Hsp47 promotes biogenesis of multi-subunit neuroreceptors in the endoplasmic reticulum.

Protein homeostasis (proteostasis) deficiency is an important contributing factor to neurological and metabolic diseases. However, how the proteostasis network orchestrates the folding and assembly of multi-subunit membrane proteins is poorly understood. Previous proteomics studies identified Hsp47 (Gene: SERPINH1), a heat shock protein in the endoplasmic reticulum lumen, as the most enriched interacting chaperone for gamma-aminobutyric acid type A (GABAA) receptors. Here, we show that Hsp47 enhances the functional surface expression of GABAA receptors in rat neurons and human HEK293T cells. Furthermore, molecular mechanism study demonstrates that Hsp47 acts after BiP (Gene: HSPA5) and preferentially binds the folded conformation of GABAA receptors without inducing the unfolded protein response in HEK293T cells. Therefore, Hsp47 promotes the subunit-subunit interaction, the receptor assembly process, and the anterograde trafficking of GABAA receptors. Overexpressing Hsp47 is sufficient to correct the surface expression and function of epilepsy-associated GABAA receptor variants in HEK293T cells. Hsp47 also promotes the surface trafficking of other Cys-loop receptors, including nicotinic acetylcholine receptors and serotonin type 3 receptors in HEK293T cells. Therefore, in addition to its known function as a collagen chaperone, this work establishes that Hsp47 plays a critical and general role in the maturation of multi-subunit Cys-loop neuroreceptors.

Carbon confined GeO2 hollow spheres for stable rechargeable Na ion batteries.

Germanium (Ge) based nanomaterials are regarded as promising high-capacity anode materials for Na ion batteries, but suffer fast capacity fading problems caused by the alloying/de-alloying reactions of Na-Ge. Herein, we report a new method for preparing highly dispersed GeO2 by using molecular-level ionic liquids (ILs) as carbon sources. In the obtained GeO2@C composite material, GeO2 exhibits hollow spherical morphology and is uniformly distributed in the carbon matrix. The as-prepared GeO2@C exhibits improved Na ion storage performances including high reversible capacity (577 mA h g-1 at 0.1C), rate property (270 mA h g-1 at 3C), and high capacity retention (82.3% after 500 cycles). The improved electrochemical performance could be attributed to the unique nanostructure of GeO2@C, the synergistic effect between GeO2 hollow spheres and the carbon matrix ensures the anode material effectively alleviates the volume expansion and the particle agglomeration problems.

HisVA: A Visual Analytics System for Studying History.

Studying history involves many difficult tasks. Examples include searching for proper data in a large event space, understanding stories of historical events by time and space, and finding relationships among events that may not be apparent. Instructors who extensively use well-organized and well-argued materials (e.g., textbooks and online resources) can lead students to a narrow perspective in understanding history and prevent spontaneous investigation of historical events, with the students asking their own questions. In this article, we proposed HisVA, a visual analytics system that allows the efficient exploration of historical events from Wikipedia using three views: event, map, and resource. HisVA provides an effective event exploration space, where users can investigate relationships among historical events by reviewing and linking them in terms of space and time. To evaluate our system, we present two usage scenarios, a user study with a qualitative analysis of user exploration strategies, and in-class deployment results.

Phytoplankton community structural reshaping as response to the thermal effect of cooling water discharged from power plant.

The increase of water temperature caused by the thermal effect of cooling water discharged from power plants has become a major environmental problem, especially its influence on phytoplankton community. The change of water temperature usually reshapes the structure of phytoplankton community. A research combining phytoplankton community and thermal discharge of power plants was conducted to identify the potential influences. Results indicated the average annual water temperature of the reservoir increased gradually by 5-11 °C because of the thermal discharge. Through annual diversity analysis, 139 species or taxa from 6 phyla (i.e., Bacillariophyta, Chlorophyta, Cyanobacteria, Euglenophyta, Dinoflagellata, and Cryptophyta) were found in different sampling sites, among which Bacillariophyta was the dominant community. Preliminary experimental results revealed the increasing temperature completely reshaped the phytoplankton community structure, especially during the cold season, and this was confirmed by the results of redundancy analysis. In addition, lots of thermophilic genera (i.e., Synedra, Nitzschia, and Navicula) were detected at sampling station 1 (Spt1) and sampling station 2 (Spt2) where the effect of thermal discharge was the most obvious. The increase in biomass and cell count of Bacillariophyta was the result of thermal effect, especially in cold season. Besides, consequences also revealed some environmental parameters (i.e., dissolved oxygen concentration, chlorophyll a concentration, and transparency) were affected by the thermal discharge. Chlorophyll a concentration exhibited a slow rising trend while dissolved oxygen concentration and transparency gradually decreased.

