Molecular dynamics simulation of TMEM16A channel: Linking structure with gating

TMEM16A, the calcium-activated chloride channel, is broadly expressed and plays pivotal roles in diverse physiological processes. To understand the structural and functional relationships of TMEM16A, it is necessary to fully clarify the structural basis of the gating of the TMEM16A channel. Herein, we performed the protein electrostatic analysis and molecular dynamics simulation on the TMEM16A in the presence and absence of Ca²⁺. Data showed that the separation of TM4 and TM6 causes pore expansion, and Q646 may be a key residue for the formation of π-helix in the middle segment of TM6. Moreover, E705 was found to form a group of H-bond interactions with D554/K588/K645 below the hydrophobic gate to stabilize the closed conformation of the pore in the Ca²⁺-free state. Interestingly, in the Ca²⁺ bound state, the E705 side chain swings 100^o to serve as Ca²⁺-binding coordination and released K645. K645 is closer to the hydrophobic gate in the calcium-bound state, which facilitates the provision of electrostatic forces for chloride ions as the ions pass through the hydrophobic gate. Our findings provide the structural-based insights to understanding the mechanisms of gating of TMEM16A.     

Regulation of the Ca2+-activated chloride channel Anoctamin-1 (TMEM16A) by Ca2+-induced interaction with FKBP12 and calcineurin

Chloride fluxes through the calcium-gated chloride channel Anoctamin-1 (TMEM16A) control blood pressure, secretion of saliva, mucin, insulin, and melatonin, gastrointestinal motility, sperm capacitation and motility, and pain sensation. Calcium activates a myriad of regulatory proteins but how these proteins affect TMEM16A activity is unresolved. Here we show by co-immunoprecipitation that increasing intracellular calcium with ionomycin or by activating sphingosine-1-phosphate receptors, induces coupling of calcium/calmodulin-dependent phosphatase calcineurin and prolyl isomerase FK506-binding protein 12 (FKBP12) to TMEM16A in HEK-293 cells. Application of drugs that target either calcineurin (cyclosporine A) or FKBP12 (tacrolimus known as FK506 and sirolimus known as rapamycin) caused a decrease in TMEM16A activity. In addition, FK506 and BAPTA-AM prevented co-immunoprecipitation between FKBP12 and TMEM16A. FK506 rendered the channel insensitive to cyclosporine A without altering its apparent calcium sensitivity whereas zero intracellular calcium blocked the effect of FK506. Rapamycin decreased TMEM16A activity in cells pre-treated with cyclosporine A or FK506. These results suggest the formation of a TMEM16A-FKBP12-calcineurin complex that regulates channel function. We conclude that upon a cytosolic calcium increase the TMEM16A-FKPB12-calcineurin trimers are assembled. Such hetero-oligomerization enhances TMEM16A channel activity but is not mandatory for activation by calcium.     

Phosphate position is key in mediating transmembrane ion channel TMEM16A–phosphatidylinositol 4,5-bisphosphate interaction

TransMEMbrane 16A (TMEM16A) is a Ca²⁺-activated Cl⁻ channel that plays critical roles in regulating diverse physiologic processes, including vascular tone, sensory signal transduction, and mucosal secretion. In addition to Ca²⁺, TMEM16A activation requires the membrane lipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂). However, the structural determinants mediating this interaction are not clear. Here, we interrogated the parts of the PI(4,5)P₂ head group that mediate its interaction with TMEM16A by using patch- and two-electrode voltage-clamp recordings on oocytes from the African clawed frog Xenopus laevis, which endogenously express TMEM16A channels. During continuous application of Ca²⁺ to excised inside–out patches, we found that TMEM16A-conducted currents decayed shortly after patch excision. Following this rundown, we show that the application of a synthetic PI(4,5)P₂ analog produced current recovery. Furthermore, inducible dephosphorylation of PI(4,5)P₂ reduces TMEM16A-conducted currents. Application of PIP₂ analogs with different phosphate orientations yielded distinct amounts of current recovery, and only lipids that include a phosphate at the 4′ position effectively recovered TMEM16A currents. Taken together, these findings improve our understanding of how PI(4,5)P₂ binds to and potentiates TMEM16A channels.     

