Gating of the CFTR Cl− channel by ATP-driven nucleotide-binding domain dimerisation

The cystic fibrosis transmembrane conductance regulator (CFTR) plays a fundamental role in fluid and electrolyte transport across epithelial tissues. Based on its structure, function and regulation, CFTR is an ATP-binding cassette (ABC) transporter. These transporters are assembled from two membrane-spanning domains (MSDs) and two nucleotide-binding domains (NBDs). In the vast majority of ABC transporters, the NBDs form a common engine that utilises the energy of ATP hydrolysis to pump a wide spectrum of substrates through diverse transmembrane pathways formed by the MSDs. By contrast, in CFTR the MSDs form a pathway for passive anion flow that is gated by cycles of ATP binding and hydrolysis by the NBDs. Here, we consider how the interaction of ATP with two ATP-binding sites, formed by the NBDs, powers conformational changes in CFTR structure to gate the channel pore. We explore how conserved sequences from both NBDs form ATP-binding sites at the interface of an NBD dimer and highlight the distinct roles that each binding site plays during the gating cycle. Knowledge of how ATP gates the CFTR Cl⁻ channel is critical for understanding CFTR's physiological role, its malfunction in disease and the mechanism of action of small molecules that modulate CFTR channel gating.     

Further evidence for the existence of a volume-activated taurine efflux pathway in rat mammary tissue independent from volume-sensitive Cl- channels

The effect of cell-swelling, induced by a hyposmotic shock, on the fractional loss of taurine, D-aspartate and iodide from lactating rat mammary tissue explants has been examined. In paired experiments, cell-swelling markedly increased the fractional efflux of taurine but not that of D-aspartate. Similarly, in paired experiments, a hyposmotic challenge stimulated taurine release but not iodide efflux. The results suggest that volume-activated taurine efflux from mammary tissue explants is via a pathway independent from volume-sensitive anion channels. It is apparent that the volume-activated taurine efflux pathway in mammary tissue is not the volume-sensitive organic osmolyte-anion channel which has been described in other cells. Therefore, the results of this study together with others in the literature constitute prima facie evidence for the existence of more than one type of swelling-activated pathway which accepts taurine as a substrate.     

Single-channel properties of volume-sensitive Cl- channel in ClC-3-deficient cardiomyocytes

It is controversial whether the ClC-3 protein, which is one of the voltage-dependent chloride channel ClC family members, is a candidate for the volume-sensitive outwardly rectifying (VSOR) Cl(-) channel per se or its regulator. Here, for the first time, we examined the single-channel properties of the VSOR Cl(-) channel in ventricular myocytes isolated from ClC-3-deficient mice. The single-channel current induced by cell swelling exhibited Cl(-) selectivity, mild outward rectification, and an intermediate unitary conductance (around 38 pS). A Cl(-) channel blocker, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), reversibly inhibited the outward current. These single-channel properties were identical with those in ClC-3 expressing wild-type ventricular myocytes. These results indicate that the single-channel activity of the VSOR Cl(-) channel is independent of the expression of ClC-3 proteins in mouse ventricular myocytes.     

Cell volume-sensitive sodium channels upregulated by glucocorticoids in U937 macrophages

Glucocorticoids exert their anti-inflammatory action in part by influencing macrophages. As regulation of macrophage function involves ion channels, the present study was performed to elucidate the influence of glucocorticoids on macrophage ion channel activity. To this end, the effects of corticosteroids on the sodium conductance in human monocytic cells (U937) was studied using whole-cell and outside-out patch-clamp techniques. Increasing extracellular osmolarity from 310 to 420 mosmol/kg led to cell shrinkage followed by marked activation of inward whole-cell current from -36+/-2 to -72+/-9 pA (n=13; recorded at -150 mV voltage with CsCl intracellular solution, NaCl extracellular solution) while outward current remained unchanged. The increase of inward current was accompanied by a positive shift of reversal potential and was sensitive to amiloride (100 microM). The activation of inward current by shrinkage was not observed when external sodium was replaced by potassium, indicating that the shrinkage-stimulated conductance is sodium selective. Outside-out single-channel measurements revealed a unitary conductance of 6+/-1 pS (n=5) for the sodium-selective amiloride-sensitive current. Pretreating the cells with deoxycorticosterone (100 nM/6 h) markedly upregulated the shrinkage-activated Na+ current. In conclusion, human macrophage-like U937 cells express a sodium-selective shrinkage-activated channel which is upregulated by corticosteroids. Activation of the channel may increase cell volume, an effect of glucocorticoids in other cells.     