Growth inhibition and oxidative damage of Microcystis aeruginosa induced by aqueous extract of different submerged macrophytes.

With the increasing eutrophication of the aquatic environments, cyanobacteria blooms caused certain damage to the animals and plants in the aquatic environments. In this experiment, two species were selected from six species of submerged macrophytes, the experimental conditions were changed to achieve the best inhibitory effect on Microcystis aeruginosa, and oxidative damage analysis was carried out. The experiment results demonstrated that the inhibition rate of Vallisneria natans and Ceratophyllum demersum was nearly 100% at the concentration of 3 g/L after 15 days of anaerobic soaking extract. In addition, the longer the soaking time of the two submerged macrophytes, the weaker the photosynthesis effect, and the lower the chlorophyll fluorescence parameters, the more obvious the inhibition effect on M. aeruginosa. Lipid peroxidation injury of M. aeruginosa could be reflected by malondialdehyde (MDA) concentration. The MDA concentration in the experimental group was significantly higher than the control group. Results showed that V. natans and C. demersum could induce oxidative damage in M. aeruginosa. It was also observed that the secondary metabolites produced by V. natans were mainly fatty acids (e.g., the oxidative acid was 6.92 w/%, and the successful acid was 9.85 w/%) which inhibited M. aeruginosa in gas chromatography-mass spectrometry (GC-MS). The main secondary metabolites in C. demersum were hydroxyl acids (e.g., the 4-hydroxy-3-methoxyphenylacetic acid was 24.33 w/%), which could inhibit the algae through allelopathy. This study provided reference for submerged macrophytes to inhibit M. aeruginosa under different conditions.

Proteostasis Regulators Restore Function of Epilepsy-Associated GABAA Receptors.

Proteostasis deficiency in mutated ion channels leads to a variety of ion channel diseases that are caused by excessive endoplasmic reticulum-associated degradation (ERAD) and inefficient membrane trafficking. We investigated proteostasis maintenance of γ-aminobutyric acid type A (GABAA) receptors, the primary mediators of neuronal inhibition in the mammalian central nervous system. We screened a structurally diverse, Food and Drug Administration-approved drug library and identified dinoprost (DNP) and dihydroergocristine (DHEC) as highly efficacious enhancers of surface expression of four epilepsy-causing trafficking-deficient mutant receptors. Furthermore, DNP and DHEC restore whole-cell and synaptic currents by incorporating mutated subunits into functional receptors. Mechanistic studies revealed that both drugs reduce subunit degradation by attenuating the Grp94/Hrd1/Sel1L/VCP-mediated ERAD pathway and enhance the subunit folding by promoting subunit interactions with major GABAA receptors-interacting chaperones, BiP and calnexin. In summary, we report that DNP and DHEC remodel the endoplasmic reticulum proteostasis network to restore the functional surface expression of mutant GABAA receptors.

Combined effects of green manure returning and addition of sewage sludge compost on plant growth and microorganism communities in gold tailings.

