Targeting of ion channels to membrane microdomains: localization of KV channels to lipid rafts

Voltage-gated K(+) channels are an important determinant of cellular excitability and key components of multiple signal transduction pathways. However, relatively little is known about the mechanisms of K(V) channel localization or their membrane partitioning. Lipid rafts are specialized membrane microdomains that are rich in sphingolipids and cholesterol. These rafts have been implicated in the organization of many membrane-associated signaling pathways and are currently the focus of intense interest in the scientific community. Biochemical and functional evidence indicate that K(V) channels, in addition to other ion channels, localize to lipid raft microdomains on the cell surface. Although several important questions regarding specific mechanisms of channel localization remain, emerging data indicate that protein-lipid interactions should be considered as a new mechanism of ion channel localization and compartmentation that might permit the therapeutic modulation of channel properties via alteration in membrane lipids.     

Signaling and ligand binding by recombinant neuromedin U receptors: evidence for dual coupling to Galphaq/11 and Galphai and an irreversible ligand-receptor interaction

The neuropeptide neuromedin U (NmU) shows considerable structural conservation across species. Within the body, it is widely distributed and in mammals has been implicated in physiological roles, including the regulation of feeding, anxiety, pain, blood flow, and smooth muscle contraction. Human NmU-25 (hNmU-25) and other NmU analogs were recently identified as ligands for two human orphan G protein-coupled receptors, subsequently named hNmU-R1 and hNmU-R2. These receptors have approximately 50% amino acid homology, and, at least in mammalian species, NmU-R1 and NmU-R2 are expressed predominantly in the periphery and central nervous system, respectively. Here, we have characterized signaling mediated by hNmU-R1 and hNmU-R2 expressed as recombinant proteins in human embryonic kidney 293 cells, particularly to define their G protein coupling and the activation and regulation of signal transduction pathways. We show that these receptors couple to both Galpha(q/11) and Galpha(i). Activation of either receptor type causes a pertussis toxin-insensitive activation of both phospholipase C and mitogen activated-protein kinase and a pertussis toxin-sensitive inhibition of adenylyl cyclase with subnanomolar potency for each. Activation of phospholipase C is sustained, but despite this capacity for prolonged receptor activation, repetitive application of hNmU-25 does not cause repetitive intracellular Ca2+ signaling by either recombinant receptors or those expressed endogenously in isolated smooth muscle cells from rat fundus. Using several strategies, we show this to be a consequence of essentially irreversible binding of hNmU-25 to its receptors and that this is followed by ligand internalization. Despite structural differences between receptors, there were no apparent differences in their activation, coupling, or regulation.     

Interaction of proteins with lipid rafts through glycolipid-binding domains: biochemical background and potential therapeutic applications

The wide biochemical diversity of glycolipids in membranes explains why these molecules are often selected by pathogens (viruses, bacteria, prions) as primary sites of interactions with the cell surface. Moreover, glycolipids concentrate into cholesterol/glycolipid-rich microdomains where they can reach high local concentrations consistent with the multivalent attachment of pathogens on the cell surface. Finally, recent studies have shown that glycolipids could also modulate protein conformation. This chaperone activity of glycolipids has been associated with various pathogenic processes including HIV infection, prion propagation, and amyloid aggregation in Alzheimer's and Creutzfeldt-Jakob's diseases. Despite the potential interest for drugs mimicking glycolipid structure and function, the physicochemical properties of authentic glycolipids suggested that it might be difficult to obtain synthetic glycolipid analogues able to neutralise those pathogens before they could reach the cell surface. Recent data obtained with mono-, di-, and tri-hexosylceramide (GalCer, LacCer and Gb(3)) have proven that this was absolutely not the case and that highly active inhibitors could be designed through slight modifications of glycolipid structure. Biochemical studies of glycolipid-protein interactions have highlighted the importance of CH-pi stacking interactions between galactosyl head groups of the glycolipid and aromatic amino acids of the protein. The discovery of this unique mechanism of interaction may allow a rational strategy for the design and synthesis of glycolipid-based molecules as new anti-infectious and/or anti-amyloidogenesis compounds. This strategy, which takes into account the hierarchical organisation of glycolipids into discrete membrane microdomains as well as their association with cholesterol, is discussed in the present review.     