CFTR氯通道在硫化氢诱导的心肌保护及细胞增殖中的作用

目的： 探讨囊性纤维化跨膜传导调节因子（CFTR）氯通道在硫化氢（H2S）诱导的心肌保护及细胞增殖中的作用。方法：应用氯化钴（CoCl2）在大鼠H9c2心肌细胞建立化学性缺氧损伤心肌细胞实验模型;CCK-8试剂盒检测心肌细胞存活率;Hoechst 33342核染色法检测心肌细胞凋亡。结果：在400-2 000 μmol/L浓度范围内,CoCl2呈剂量依赖性地抑制H9c2心肌细胞的存活率,600 μmol/L CoCl2 能诱导H9c2心肌细胞产生明显的凋亡;在100-800 μmol/L浓度范围内,硫氢化钠（NaHS）呈剂量依赖性地促进H9c2心肌细胞增殖;NaHS能保护H9c2心肌细胞对抗CoCl2引起的细胞损伤作用,使细胞存活率升高,凋亡率降低;100 μmol/L CFTR氯通道拮抗剂5-硝基-2-（3-苯丙胺）-苯甲酸（NPPB)能明显地阻断NaHS对CoCl2的细胞毒性的抑制作用,但不能阻断NaHS抗心肌细胞凋亡作用及促进心肌细胞增殖作用。结论：CFTR氯通道可能参与H2S的抗CoCl2引起的心肌细胞毒性作用。     

Modification of the structure of peptidoglycan is a strategy to avoid detection by nucleotide-binding oligomerization domain protein 1

Nucleotide-binding oligomerization domain (NOD) protein 1 (NOD1) and NOD2 are pathogen recognition receptors that sense breakdown products of peptidoglycan (PGN) (muropeptides). It is shown that a number of these muropeptides can induce tumor necrosis factor alpha (TNF-alpha) gene expression without significant TNF-alpha translation. This translation block is lifted when the muropeptides are coincubated with lipopolysaccharide (LPS), thereby accounting for an apparently synergistic effect of the muropeptides with LPS on TNF-alpha protein production. The compounds that induced synergistic effects were also able to activate NF-kappaB in a NOD1- or NOD2-dependent manner, implicating these proteins in synergistic TNF-alpha secretion. It was found that a diaminopimelic acid (DAP)-containing muramyl tetrapeptide could activate NF-kappaB in a NOD1-dependent manner, demonstrating that an exposed DAP is not essential for NOD1 sensing. The activity was lost when the alpha-carboxylic acid of iso-glutamic acid was modified as an amide. However, agonists of NOD2, such as muramyl dipeptide and lysine-containing muramyl tripeptides, were not affected by amidation of the alpha-carboxylic acid of iso-glutamic acid. Many pathogens modify the alpha-carboxylic acid of iso-glutamic acid of PGN, and thus it appears this is a strategy to avoid recognition by the host innate immune system. This type of immune evasion is in particular relevant for NOD1.     

Glucose-induced excitation of hypothalamic neurones is mediated by ATP-sensitive K+ channels

Intracellular recordings were made from neurones located in the ventromedial hypothalamic nucleus (VMHN) of slices from rat hypothalamus. These neurones were hyperpolarized on removal of extracellular glucose, resulting in an inhibition of firing, actions which were reversed on the re-introduction of glucose. No reversal of the inhibition of firing was observed when 10 mM mannoheptulose an inhibitor of glucose metabolism, was present in addition to glucose. Increasing the mannoheptulose concentration to 20 mM resulted in further hyperpolarization. Cell-attached recordings from isolated neurones revealed that an increase in extracellular glucose inhibited a K⁺ channel and increased action current activity. ATP induced closure of this K⁺ channel when applied to inside-out membrane patches. Closure was also induced by Mg-free ATP or the nonhydrolysable ATP-analogue, adenylylimidodiphosphate indicating no requirement for ATP metabolism. We suggest that the closure of ATP-sensitive potassium channels underlies increased hypothalamic firing following an increase in extracellular glucose.     