Remediation of gold tailings is often difficult due to their extremely barren nature and highly heavy metal concentrations. Returning green manure and applying sewage sludge compost have the beneficial effects of providing nutrients and improving the soil environment. The effects of green manure plants, alfalfa (Medicago sativa L.), ryegrass (Lolium perenne Linn.), and tall fescue (Festuca arundinacea), returning in situ on nutrients, bioavailability of trace metals, and community structure of microorganism in gold tailings amended with 0%, 5%, and 10% (weight/weight) sewage sludge compost on the top 4 cm of tailings (SSC-5, SSC-10) were investigated in a pot experiment. The results showed that the plant biomass and microbial biomass carbon in tailings significantly increased in the treatments with sewage sludge compost. The available N and available P and the availability of Zn decreased markedly with the returning of alfalfa and ryegrass. Moreover, through high-throughput sequencing, it was found that the returning of alfalfa had positive effects on the bacterial community richness but a negative impact on the fungal community richness. The microbial community diversity was reduced in the treatment without sewage sludge compost amendment and with alfalfa returning. However, the microbial community diversity was enriched in the treatment of alfalfa returning with sewage sludge compost. In each plant species, 9 dominant bacterial phyla and 10 dominant fungi phyla could be detected. Returning alfalfa green manure and applying sewage sludge compost led to a relative increase in the abundance of Proteobacteria and Ascomycota. These results demonstrated that returning alfalfa and applying sewage sludge compost could be effective in the ecological restoration of gold tailings.

Remodeling the endoplasmic reticulum proteostasis network restores proteostasis of pathogenic GABAA receptors.

Biogenesis of membrane proteins is controlled by the protein homeostasis (proteostasis) network. We have been focusing on protein quality control of γ-aminobutyric acid type A (GABAA) receptors, the major inhibitory neurotransmitter-gated ion channels in mammalian central nervous system. Proteostasis deficiency in GABAA receptors causes loss of their surface expression and thus function on the plasma membrane, leading to epilepsy and other neurological diseases. One well-characterized example is the A322D mutation in the α1 subunit that causes its extensive misfolding and expedited degradation in the endoplasmic reticulum (ER), resulting in autosomal dominant juvenile myoclonic epilepsy. We aimed to correct misfolding of the α1(A322D) subunits in the ER as an approach to restore their functional surface expression. Here, we showed that application of BIX, a specific, potent ER resident HSP70 family protein BiP activator, significantly increases the surface expression of the mutant receptors in human HEK293T cells and neuronal SH-SY5Y cells. BIX attenuates the degradation of α1(A322D) and enhances their forward trafficking and function. Furthermore, because BiP is one major target of the two unfolded protein response (UPR) pathways: ATF6 and IRE1, we continued to demonstrate that modest activations of the ATF6 pathway and IRE1 pathway genetically enhance the plasma membrane trafficking of the α1(A322D) protein in HEK293T cells. Our results underlie the potential of regulating the ER proteostasis network to correct loss-of-function protein conformational diseases.

Grp94 Protein Delivers γ-Aminobutyric Acid Type A (GABAA) Receptors to Hrd1 Protein-mediated Endoplasmic Reticulum-associated Degradation.

Proteostasis maintenance of γ-aminobutyric acid type A (GABAA) receptors dictates their function in controlling neuronal inhibition in mammalian central nervous systems. However, as a multisubunit, multispan, integral membrane protein, even wild type subunits of GABAA receptors fold and assemble inefficiently in the endoplasmic reticulum (ER). Unassembled and misfolded subunits undergo ER-associated degradation (ERAD), but this degradation process remains poorly understood for GABAA receptors. Here, using the α1 subunits of GABAA receptors as a model substrate, we demonstrated that Grp94, a metazoan-specific Hsp90 in the ER lumen, uses its middle domain to interact with the α1 subunits and positively regulates their ERAD. OS-9, an ER-resident lectin, acts downstream of Grp94 to further recognize misfolded α1 subunits in a glycan-dependent manner. This delivers misfolded α1 subunits to the Hrd1-mediated ubiquitination and the valosin-containing protein-mediated extraction pathway. Repressing the initial ERAD recognition step by inhibiting Grp94 enhances the functional surface expression of misfolding-prone α1(A322D) subunits, which causes autosomal dominant juvenile myoclonic epilepsy. This study clarifies a Grp94-mediated ERAD pathway for GABAA receptors, which provides a novel way to finely tune their function in physiological and pathophysiological conditions.