Prevention of ginsenoside-induced desensitization of Ca2+-activated Cl- current by microinjection of inositol hexakisphosphate in Xenopus laevis oocytes: involvement of GRK2 and beta-arrestin I

We demonstrated that ginsenosides, the active ingredient of Panax ginseng, enhance endogenous Ca(2+)-activated Cl(-) currents via Galpha(q/11)-phospholipase C-beta3 pathway in Xenopus laevis oocytes. Moreover, prolonged treatment of ginsenosides induced Cl(-) channel desensitization. However, the molecular mechanisms involved in ginsenoside-induced Cl(-) channel desensitization have not yet been determined precisely. To provide answers to these questions, we investigated the changes in ginsenoside-induced Cl(-) channel desensitization after intraoocyte injection of inositol hexakisphosphate (InsP(6)), which is known to bind beta-arrestins and interfere with beta-arrestin-induced receptor down-regulation, and cRNAs coding beta-arrestin I/II and G-protein receptor kinase 2 (GRK2), which is known to phosphorylate G protein-coupled receptors and attenuate agonist stimulations. When control oocytes were stimulated with ginsenosides, the second, third, and fourth responses to ginsenosides were 69.6 +/- 4.1, 9.2 +/- 2.3, and 2.6 +/- 2.2% of the first responses, respectively. Preintraoocyte injection of InsP(6) before ginsenoside treatment restored ginsenoside effect to initial response levels in a concentration-, time-, and structurally specific manner, in that inositol hexasulfate had no effect. The EC(50) was 13.9 +/- 8.7 microM. Injection of cRNA coding beta-arrestin I but not beta-arrestin II blocked InsP(6) effect on prevention of ginsenoside-induced Cl(-) channel desensitization. Injection of cRNA coding GRK2 abolished ginsenoside effect enhancing Cl(-) current. However, the GRK2-caused loss of ginsenoside effect on Cl(-) current was prevented by coinjection of GRK2 with GRK2-K220R, a dominant-negative mutant of GRK. These results indicate that ginsenoside-induced Cl(-) channel desensitization is mediated via activation of GRK2 and beta-arrestin I.     

G alpha(q/11) coupled to mammalian phospholipase C beta 3-like enzyme mediates the ginsenoside effect on Ca(2+)-activated Cl(-) current in the Xenopus oocyte.

Recently we demonstrated that ginsenosides, the active ingredients of Panax ginseng, enhanced Ca(2+)-activated Cl(-) current in the Xenopus oocyte through a signal transduction mechanism involving the activation of pertussis toxin-insensitive G protein and phospholipase C (PLC). However, it has not yet been determined precisely which G protein subunit(s) and which PLC isoform(s) participate in the ginsenoside signaling. To provide answers to these questions, we investigated the changes in ginsenoside effect on the Cl(-) current after intraoocyte injections of the cRNAs coding various G protein subunits, a regulator of G protein signaling (RGS2), and G beta gamma-binding proteins. In addition, we examined which of mammalian PLC beta 1-3 antibodies injected into the oocyte inhibited the action of ginsenosides on the Cl(-) current. Injection of G alpha(q) or G alpha(11) cRNA increased the basal Cl(-) current recorded 48 h after, and it further prevented ginsenosides from enhancing the Cl(-) current, whereas G alpha(i2) and G alpha(oA) cRNA injection had no significant effect. The changes following G alpha(q) cRNA injection were prevented when G beta(1)gamma(2) and G alpha(q) subunits were co-expressed by simultaneous injection of the cRNAs coding these subunits. Injection of cRNA coding G alpha(q)Q209L, a constitutively active mutant that does not bind to G beta gamma, produced effects similar to those of G alpha(q) cRNA injection. The effects of G alpha(q)Q209L cRNA injection, however, were not prevented by co-injection of G beta(1)gamma(2) cRNA. Injection of the cRNA coding RGS2, which interacts most selectively with G alpha(q/11) among various identified RGS isoforms and stimulates the hydrolysis of GTP to GDP in active GTP-bound G alpha subunit, resulted in a severe attenuation of ginsenoside effect on the Cl(-) current. Finally, antibodies against PLC beta 3, but not -beta 1 and -beta 2, markedly attenuated the ginsenoside effect examined at 3-h postinjection. These results suggest that G alpha(q/11) coupled to mammalian PLC beta 3-like enzyme mediates ginsenoside effect on Ca(2+)-activated Cl(-) current in the Xenopus oocyte.     