Cross talk between the GABA(A) receptor and the Na-K-Cl cotransporter is mediated by intracellular Cl-

It has been suggested that the GABA(A) receptor-mediated depolarization in immature neurons depends on a high intracellular Cl(-) concentration maintained by Na-K-Cl cotransporter isoform1 (NKCC1). We previously found that activation of the GABA(A) receptor led to stimulation of NKCC1. This stimulation could be a result of GABA(A) receptor-mediated Cl(-) efflux. However, a loss of intracellular Cl(-) is associated with cell shrinkage, membrane depolarization, as well as a rise of intracellular Ca(2+) concentration ([Ca(2+)](i)). To determine which cellular mechanism is underlying NKCC1 stimulation, we investigated changes of intracellular Cl(-) content, [Ca(2+)](i), cell volume, and NKCC1 activity following GABA(A) receptor activation. The basal levels of intracellular (36)Cl were 0.70 +/- 0.04 micromol/mg protein. The intracellular (36)Cl content decreased to 0.53 +/- 0.03 micromol/mg protein in response to 30 microM muscimol (P < 0.05). The loss of intracellular (36)Cl was blocked by 10 microM bicuculline. Muscimol triggered a rise in [Ca(2+)](i), but did not cause cell shrinkage. In contrast, 10-50 mM [Cl(-)](o) or hypertonic HEPES-MEM resulted in reversible cell shrinkage (P < 0.05). Moreover, the GABA-mediated stimulation of NKCC1 activity was not abolished either by removal of extracellular Ca(2+) or BAPTA-AM. An increase in phosphorylation of NKCC1 was detected under both 10 mM [Cl(-)](o) and muscimol conditions. These results suggest that a GABA-mediated loss of intracellular Cl(-), but not a subsequent rise in [Ca(2+)](i) or shrinkage, leads to stimulation of NKCC1. This stimulation may be an important positive feedback mechanism to maintain intracellular Cl(-) level and GABA function in immature neurons.     

Endocytic adaptor complexes bind the C-terminal domain of CFTR

The cystic fibrosis transmembrane conductance regulator (CFTR) functions at the apical membrane of epithelial cells to regulate chloride permeability. Recent studies have shown that CFTR is rapidly and efficiently internalized from the plasma membrane. We have shown that such internalization is mediated solely by clathrin-coated pathways, and that other pathways, such as caveolae, exclude CFTR. Moreover, CFTR co-precipitates with alpha-adaptin, a component of the endocytic adaptor complex (AP-2). The goal of our current studies was to elucidate further the molecular mechanisms that facilitate entry of CFTR into endocytic clathrin-coated vesicles. Protein-protein interactions generated by incubation of full-length in-vitro-translated CFTR with partially purified bovine brain adaptor complexes were evaluated following immunoprecipitation using an antibody against the alpha-adaptin subunit of the AP-2 complex. Such studies revealed co-immunoprecipitation of alpha-adaptin with full-length but not partially translated CFTR, suggesting that the C-terminus of CFTR may be responsible for this interaction. To test this hypothesis a C-terminal GST fusion protein (amino acids 1404-1480; CF-GST) was used in a "pull-down" assay with purified adaptor complexes. CF-GST sepharose was able to pull-down AP-2 endocytic adaptor complexes, as determined by immunoblot analyses of the precipitates using antibodies directed against alpha-adaptin. In contrast, CF-GST sepharose was unable to pull-down gamma-adaptin, a component of the Golgi-derived AP-1 clathrin adaptor complex. Thus, we demonstrate that CFTR is endocytosed via clathrin-coated vesicles, and that targeting of CFTR to these structures is mediated by binding of the AP-2 adaptor complex to the C-terminal domain of CFTR.     

NLR, the nucleotide-binding domain leucine-rich repeat containing gene family

The NLR (nucleotide-binding domain leucine-rich repeat containing) family is found in plants and animals, and serves as crucial regulators of inflammatory and innate immune response, though its functions are likely to extend greatly beyond innate immunity, and even beyond the immune system. This review discusses recent findings regarding the function of NLR proteins in the control of IL-1, NF-kappaB, and host response to pathogens including distinct forms of cell death. The review also covers recent advances regarding the biochemical nature of NLRs, its regulation by intracellular nucleotides and extracellular ATP, by the chaperone protein HSP90, and the ubiquitin ligase-associated protein SGT1. Its role in inflammation is linked to the formation of biochemical complexes such as the inflammasome, and its roles in cell death might be linked to the proposed formation of pyroptosome and necrosome.     