L-type Calcium Channel Blockers Enhance Trafficking and Function of Epilepsy-associated α1(D219N) Subunits of GABA(A) Receptors.

Gamma-aminobutyric acid type A (GABAA) receptors are the primary inhibitory ion channels in the mammalian central nervous system and play an essential role in regulating inhibition-excitation balance in neural circuits. The α1 subunit harboring the D219N mutation of GABAA receptors was reported to be retained in the endoplasmic reticulum (ER) and traffic inefficiently to the plasma membrane, leading to a loss of function of α1(D219N) subunits and thus idiopathic generalized epilepsy (IGE). We present the use of small molecule proteostasis regulators to enhance the forward trafficking of α1(D219N) subunits to restore their function. We showed that treatment with verapamil (4 µM, 24 h), an L-type calcium channel blocker, substantially increases the α1(D219N) subunit cell surface level in both HEK293 cells and neuronal SH-SY5Y cells and remarkably restores the GABA-induced maximal chloride current in HEK293 cells expressing α1(D219N)ß2γ2 receptors to a level that is comparable to wild type receptors. Our drug mechanism study revealed that verapamil treatment promotes the ER to Golgi trafficking of the α1(D219N) subunits post-translationally. To achieve that, verapamil treatment enhances the interaction between the α1(D219N) subunit and ß2 subunit and prevents the aggregation of the mutant protein by shifting the protein from the detergent-insoluble fractions to detergent-soluble fractions. By combining (35)S pulse-chase labeling and MG-132 inhibition experiments, we demonstrated that verapamil treatment does not inhibit the ER-associated degradation of the α1(D219N) subunit. In addition, its effect does not involve a dynamin-1 dependent endocytosis. To gain further mechanistic insight, we showed that verapamil increases the interaction between the mutant protein and calnexin and calreticulin, two major lectin chaperones in the ER. Moreover, calnexin binding promotes the forward trafficking of the mutant subunit. Taken together, our data indicate that verapamil treatment enhances the calnexin-assisted forward trafficking and subunit assembly, which leads to substantially enhanced functional surface expression of the mutant receptors. Since verapamil is an FDA-approved drug that crosses blood-brain barrier and has been used as an additional medication for some epilepsies, our findings suggest that verapamil holds great promise to be developed to ameliorate IGE resulting from α1(D219N) subunit trafficking deficiency.

Combining valosin-containing protein (VCP) inhibition and suberanilohydroxamic acid (SAHA) treatment additively enhances the folding, trafficking, and function of epilepsy-associated γ-aminobutyric acid, type A (GABAA) receptors.

GABAA receptors are the primary inhibitory ion channels in the mammalian central nervous system. The A322D mutation in the α1 subunit results in its excessive endoplasmic reticulum-associated degradation at the expense of plasma membrane trafficking, leading to autosomal dominant juvenile myoclonic epilepsy. Presumably, valosin-containing protein (VCP)/p97 extracts misfolded subunits from the endoplasmic reticulum membrane to the cytosolic proteasome for degradation. Here we showed that inhibiting VCP using Eeyarestatin I reduces the endoplasmic reticulum-associated degradation of the α1(A322D) subunit without an apparent effect on its dynamin-1 dependent endocytosis and that this treatment enhances its trafficking. Furthermore, coapplication of Eeyarestatin I and suberanilohydroxamic acid, a known small molecule that promotes chaperone-assisted folding, yields an additive restoration of surface expression of α1(A322D) subunits in HEK293 cells and neuronal SH-SY5Y cells. Consequently, this combination significantly increases GABA-induced chloride currents in whole-cell patch clamping experiments than either chemical compound alone in HEK293 cells. Our findings suggest that VCP inhibition without stress induction, together with folding enhancement, represents a new strategy to restore proteostasis of misfolding-prone GABAA receptors and, therefore, a potential remedy for idiopathic epilepsy.

Docetaxel modulates the delayed rectifier potassium current (IK) and ATP-sensitive potassium current (IKATP) in human breast cancer cells.