Direct or Indirect Regulation of Calcium-Activated Chloride Channel by Calcium

Calcium-activated chloride channels (CaCCs) play fundamental roles in numerous physiological processes. Despite their physiological importance, the molecular identity of CaCCs has not been fully investigated until now. Recently, transmembrane 16A (TMEM16A) was demonstrated by three independent research groups to be a strong candidate for the CaCC molecular basis. To further investigate the electrophysiological characteristics, we constructed TMEM16A (abcd) stably transfected HEK293 cell lines and carried out whole-cell and excised inside–out patch-clamp experiments. The TMEM16A channel was Ca²⁺-dependent in both patch configurations. The TMEM16A current could be strongly inhibited by niflumic acid, and when Cl⁻ was substituted by gluconate ions, the current was reduced considerably. In inside–out configuration, TMEM16A channel was time-independent but voltage-dependent, in which the half-maximum activating free Ca²⁺ concentration was 63 nM at 80 mV. While in whole-cell configuration, the current was both time- and voltage-dependent. About the rectification feature, the TMEM16A current also showed distinct characteristics in the two patch configurations. In whole cells, the TMEM16A channel expressed outward rectification at low Ca²⁺ concentration but when the Ca²⁺ concentration was high it became linear. On the contrary, in inside–out configuration, it always expressed outward rectification. Comparing the different characteristics in the two configurations, some underlying mechanisms remain to be identified, which is discussed with respect to direct or indirect activation. There was irreversible rundown in this channel.     

CFTR and TMEM16A are separate but functionally related Cl- channels

Previous reports point out to a functional relationship of the cystic fibrosis transmembrane conductance regulator (CFTR) and Ca(2+) activated Cl(-) channels (CaCC). Recent findings showing that TMEM16A forms the essential part of CaCC, prompted us to examine whether CFTR controls TMEM16A. Inhibition of endogenous CaCC by activation of endogenous CFTR was found in 16HBE human airway epithelial cells, which also express TMEM16A. In contrast, CFBE airway epithelial cells lack of CFTR expression, but express TMEM16A along with other TMEM16-proteins. These cells produce CaCC that is inhibited by overexpression and activation of CFTR. In HEK293 cells coexpressing TMEM16A and CFTR, whole cell currents activated by IMBX and forskolin were significantly reduced when compared with cells expressing CFTR only, while the halide permeability sequence of CFTR was not changed. Expression of TMEM16A, but not of TMEM16F, H or J, produced robust CaCC, which that were inhibited by CaCCinh-A01 and niflumic acid, but not by CFTRinh-172. TMEM16A-currents were attenuated by additional expression of CFTR, and were completely abrogated when additionally expressed CFTR was activated by IBMX and forskolin. On the other hand, CFTR-currents were attenuated by additional expression of TMEM16A. CFTR and TMEM16A were both membrane localized and could be coimmunoprecipitated. Intracellular Ca(2+) signals elicited by receptor-stimulation was not changed during activation of CFTR, while ionophore-induced rise in [Ca(2+)](i) was attenuated after stimulation of CFTR. The data indicate that both CFTR and TMEM16 proteins are separate molecular entities that show functional and molecular interaction.     