Differential regulation of cell death in head and neck cell carcinoma through alteration of cholesterol levels in lipid rafts microdomains

Lipid rafts are cholesterol-enriched microdomains in the plasma membrane. They act as molecular platforms that spatially organize membrane receptor molecules and are involved in the transduction of various signaling pathways. We recently reported that in the radiosensitive squamous cell carcinoma SCC61 line, gamma-irradiation results in a rearrangement of the plasma membrane rafts and signaling platforms leading to radiation-induced apoptosis in a ceramide-dependent pathway. By contrast, this reorganization was found to be defective in the radioresistant counterpart cell line, SQ20B. As the cholesterol content of lipid rafts is two times higher in SQ20B compared with SCC61 cells, we investigated the modulation of these microdomains using methyl-beta-cyclodextrin (MbetaCDX), a widely used cholesterol-depleting agent, in order to disrupt raft organization in both cells. Here, we report that MbetaCDX treatment resulted in the triggering of apoptosis in SCC61 cells involving mitochondrial events and associated with the clustering of Fas, the formation of Fas-FADD complexes and the cleavage of procaspase 8. The ligand-independent activation of this death receptor was totally absent in SQ20B cells, which remained resistant to MbetaCDX-triggered apoptosis. However, treatment of SQ20B with MbetaCDX resulted in a ligand-independent activation of the epidermal growth factor receptor (EGFR) survival pathway, as evidenced by an increased tyrosine phosphorylation of EGFR. Taken altogether, our results indicate that lipid raft integrity is intimately involved in the triggering of apoptotic cell death and/or survival pathways in head and neck carcinoma cells.     

Effect of YM-254890, a specific Galphaq/11 inhibitor, on experimental peripheral arterial disease in rats

The protective effect of YM-254890, a specific Galphaq/11 inhibitor, on laurate-induced peripheral arterial disease in rats was compared with those of prostaglandin E1 (PGE1), beraprost, and clopidogrel. YM-254890 inhibited ADP-induced ex vivo rat platelet aggregation at a dose of 3 microg/kg. Furthermore, YM-254890 strongly inhibited phenylephrine-, serotonin- and endothelin-1-induced contractions in the rat aorta, and improved dermal blood flow after the laurate injection. The intra-arterial single bolus administration of YM-254890 15 min after the laurate injection dose-dependently inhibited the progression of the lesion, with significance, at 3 microg/kg without affecting systemic blood pressure. PGE1 and beraprost, when administered before the laurate injection, were effective, but their potencies were less than that of YM-254890. Clopidogrel significantly suppressed lesion progression when administered at 30 mg/kg twice a day for 3 days, which completely inhibited platelet aggregation. These results suggest that the local administration of YM-254890 may be useful for treating peripheral arterial disease.     

Endogenous regulator of G-protein signaling proteins regulate the kinetics of Galphaq/11-mediated modulation of ion channels in central nervous system neurons