Forskolin interaction with voltage-dependent K channels in Helix is not mediated by cyclic nucleotides

The effects of the adenylate cyclase activator forskolin upon voltage-activated K(V) currents were investigated in Helix nerve cells. Forskolin induced a fast concentration-dependent inactivation of K(V) currents without modifying the activation kinetics. The induced fast inactivation did not result from a speeding up of the voltage-controlled slow inactivation; it could not be mimicked by intracellular injection of cAMP and it was not potentiated by inhibition of the phosphodiesterase. The forskolin induced K current relaxation can be simulated by a model assuming that the drug binds reversibly to open K channels with a dissociation constant of 16.4 microM.     

Susceptibility to multiple sclerosis mediated by HLA-DRB1 is influenced by a second gene telomeric of the TNF cluster

Susceptibility to multiple sclerosis (MS) is clearly associated with human leukocyte antigen (HLA)-DRB1*1501, but some studies show associations with HLA-B7 and -B18. These are often co-expressed with DRB1*1501 in the ancestral haplotypes (AH) denoted 7.1 (HLA-A3, B7, tumor necrosis factor [TNF]a11b4, DRB1*1501) and 18.1 (HLA-A25, B18, TNFa10b4, DRB1*1501). Here we present a systematic study of 218 patients and 274 controls typed at all standard class II and TNF microsatellite loci, and a novel non-synonymous polymorphism in the central major histocompatibility complex gene, inhibitor of κ B-like protein (IKBL). The C allele at IKBL+738 is only found on the 7.1 haplotype. HLA-DRB1*1501 was associated with disease, as expected. When subjects expressing DRB1*1501 were analyzed separately, TNFa11b4 and IKBL+738C were less common in the patients and, hence, mark an allele that mediates resistance which lies telomeric of IKBL.

TNFa10b4 and TNFa1b5 were more common in DRB1*1501 patients than in controls. These alleles have been associated with the 18.1 and 18.2 AH, respectively. Since no component of these haplotypes was an independent risk factor in this study, it appears likely that a gene linked to TNFa10b4 and TNFa1b5 modifies the effect of the susceptibility locus marked by HLA-DRB1*1501. Potential candidate genes telomeric of the TNF cluster are discussed.     

The role of volume-sensitive CI channels in the stimulation of chloride absorption by short-chain fatty acids in the rat colon

The short-chain fatty acids acetate, propionate and butyrate induced a concentration-dependent decrease in short-circuit current (I_sc) of the rat colon in vitro. The decrease in I_sc, being more pronounced in the distal than in the proximal colon, was dependent on the presence of CI^- ions and partly on the presence of HCO^-₃. In the distal colon, the fall in I_sc could be inhibited by amiloride, indicating that the activity of the Na⁺/H⁺ exchanger is necessary for the induction of this response. The decrease in I_sc was diminished by the CI^- channel blocker, 5-nitro-2-(3-phenylpropylamino)-benzoate, and the lipoxygenase inhibitor, nordihydroguaiaretic acid. In contrast, inhibitors of the leukotriene pathway or a CI^- channel blocker did not affect the I_sc response in the proximal colon. Measurements of unidirectional fluxes revealed that butyrate caused a stimulation of the mucosa to serosa fluxes (J_ms) of Na⁺ and CI^- in the distal, but only of j_ms^Na in the proximal colon. Unidirectional Rb⁺ fluxes were not altered. The stimulation of j_ms^cl correlated with the degree of metabolism of the short-chain fatty acid. The increase in j_ms^cl was most pronounced for butyrate, smaller for acetate and not observed with the poorly metabolizable short-chain fatty acid, isobutyrate. Consequently, two factors seem to be responsible for the stimulation of CI^- absorption by short-chain fatty acids in the distal colon: (1) the intracellular production of HCO^-₃ during the oxidation of short-chain fatty acids as substrate for the apical Cr/HCO^-₃ exchanger, and (2) the activation of volume-sensitive basolateral CI^- channels.     