In vitro metabolism of ginsenosides by the ginseng root pathogen Pythium irregulare

The role of ginseng saponins (ginsenosides) as modulators or inhibitors of disease is vague, but our earlier work supports the existence of an allelopathic relationship between ginsenosides and soilborne microbes. Interestingly, this allelopathy appears to significantly promote the growth of the important ginseng pathogen, Pythium irregulare while inhibiting that of an antagonistic non-pathogenic fungus, Trichoderma hamatum. Herein we report on the apparent selective metabolism of 20(S)-protopanaxadiol ginsenosides by an extracellular glycosidase from P. irregulare. Thus, when P. irregulare was cultured in the presence of a purified (> 90%) ginsenoside mixture, nearly all of the 20(S)-protopanaxadiol ginsenosides (Rb1, Rb2, Rc, Rd, and to a limited extent G-XVII) were metabolized into the minor ginsenoside F2, at least half of which appears to be internalized by the organism. No metabolism of the 20(S)-protopanaxatriol ginsenosides (Rg1 and Re) was evident. By contrast, none of the ginsenosides added to the culture medium of the non-pathogenic fungus T. hamatum were metabolized. The metabolism of 20(S)-protopanaxadiol ginsenosides by P. irregulare appears to occur through the hydrolysis of terminal monosaccharide units from disaccharides present at C-3 and/or C-20 of ginsenosides Rb1, Rc, Rb2, Rd and G-XVII to yield one major product, ginsenoside F2 and one minor product (possibly G-III). A similar transformation of ginsenosides was observed using a crude protein preparation isolated from the spent medium of P. irregulare cultures.     

Identification of the Novel TMEM16A Inhibitor Dehydroandrographolide and Its Anticancer Activity on SW620 Cells

TMEM16A, a calcium-activated chloride channel (CaCC), is highly amplified and expressed in human cancers and is involved in the growth and metastasis of some malignancies. Inhibition of TMEM16A represents a novel pharmaceutical approach for the treatment of cancers and metastases. The purpose of this study is to identify a new TMEM16A inhibitor, investigate the effects of this inhibitor on the proliferation and metastasis of TMEM16A-amplified SW620 cells, and to elucidate the underlying molecular mechanism in vitro. We identified a novel small-molecule TMEM16A inhibitor dehydroandrographolide (DP). By using patch clamp electrophysiology, we showed that DP inhibited TMEM16A chloride currents in Fisher rat thyroid (FRT) cells that were transfected stably with human TMEM16A and in TMEM16A-overexpressed SW620 cells but did not alter cystic fibrosis transmembrane conductance regulator (CFTR) chloride currents. Further functional studies showed that DP suppressed the proliferation of SW620 cells in a dose- and time-dependent manner using MTT assays. Moreover, DP significantly inhibited migration and invasion of SW620 cells as detected by wound-healing and transwell assays. Further mechanistic study demonstrated that knockdown of human TMEM16A decreased the inhibitory effect of DP on the proliferation of SW620 cells and that TMEM16A-dependent cells (SW620 and HCT116) were more sensitive to DP than TMEM16A-independent cells (SW480 and HCT8). In addition, we found that treatment of SW620 cells with DP led to a decrease in TMEM16A protein levels but had no effect on TMEM16A mRNA levels. The current work reveals that DP, a novel TMEM16A inhibitor, exerts its anticancer activity on SW620 cells partly through a TMEM16A-dependent mechanism, which may introduce a new targeting approach for an antitumour therapy in TMEM16A-amplified cancers.     

Systems Approaches to Unravel Molecular Function: High-content siRNA Screen Identifies TMEM16A Traffic Regulators as Potential Drug Targets for Cystic Fibrosis