Slow synaptic potentials are generated when metabotropic G-protein-coupled receptors activate heterotrimeric G-proteins, which in turn modulate ion channels. Many neurons generate excitatory postsynaptic potentials mediated by G-proteins of the Galphaq/11 family, which in turn activate phospholipase C-beta. Accessory GTPase-activating proteins (GAPs) are thought to be required to accelerate GTP hydrolysis and rapidly turn off G-proteins, but the involvement of GAPs in neuronal Galphaq/11 signaling has not been examined. Here, we show that regulator of G-protein signaling (RGS) proteins provide necessary GAP activity at neuronal Galphaq/11 subunits. We reconstituted inhibition of native 2-pore domain potassium channels in cerebellar granule neurons by expressing chimeric Galpha subunits that are activated by Galphai/o-coupled receptors, thus bypassing endogenous Galphaq/11 subunits. RGS-insensitive variants of these chimeras mediated inhibition of potassium channels that developed and recovered more slowly than inhibition mediated by RGS-sensitive (wild-type) chimeras or native Galphaq/11 subunits. These changes were not accompanied by a change in agonist sensitivity, as might be expected if RGS proteins acted primarily as effector antagonists. The slowed recovery from potassium channel inhibition was largely reversed by an additional mutation that mimics the RGS-bound state. These results suggest that endogenous RGS proteins regulate the kinetics of rapid Galphaq/11-mediated signals in central nervous system neurons by providing GAP activity.     

Characterization of the binding of cholera toxin to ganglioside GM1 immobilized onto microtitre plates

Ganglioside GM1 is the receptor for cholera toxin on cell surfaces, and the binding of cholera toxin to GM1 immobilized on microtitre plates has been reported previously by several authors as an assay for the toxin (GM1-ELISA). This assay has been examined in detail. Results were independent of the adsorption solvent for GM1 (methanol or phosphate-buffered saline), the pH of aqueous solvents (7.4-10.2) and the temperature (4-37 degrees C). High and near-maximal rates of absorbance change in the assay were found for lower concentrations of GM1 (100 ng ml(-1)) and for shorter incubation times (a few hours) than reported in the literature. A method was devised to provide a semi-quantitative estimate of the amount of GM1 bound to the plate; this was found to be in the low nanogram range. Binding of cholera toxin to the immobilized GM1 required > or =1.5 h for maximal assay results. The failure of free GM1 in solution to displace cholera toxin once bound to immobilized GM1 indicated that binding to immobilized GM1 is irreversible in the time frame of the experiment. Data from the literature support the very slow dissociation rates of the toxin-GM1 complex.     

Brain lipid changes in rats exposed to xylene and toluene

Rats were continuously exposed to vapors of xylene (320 ppm) for 30 or 90 days, other groups were exposed to toluene (320 ppm) for 30 days. After termination of exposure, different brain regions were removed for the determination of their lipid contents and ethanolamine phosphoglyceride fatty acid patterns. The 2 solvents had different effects on the animals. Xylene exposure resulted in limited transient changes. After 30 days exposure an increase in the liver to body weight ratio and a decrease in linoleic acid of ethanolamine phosphoglyceride in the cerebral cortex were observed. These changes were normalized after 90 days exposure. Toluene exposure, on the other hand, resulted in decreased weights of the body, of the brain as a whole and of the cerebral cortex. Liver weights were unchanged. Total phospholipids were found to be reduced in the cerebral cortex where also a slight increase in phosphatidic acid was observed. In this brain region a minor fatty acid of ethanolamine phosphoglyceride, 22:5 (n-3), was decreased. No changes were observed in the brainstem. The data on brain weights and lipid composition after exposure to toluene indicates a breakdown of phospholipids resulting in a loss of gray matter. The mechanism for these changes is uncertain but may involve degradation of phospholipids by phospholipase D. The effective metabolism of xylene and toluene seem to protect from fatty acid changes of brain phospholipids previously observed after exposure to chlorinated ethylenes.     

Constitutively active Src tyrosine kinase changes gating of HCN4 channels through direct binding to the channel proteins