Disrupted recycling of the low density lipoprotein receptor by PCSK9 is not mediated by residues of the cytoplasmic domain

Proprotein convertase subtilisin/kexin type 9 (PCSK9) post-translationally regulates the number of cell-surface low density lipoprotein receptors (LDLR). This is accomplished by the ability of PCSK9 to mediate degradation of the LDLR. The underlying mechanism involves binding of secreted PCSK9 to the epidermal growth factor-like repeat A of the extracellular domain of the LDLR at the cell surface, followed by lysosomal degradation of the internalized LDLR:PCSK9 complex. However, the mechanism by which the normal recycling of the LDLR is disrupted by PCSK9, remains to be determined. In this study we have investigated the role of the cytoplasmic domain of the LDLR for this process. This has been done by studying the ability of a mutant LDLR (K811X-LDLR) which lacks the cytoplasmic domain, to be degraded by PCSK9. We show that this mutant receptor is degraded by PCSK9. Thus, the machinery which directs the LDLR:PCSK9 complex to the lysosomes for degradation, does not interact with the cytoplasmic domain of the LDLR.     

HLA-G mediated immune regulation is impaired by a single amino acid exchange in the alpha 2 domain

The trade-off from HLA class I expression to HLA-G expression support the immune evasion of malignant cells. The essential role of the virtually invariant HLA-G in immune tolerance, tumor immunology and its expression frequency in immune privileged tissues is known; however the specific importance of allelic subtypes in immune responses is still not well understood. HLA-G¹01:01, ¹01:03 and ¹01:04 are the most prevalent allelic variants differing at residues 31 and 110, respectively. In cytotoxicity assays applying K562 cells transduced with the HLA-G variants as targets and NK cells as effectors the differential protective potential of HLA-G variants was analyzed. Their peptide profiles were determined utilizing soluble HLA technology. An increased protective potential of HLA-G¹01:04 could be observed. All variants exhibit a unique peptide repertoire with marginal overlap, while G¹01:04 differs in its peptide anchor profile substantially. The functional differences between HLA-G subtypes could be explained by the constraint of the bound peptides, modifying the pHLA-G accessible surface. For the first time a contribution of amino acid alterations within the HLA-G heavy chain for peptide selection and NK cell recognition could be observed. These results will be a step towards understanding immune tolerance and will guide towards personalized immune therapeutic strategies.     

RNA干扰介导的端锚聚合酶1表达下调诱导人神经母细胞瘤细胞的凋亡

目的探讨RNA干扰(RNAi)介导的端锚聚合酶1(TNKS1)的表达下调对人神经母细胞瘤(NB)细胞SH-SY5Y体外增殖能力和凋亡的影响及机制,为开发治疗NB的新靶点提供一定的实验基础。方法根据TNKS1基因序列,设计合成3个TNKS1基因的特异性短发夹环(shRNA)干扰片段,利用慢病毒作为载体,构建目的基因的RNAi序列。慢病毒感染SH-SY5Y细胞,实时定量PCR(Real-time PCR)法检测TNKS1基因的表达,筛选出最佳干扰序列进行后续实验。然后进行克隆形成实验,检测RNAi-TNKS1后细胞增殖能力的改变。并进一步用Western blotting法检测抗凋亡蛋白Bcl-2、促凋亡蛋白Caspase-3、Wnt/β-连环蛋白(β-catenin)通路的关键蛋白β-catenin及其下游靶蛋白Cyclin D1和c-Myc的表达,最后用透射电镜观察凋亡形态的改变,探讨RNAi-TNKS1对细胞凋亡的影响及其作用机制。结果慢病毒载体构建成功,病毒滴度为5×1011TU/L。Real-time PCR检测结果显示,#3 shRNA为最佳的有效靶点。克隆形成实验的结果显示,RNAi-TNKS1后SH-SY5Y细胞的克隆形成率较对照组明显下降。Western blotting结果显示,RNAi-TNKS1可显著抑制Bcl-2蛋白的表达,促进Caspase-3蛋白的表达。此外,Wnt/β-catenin通路的关键蛋白β-catenin及其下游靶蛋白Cyclin D1和c-Myc的表达也降低。透射电镜结果显示,RNAi-TNKS1后细胞呈现明显的凋亡结构和形态。结论 RNAi介导的TNKS1的表达下调可降低SH-SY5Y细胞的体外增殖能力,诱导其凋亡,可能部分是通过抑制Wnt/β-catenin通路发挥作用。