice Variants by Membrane PI(4,5)P2

TMEM16A is a Ca²⁺-activated Cl⁻ channel that controls broad cellular processes ranging from mucus secretion to signal transduction and neuronal excitability. Recent studies have reported that membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) is an important cofactor that allosterically regulates TMEM16A channel activity. However, the detailed regulatory actions of PIP₂ in splice variants of TMEM16A remain unclear. Here, we demonstrated that the attenuation of membrane phosphoinositide levels selectively inhibited the current amplitude of the TMEM16A(ac) isoform by decreasing the slow, but not instantaneous, Cl⁻ currents, which are independent of the membrane potential and specific to PI(4,5)P₂ depletion. The attenuation of endogenous PI(4,5)P₂ levels by the activation of Danio rerio voltage-sensitive phosphatase (Dr-VSP) decreased the Cl⁻ currents of TMEM16A(ac) but not the TMEM16A(a) isoform, which was abolished by the co-expression of PIP 5-kinase type-1γ (PIPKIγ). Using the rapamycin-inducible dimerization of exogenous phosphoinositide phosphatases, we further revealed that the stimulatory effects of phosphoinositide on TMEM16A(ac) channels were similar in various membrane potentials and specific to PI(4,5)P₂, not PI4P and PI(3,4,5)P₃. Finally, we also confirmed that PI(4,5)P₂ resynthesis is essential for TMEM16A(ac) recovery from Dr-VSP-induced current inhibition. Our data demonstrate that membrane PI(4,5)P₂ selectively modulates the gating of the TMEM16A(ac) channel in an agonistic manner, which leads to the upregulation of TMEM16A(ac) functions in physiological conditions.