Ion channel expression and activity may be affected during tumor development and cancer growth. Activation of potassium (K(+)) channels in human breast cancer cells is reported to be involved in cell cycle progression. In this study, we investigated the effects of docetaxel on the delayed rectifier potassium current (I K) and the ATP-sensitive potassium current (I KATP) in two human breast cancer cell lines, MCF-7 and MDA-MB-435S, using the whole-cell patch-clamp technique. Our results show that docetaxel inhibited the I K and I KATP in both cell lines in a dose-dependent manner. Compared with the control at a potential of +60 mV, treatment with docetaxel at doses of 0.1, 1, 5, and 10 µM significantly decreased the I K in MCF-7 cells by 16.1 ± 3.5, 30.2 ± 5.2, 42.5 ± 4.3, and 46.4 ± 9% (n = 5, P < 0.05), respectively and also decreased the I KATP at +50 mV. Similar results were observed in MDA-MB-435S cells. The G-V curves showed no significant changes after treatment of either MCF-7 or MDA-MB-435S cells with 10 µM docetaxel. The datas indicate that the possible mechanisms of I K and I KATP inhibition by docetaxel may be responsible for its effect on the proliferation of human breast cancer cells.

SAHA enhances Proteostasis of epilepsy-associated α1(A322D)ß2γ2 GABA(A) receptors.

GABA(A) receptors are the primary inhibitory ion channels in the mammalian central nervous system. The A322D mutation in the α1 subunit of GABA(A) receptors is known to result in its degradation and reduce its cell surface expression, leading to loss of GABAA receptor function in autosomal dominant juvenile myoclonic epilepsy. Here, we show that SAHA, a FDA-approved drug, increases the transcription of the α1(A322D) subunit, enhances its folding and trafficking posttranslationally, increases its cell surface level, and restores the GABA-induced maximal current in HEK293 cells expressing α1(A322D)ß2γ2 receptors to 10% of that for wild-type receptors. To enhance the trafficking efficiency of the α1(A322D) subunit, SAHA increases the BiP protein level and the interaction between the α1(A322D) subunit and calnexin. SAHA is a drug that enhances epilepsy-associated GABAA receptor proteostasis.

Identification of GABA(C) receptor protein homeostasis network components from three tandem mass spectrometry proteomics approaches.

γ-Amino butyric acid type C (GABA(C)) receptors inhibit neuronal firing primarily in retina. Maintenance of GABA(C) receptor protein homeostasis in cells is essential for its function. However, a systematic study of GABA(C) receptor protein homeostasis (proteostasis) network components is absent. Here coimmunoprecipitation of human GABA(C)-ρ1-receptor complexes was performed in HEK293 cells overexpressing ρ1 receptors. To enhance the coverage and reliability of identified proteins, immunoisolated ρ1-receptor complexes were subjected to three tandem mass spectrometry (MS)-based proteomic analyses, namely, gel-based tandem MS (GeLC-MS/MS), solution-based tandem MS (SoLC-MS/MS), and multidimensional protein identification technology (MudPIT). From the 107 identified proteins, we assembled GABA(C)-ρ1-receptor proteostasis network components, including proteins with protein folding, degradation, and trafficking functions. We studied representative individual ρ1-receptor-interacting proteins, including calnexin, a lectin chaperone that facilitates glycoprotein folding, and LMAN1, a glycoprotein trafficking receptor, and global effectors that regulate protein folding in cells based on bioinformatics analysis, including HSF1, a master regulator of the heat shock response, and XBP1, a key transcription factor of the unfolded protein response. Manipulating selected GABA(C) receptor proteostasis network components is a promising strategy to regulate GABA(C) receptor folding, trafficking, degradation and thus function to ameliorate related retinal diseases.

Characterization of a novel splice variant of δ ENaC subunit in human lungs.