Cardiac pacemaker current, if, is generated by hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. Our previous studies demonstrated that altered tyrosine phosphorylation can modulate the properties of both if and HCN channels. To assess a hypothesis that the intracellular tyrosine kinase Src may play a role in modulation by tyrosine phosphorylation of if, we cotransfected HEK293 cells with HCN4 and Src proteins. When HCN4 was cotransfected with a constitutively activated Src protein (Src529), the resultant voltage-dependent HCN4 activation was positively shifted (HCN4: V1/2 = -93 mV; Src529: V1/2 = -80 mV). The activation kinetics were accelerated at some potentials but not over the entire voltage range tested (eg, at -95 mV, tau_act(HCN4) = 3,243 ms; tau_act(Src529) = 1,113 ms). When HCN4 was cotransfected with a dominant negative Src protein (Src296), the HCN4 activation was shifted more negative to a smaller degree (HCN4: V1/2 = -93 mV; Src296: V1/2 = -98 mV; statistically insignificant) and the activation kinetics were slowed at most test potentials (eg, at -95 mV, tau_act(Src296) = 7,396 ms). Neither Src529 nor Src296 significantly altered HCN4 current density. Coimmunoprecipitation experiments revealed that Src forms a complex with HCN4 in HEK293 cells and in rat ventricular myocytes. Our data provide a novel mechanism of if regulation by Src tyrosine phosphorylation.     

Theoretical changes in drug distribution resulting from changes in binding to plasma proteins and to tissues

Concentrations of chlorpromazine fluctuate in the plasma of dogs and man after intravenous doses. The possibility that the fluctuations could arise from movement of the drug between tissue and plasma stores is examined theoretically. Calculations show that small changes in protein binding of drugs in plasma and tissues could cause redistribution of highly bound drugs between tissues and plasma. Redistribution would be greatest after changes in tissue binding of highly bound drugs. Fluctuations in chlorpromazine concentrations could be caused in this way.     

Pharmacodynamic modeling of the effect of changes in the environment on cellular lifespan and cellular response

Lifespan-based pharmacodynamic (PD) models of cellular response assume that the lifespan of cells is predetermined at the time of cellular production, despite recognized changes in the cellular environment following production that may alter the survival of the cells. This work extends previously proposed cellular lifespan PD models to incorporate environmental effects on the cell lifespan by considering two basic classes of models from survival analysis: accelerated life and relative risk models. Cellular responses using both model classes were simulated using a steady-state cellular production rate with changes in the environmental effects resulting from three different basic profiles. The environmental effect models were also fitted to the red blood cell (RBC) and hemoglobin concentration data from six sheep following hematopoietic ablation by busulfan administration. The simulations indicated that the basic shapes of the cellular responses were different between the accelerated life and relative risk models. Due to the more direct physical interpretation, relatively simple steady-state relationship between the cellular response and environmental effects, and the ability to reduce the model to a “point” baseline lifespan distribution, the accelerated life model appears to be a more realistic and flexible model. The analysis of the sheep RBC and hemoglobin data indicated that the environmental effect began to decrease the survival of cells 1–2 weeks following initiation of ablation and that the average “severity” of the environmental effect increased 3.49 (29.5%) (mean (C.V.)) fold under the accelerated life model. Alternative models without an environmental effect did not describe the observed data as well. The proposed environmental effect cellular lifespan PD models allow for the incorporation of arbitrary changes in the conditions of the cellular environment and modeling of environmentally dependent cellular survival. These PD models have potential applications in hematological management of end-stage renal disease, transfusion medicine, and patients undergoing chemotherapy, among other diseases and therapies.     

Tubulin binding, protein-bound conformation in solution, and antimitotic cellular profiling of noscapine and its derivatives

Noscapine and its 7-hydroxy and 7-amino derivatives were characterized for their binding to tubulin. A solution NMR structure of these compounds bound to tubulin shows that noscapine and its 7-aniline derivative do not compete for the same binding site nor does its small molecule crystal structure match its tubulin-bound conformation. These compounds were also tested for their antiproliferative effects on a panel hepatocellular carcinoma cell lines.     