An attractive approach to treat people with Cystic Fibrosis (CF), a life-shortening disease caused by mutant CFTR, is to compensate for the absence of this chloride/bicarbonate channel by activating alternative (non-CFTR) chloride channels. One obvious target for such “mutation-agnostic” therapeutic approach is TMEM16A (anoctamin-1/ANO1), a calcium-activated chloride channel (CaCC) which is also expressed in the airways of people with CF, albeit at low levels. To find novel TMEM16A regulators of both traffic and function, with the main goal of identifying candidate CF drug targets, we performed a fluorescence cell-based high-throughput siRNA microscopy screen for TMEM16A trafficking using a double-tagged construct expressed in human airway cells. About 700 genes were screened (2 siRNAs per gene) of which 262 were identified as candidate TMEM16A modulators (179 siRNAs enhanced and 83 decreased TMEM16A traffic), being G-protein coupled receptors (GPCRs) enriched on the primary hit list. Among the 179 TMEM16A traffic enhancer siRNAs subjected to secondary screening 20 were functionally validated. Further hit validation revealed that siRNAs targeting two GPCRs – ADRA2C and CXCR3 – increased TMEM16A-mediated chloride secretion in human airway cells, while their overexpression strongly diminished calcium-activated chloride currents in the same cell model. The knockdown, and likely also the inhibition, of these two TMEM16A modulators is therefore an attractive potential therapeutic strategy to increase chloride secretion in CF.     

A minimal isoform of the TMEM16A protein associated with chloride channel activity

TMEM16A protein, also known as anoctamin-1, has been recently identified as an essential component of Ca(2+)-activated Cl(-) channels. We previously reported the existence of different TMEM16A isoforms generated by alternative splicing. In the present study, we have determined the functional properties of a minimal TMEM16A protein. This isoform, called TMEM16A(0), has a significantly shortened amino-terminus and lacks three alternative segments localized in the intracellular regions of the protein (total length: 840 amino acids). TMEM16A(0) expression is associated with Ca(2+)-activated Cl(-) channel activity as measured by three different functional assays based on the halide-sensitive yellow fluorescent protein, short-circuit current recordings, and patch-clamp technique. However, compared to a longer isoform, TMEM16(abc) (total length: 982 amino acids), TMEM16A(0) completely lacks voltage-dependent activation. Furthermore, TMEM16A(0) and TMEM16A(abc) have similar but not identical responses to extracellular anion replacement, thus suggesting a difference in ion selectivity and conductance. Our results indicate that TMEM16A(0) has the basic domains required for anion transport and Ca(2+)-sensitivity. However, the absence of alternative segments, which are present in more complex isoforms of TMEM16A, modifies the channel gating and ion transport ability.     

一种真菌对人参皂苷Rg3的转化

[目的]筛选长白山人参土壤中的活性微生物,转化人参总皂苷及单体人参皂苷产生稀有抗肿瘤成份.[方法]从长白山人参根际土壤中分离各类菌株,对人参总皂苷及单体人参皂苷进行微生物转化,并通过硅胶柱层析等方法对转化产物进行分离纯化,采用波谱解析及理化常数对其进行结构鉴定;结合菌落形态、产孢结构、孢子形态特征以及菌株ITS rDNA核酸序列分析,对活性菌株进行鉴定.[结果]从长白山人参根际土壤中分离各类真菌菌株68株,有12株菌株对人参总皂苷有转化活性,其中菌株SYP2353对二醇组人参皂苷Rg3具有较强的转化活性.[结论]阳性菌株SYP2353被鉴定为疣孢漆斑菌(Myrothecium verrucaria),能将人参皂苷Rg3转化为稀有人参皂苷Rh2及二醇组人参皂苷苷元PPD,为稀有人参皂苷Rh2的制备提供了新的方法.     