Salt absorption via apical epithelial sodium channels (ENaC) is a critical rate-limiting process in maintaining airway and lung lining fluid at the physiological level. δ ENaC (termed δ1 in this article) has been detected in human lung epithelial cells in addition to α, ß, and γ subunits (Ji HL, Su XF, Kedar S, Li J, Barbry P, Smith PR, Matalon S, Benos DJ. J Biol Chem 281: 8233-8241, 2006; Nie HG, Chen L, Han DY, Li J, Song WF, Wei SP, Fang XH, Gu X, Matalon S, Ji HL, J Physiol 587: 2663-2676, 2009) and may contribute to the differences in the biophysical properties of amiloride-inhibitable cation channels in pulmonary epithelial cells. Here we cloned a splicing variant of the δ1 ENaC, namely, δ2 ENaC in human bronchoalveolar epithelial cells (16HBEo). δ2 ENaC possesses 66 extra amino acids attached to the distal amino terminal tail of the δ1 ENaC. δ2 ENaC was expressed in both alveolar type I and II cells of human lungs as revealed by in situ hybridization and real-time RT-PCR. To characterize the biophysical and pharmacological features of the splicing variant, we injected Xenopus oocytes with human ENaC cRNAs and measured whole cell and single channel currents of δ1ßγ, δ2ßγ, and αßγ channels. Oocytes injected with δ2ßγ cRNAs exhibited whole cell currents significantly greater than those expressing δ1ßγ and αßγ channels. Single channel activity, unitary conductance, and open probability of δ2ßγ channels were significantly greater compared with δ1ßγ and αßγ channels. In addition, δ2ßγ and δ1ßγ channels displayed significant differences in apparent Na(+) affinity, dissociation constant for amiloride (K(i)(amil)), the EC(50) for capsazepine activation, and gating kinetics by protons. Channels comprising of this novel splice variant may contribute to the diversities of native epithelial Na(+) channels.

8-(4-chlorophenylthio)-guanosine-3',5'-cyclic monophosphate-Na stimulates human alveolar fluid clearance by releasing external Na+ self-inhibition of epithelial Na+ channels.

Salt absorption via alveolar epithelial Na(+) channels (ENaC) is a critical step for maintaining an airspace free of flooding. Previously, we found that 8-(4-chlorophenylthio)-guanosine-3',5'-cyclic monophosphate-Na (CPT-cGMP) activated native and heterologous ENaC. To investigate the potential pharmacological relevance, we applied this compound intratracheally to human lungs and found that ex vivo alveolar fluid clearance was increased significantly. Furthermore, this compound eliminated self-inhibition in human lung H441 cells and in oocytes expressing human αßγ but not δßγ channels. To further elucidate this novel mechanism, we constructed mutants abolishing (ß(ΔV348) and γ(H233R)) or augmenting (α(Y458A) and γ(M432G)) self-inhibition. The mutants eliminating self-inhibition lost their responses to CPT-cGMP, whereas those enhancing self-inhibition facilitated the stimulatory effects of this compound. CPT-cGMP was unable to activate a high P(o) mutant (ß(S520C)) and plasmin proteolytically cleaved channels. Our data suggest that elimination of self-inhibition of αßγ ENaC may be a novel mechanism for CPT-cGMP to stimulate salt reabsorption in human lungs.

Cpt-cAMP activates human epithelial sodium channels via relieving self-inhibition.

External Na(+) self-inhibition is an intrinsic feature of epithelial sodium channels (ENaC). Cpt-cAMP regulates heterologous guinea pig but not rat αßγ ENaC in a ligand-gated manner. We hypothesized that cpt-cAMP may eliminate the self-inhibition of human ENaC thereby open channels. Regulation of self-inhibition by this compound in oocytes was analyzed using the two-electrode voltage clamp and Ussing chamber setups. External cpt-cAMP stimulated human but not rat and murine αßγ ENaC in a dose- and external Na(+) concentration-dependent fashion. Intriguingly, cpt-cAMP activated human δßγ more potently than αßγ channels, suggesting that structural diversity in ectoloop between human α, δ, and those ENaC of other species determines the stimulating effects of cpt-cAMP. Cpt-cAMP increased the ratio of stationary and maximal currents. Mutants having abolished self-inhibition (ß(ΔV348) and γ(H233R)) almost completely eliminated cpt-cAMP mediated activation of ENaC. On the other hand, mutants both enhancing self-inhibition and elevating cpt-cAMP sensitivity increased the stimulating effects of the compound. This compound, however, could not activate already fully opened channels, e.g., degenerin mutation (αß(S520C)γ) and the proteolytically cleaved ENaC by plasmin. Cpt-cAMP activated native ENaC to the same extent as that for heterologous ENaC in human lung epithelial cells. Our data demonstrate that cpt-cAMP, a broadly used PKA activator, stimulates human αßγ and δßγ ENaC channels by relieving self-inhibition.