脂筏蛋白质组学分析揭示快速老化因素对SAMP8 小鼠海马组织的关键影响

摘要：目的 探讨快速老化小鼠SAMP8老年性痴呆的关键细胞学机制。方法 以2月龄和8月龄SAMP8小鼠 各40只为痴呆相关快速老化动物模型,以同月龄各40只正常老化小鼠SAMR1为对照,从小鼠海马组织提取脂筏蛋 白,采用高效液相色谱-串联质谱法分析。脂筏蛋白质组学检测数据导入DAVID生物信息学分析工具,进行Gene Ontology（GO）生物信息学分析和Kyoto Encyclopedia of Genes and Genomes（KEGG）代谢网络分析,并用线粒体膜电位 和Morris水迷宫方法验证生物信息学分析结果。结果 与SAMR1小鼠比较,快速老化的SAMP8小鼠出现明显的认 知障碍。GO 分析显示,老年期 SAMP8 小鼠脂筏蛋白组中线粒体相关蛋白大幅度减少。KEGG 分析显示,老年期 SAMP8小鼠海马组织线粒体的氧化磷酸化功能大幅度衰退。线粒体膜电位分析显示,老年期SAMP8小鼠海马组织 线粒体膜电位大幅度降低。结论 在老化过程中,SAMP8小鼠海马组织最关键的细胞变化是线粒体氧化磷酸化功 能的过度衰退,这可能是其痴呆发生的重要细胞学机制。     

Changed cellular membrane lipid composition and lipid peroxidation of kidney in rats with chronic fluorosis

An animal model of chronic fluorosis was produced by subjecting Wistar rats to high doses of fluoride in drinking water for a prolonged period. Phospholipid and neutral lipid contents in rat kidney were then analyzed by high-performance liquid chromatography (HPLC), and fatty acid compositions from individual phospholipids were measured by gas chromatography. Lipid peroxidation was detected by the thiobarbituric-acid-reactive substance assay. Results showed that the total phospholipid content significantly decreased in the kidney of the rats treated with high doses of fluoride and the main species influenced were phosphatidylethanolamine (PE) and phosphatidylcholine (PC). Decreased proportions of polyunsaturated fatty acids were observed in PE and PC in kidney of fluoride-treated animals compared to controls. No changes could be detected in the amounts of cholesterol and dolichol in kidneys between the rats treated with fluoride and controls. A significant decrease of ubiquinone in rat kidney was observed in the groups treated with excessive fluoride. High levels of lipid peroxidation were detected in kidney of the rats with fluorosis. It is plausible that the specific modification of lipid composition results from lipid peroxidation. The oxidative stress and modification of cellular membrane lipids may be involved in the pathogenesis of chronic fluorosis and provide a possible explanation for the gross system damage observed in the body, especially in soft tissues and organs.     

Interaction of Clostridium perfringens delta toxin with erythrocyte and liposome membranes and relation with the specific binding to the ganglioside GM2

The specific interaction of the cytolytic Clostridium perfringens delta toxin with membrane GM2 was indicated by: (i) characterization of this glycolipid in the membrane of sheep and goat erythrocytes, which are lysed by the toxin, whereas GM2 was undetectable in insensitive rabbit erythrocytes, (ii) demonstration of 125I-toxin binding to GM2, by autoradiography, following incubation with thin-layer chromatograms containing separated neuroblastoma gangliosides, and (iii) toxin fixation by phospholipid-cholesterol unilamellar vesicles containing either sheep gangliosides or GM2. In order to investigate the intramembrane events leading to membrane disruption following toxin binding, the photoreactive probe 12(4-azido-2-nitrophenoxy)stearoyl 1-14C glucosamine, which inserts into the outer layer and labels integral membrane proteins, was used to establish whether delta toxin penetrates into target cell membrane. No toxin labeling was found, suggesting that toxin action takes place at the membrane surface. This contention is supported by the observation that despite toxin binding, GM2 liposomes did not release entrapped 14C-glucose. Treatment of toxin with carboxypeptidases, but not aminopeptidases, abolished both toxin binding capacity onto erythrocytes and its combination with antitoxin neutralizing antibodies, suggesting that the carboxy terminal end of the toxin is critical for binding to cell membrane.