Molecular underpinning of intracellular pH regulation on TMEM16F

TMEM16F, a dual-function phospholipid scramblase and ion channel, is important in blood coagulation, skeleton development, HIV infection, and cell fusion. Despite advances in understanding its structure and activation mechanism, how TMEM16F is regulated by intracellular factors remains largely elusive. Here we report that TMEM16F lipid scrambling and ion channel activities are strongly influenced by intracellular pH (pH_i). We found that low pH_i attenuates, whereas high pH_i potentiates, TMEM16F channel and scramblase activation under physiological concentrations of intracellular Ca²⁺ ([Ca²⁺]_i). We further demonstrate that TMEM16F pH_i sensitivity depends on [Ca²⁺]_i and exhibits a bell-shaped relationship with [Ca²⁺]_i: TMEM16F channel activation becomes increasingly pH_i sensitive from resting [Ca²⁺]_i to micromolar [Ca²⁺]_i, but when [Ca²⁺]_i increases beyond 15 µM, pH_i sensitivity gradually diminishes. The mutation of a Ca²⁺-binding residue that markedly reduces TMEM16F Ca²⁺ sensitivity (E667Q) maintains the bell-shaped relationship between pH_i sensitivity and Ca²⁺ but causes a dramatic shift of the peak [Ca²⁺]_i from 15 µM to 3 mM. Our biophysical characterizations thus pinpoint that the pH_i regulatory effects on TMEM16F stem from the competition between Ca²⁺ and protons for the primary Ca²⁺-binding residues in the pore. Within the physiological [Ca²⁺]_i range, the protonation state of the primary Ca²⁺-binding sites influences Ca²⁺ binding and regulates TMEM16F activation. Our findings thus uncover a regulatory mechanism of TMEM16F by pH_i and shine light on our understanding of the pathophysiological roles of TMEM16F in diseases with dysregulated pH_i, including cancer.     

Divalent Cations Modulate TMEM16A Calcium-Activated Chloride Channels by a Common Mechanism

The gating of Ca²⁺-activated Cl^? channels is controlled by a complex interplay among [Ca²⁺]_i, membrane potential and permeant anions. Besides Ca²⁺, Ba²⁺ also can activate both TMEM16A and TMEM16B. This study reports the effects of several divalent cations as regulators of TMEM16A channels stably expressed in HEK293T cells. Among the divalent cations that activate TMEM16A, Ca²⁺ is most effective, followed by Sr²⁺ and Ni²⁺, which have similar affinity, while Mg²⁺ is ineffective. Zn²⁺ does not activate TMEM16A but inhibits the Ca²⁺-activated chloride currents. Maximally effective concentrations of Sr²⁺ and Ni²⁺ occluded activation of the TMEM16A current by Ca²⁺, which suggests that Ca²⁺, Sr²⁺ and Ni²⁺ all regulate the channel by the same mechanism.     

Mutations that prevent cyclic nucleotide binding to binding sites A or B of type I cyclic AMP-dependent protein kinase

Cyclic nucleotide binding and activation properties of cAMP-dependent protein kinases from five independent mutants of S49 mouse lymphoma cells were studied. These mutants were all hemizygous for expression of mutant regulatory (R) subunits of the type I kinase with lesions that altered the electrostatic charge of R subunit: lesions in three of the mutants mapped to cAMP-binding site A, and those in two of the mutants mapped to cAMP-binding site B. A nucleotide mismatch assay using 32P-labeled cRNA and ribonuclease A confirmed and refined localization of the mutations to single amino acid residues implicated in cAMP binding. R subunits from all mutants retained the ability to bind cAMP, but binding behaved as if it were entirely to nonmutated sites: 1) relative affinities of 11 adenine-modified derivatives of cAMP for mutant enzymes were identical to their relative affinities for the site of wild-type kinase that corresponded to the nonmutated site of the mutant; 2) the potencies of these analogs as activators of mutant kinases were strictly correlated with their binding affinities (for wild-type enzyme activation potencies were correlated with mean affinities of the analogs for cAMP-binding sites A and B); 3) combinations of analogs with strong preferences for opposite cAMP-binding sites in wild-type kinase showed no synergism in activating mutant kinases; 4) dissociation of cAMP from mutant kinases was monophasic; and 5) high salt accelerated dissociation of cAMP from kinases with site B lesions but retarded dissociation from those with site A lesions.     