K+ channel openers restore verapamil-inhibited lung fluid resolution and transepithelial ion transport.

BACKGROUND: Lung epithelial Na+ channels (ENaC) are regulated by cell Ca2+ signal, which may contribute to calcium antagonist-induced noncardiogenic lung edema. Although K+ channel modulators regulate ENaC activity in normal lungs, the therapeutical relevance and the underlying mechanisms have not been completely explored. We hypothesized that K+ channel openers may restore calcium channel blocker-inhibited alveolar fluid clearance (AFC) by up-regulating both apical and basolateral ion transport. METHODS: Verapamil-induced depression of heterologously expressed human alphabetagamma ENaC in Xenopus oocytes, apical and basolateral ion transport in monolayers of human lung epithelial cells (H441), and in vivo alveolar fluid clearance were measured, respectively, using the two-electrode voltage clamp, Ussing chamber, and BSA protein assays. Ca2+ signal in H441 cells was analyzed using Fluo 4AM. RESULTS: The rate of in vivo AFC was reduced significantly (40.6+/-6.3% of control, P<0.05, n=12) in mice intratracheally administrated verapamil. KCa3.1 (1-EBIO) and KATP (minoxidil) channel openers significantly recovered AFC. In addition to short-circuit current (Isc) in intact H441 monolayers, both apical and basolateral Isc levels were reduced by verapamil in permeabilized monolayers. Moreover, verapamil significantly altered Ca2+ signal evoked by ionomycin in H441 cells. Depletion of cytosolic Ca2+ in alphabetagamma ENaC-expressing oocytes completely abolished verapamil-induced inhibition. Intriguingly, KV (pyrithione-Na), K Ca3.1 (1-EBIO), and KATP (minoxidil) channel openers almost completely restored the verapamil-induced decrease in Isc levels by diversely up-regulating apical and basolateral Na+ and K+ transport pathways. CONCLUSIONS: Our observations demonstrate that K+ channel openers are capable of rescuing reduced vectorial Na+ transport across lung epithelial cells with impaired Ca2+ signal.

Calmodulin- and Ca2+-dependent facilitation and inactivation of the Cav1.2 Ca2+ channels in guinea-pig ventricular myocytes.

The L-type Ca(2+) channel (Ca(V)1.2) shows clear Ca(2+)-dependent facilitation and inactivation. Here we have examined the effects of calmodulin (CaM) and Ca(2+) on Ca(2+) channel in guinea-pig ventricular myocytes in the inside-out patch mode, where rundown of the channels was controlled. At a free [Ca(2+)] of 0.1 microM, CaM (0.15, 0.7, 1.4, 2.1, 3.5, and 7.0 microM) + ATP (2.4 mM) induced channel activities of 27%, 98%, 142%, 222%, 65%, and 20% relative to the control activity, respectively, showing a bell-shaped relationship. Similar results were observed at a free [Ca(2+)] <0.01 microM or with a Ca(2+)-insensitive mutant, CaM(1234), suggesting that apoCaM may induce facilitation and inactivation of the channel activity. The bell-shaped curve of CaM was shifted to the lower concentration side with increasing [Ca(2+)]. A simple model for CaM- and Ca(2+)-dependent modulations of the channel activity, which involves two CaM-binding sites, was proposed. We suggest that both apoCaM and Ca(2+)/CaM can induce facilitation and inactivation of Ca(V)1.2 Ca(2+) channels and that the basic role of Ca(2+) is to accelerate CaM-dependent facilitation and inactivation.