Effects of ginsenoside Rg2 on the 5-HT3A receptor-mediated ion current in Xenopus oocytes

Treatment with ginsenosides, major active ingredients of Panax ginseng, produces a variety of pharmacological or physiological responses with effects on the central and peripheral nervous systems. Recent reports showed that ginsenoside Rg2 inhibits nicotinic acetylcholine receptor-mediated Na+ influx and channel activity. In the present study, we investigated the effect of ginsenoside Rg2 on human 5-hydroxytryptamine3A (5-HT3A) receptor channel activity, which is also one of the ligand-gated ion channel families. The 5-HT3A receptor was expressed in Xenopus oocytes, and the current was measured using the two-electrode voltage clamp technique. The ginsenoside Rg2 itself had no effect on the oocytes that were injected with H2O as well as on the oocytes that were injected with the 5-HT3A receptor cRNA. In the oocytes that were injected with the 5-HT3A receptor cRNA, the pretreatment of ginsenoside Rg2 inhibited the 5-HT-induced inward peak current (I5-HT) The inhibitory effect of ginsenoside Rg2 on I5-HT was dose dependent and reversible. The half-inhibitory concentrations (IC50) of ginsenoside Rg2 was 22.3 +/- 4.6 microM. The inhibition of I5-HT by ginsenoside Rg2 was non-competitive and voltage-independent. These results indicate that ginsenoside Rg2 might regulate the 5-HT3A receptors that are expressed in Xenopus oocytes. Further, this regulation on the ligand-gated ion channel activity by ginsenosides might be one of the pharmacological actions of Panax ginseng.     

Compartmentalized crosstalk of CFTR and TMEM16A (ANO1) through EPAC1 and ADCY1

Airway epithelial cells express both Ca²⁺ activated TMEM16A/ANO1 and cAMP activated CFTR anion channels. Previous work suggested a significant crosstalk of intracellular Ca²⁺ and cAMP signaling pathways, leading to activation of both chloride channels. We demonstrate that in airway epithelial cells, stimulation of purinergic or muscarinic G-protein coupled receptors (GPCRs) activates TMEM16A and CFTR. Additional expression of G_q/11 and phospholipase C coupled GPCRs strongly enhanced the crosstalk between Ca²⁺- and cAMP-dependent signaling. Knockdown of endogenous GRCRs attenuated crosstalk and functional coupling between TMEM16A and CFTR. The number of receptors did not affect expression or membrane localization of TMEM16A or CFTR, but controlled assembly of the local signalosome. GPCRs translocate Ca²⁺-sensitive adenylate cyclase type 1 (ADCY1) and exchange protein directly activated by cAMP (EPAC1) to particular plasma membrane domains containing GPCRs, CFTR and TMEM16A, thereby producing compartmentalized Ca²⁺ and cAMP signals and significant crosstalk. While biosynthesis and membrane trafficking of CFTR requires a functional Golgi apparatus, maturation and membrane trafficking of TMEM16A may occur independent of the Golgi. Because Ca²⁺ activated TMEM16A currents are only transient, continuous Cl⁻ secretion by airway epithelial cells requires CFTR. The present data also explain why receptor-dependent activation of TMEM16A is more efficient than direct stimulation by Ca²⁺.     

Biotransformation of ginsenosides Re and Rg1 into ginsenosides Rg2 and Rh1 by recombinant β-glucosidase

Ginsenosides Re and Rg1 were transformed by recombinant β-glucosidase (Bgp1) to ginsenosides Rg2 and Rh1, respectively. The bgp1 gene consists of 2,496?bp encoding 831 amino acids which have homology to the glycosyl hydrolase families 3 protein domain. Using 0.1?mg enzyme ml(-1) in 20?mM sodium phosphate buffer at 37°C and pH 7.0, the glucose moiety attached to the C-20 position of ginsenosides Re and Rg1, was removed: 1?mg ginsenoside Re ml(-1) was transformed into 0.83?mg Rg2?ml(-1) (100% molar conversion) after 2.5?h and 1?mg ginsenoside Rg1?ml(-1) was transformed into 0.6?mg ginsenoside Rh1?ml(-1) (78% molar conversion) in 15?min. Using Bgp1 enzyme, almost all initial ginsenosides Re and Rg1 were converted completely to ginsenosides Rg2 and Rh1. This is the first report of the conversion of ginsenoside Re to ginsenoside Rg2 and ginsenoside Rg1 to ginsenoside Rh1 using the recombinant β-glucosidase.