Changing the Transport of a Cell

Abstract

The review deals with some of the transport functions of different systems that have been implicated with several pathological disorders. Membrane transport role in parasitic diseases and metal resistance is discussed as a few selected examples. Among various limitations that are encountered in recombinant technology and in heterologous expression of proteins, transport functions of the host organisms cannot be ignored. Recently, membrane transport has acquired a new emerging role in multidrug resistance. Several membrane transporters, particularly ATP binding cassette (ABC) proteins that are involved in drug resistance, have been identified throughout the evolutionary scale. The review briefly emphasizes that membranes are not only important as structural elements but are also adopted to perform diverse functions.     

Solution NMR studies of peptide-lipid interactions in model membranes

Abstract

Many important processes in life take place in or around the cell membranes. Lipids have different properties regarding their membrane-forming capacities, their mobility, shape, size and surface charge, and all of these factors influence the way that proteins and peptides interact with the membrane. In order for us to correctly understand these interactions, we need to be able to study all aspects of the interplay between lipids and peptides and proteins. Solution-state NMR offers a somewhat unique possibility to investigate structure, dynamics and location of proteins and peptides in bilayers. This review focuses on solution NMR as a tool for investigating peptide-lipid interaction, and special attention is given to the various membrane mimetics that are used to model the membrane. Examples from the field of cell-penetrating peptides and their lipid interactions will be given. The importance of studying lipid and peptide dynamics, which reflect on the effect that peptides have on bilayers, is highlighted, and in this respect, also the need for realistic membrane models.     

壳聚糖与细菌细胞膜相互作用的分子动力学研究

目的 壳聚糖（chitosan,CS）是一种天然的广谱抗菌活性物质。现有研究表明，壳聚糖与细菌细胞膜的相互作用是其发挥抗菌功能的关键。受限于传统实验技术的表征能力，壳聚糖与细菌细胞膜相互作用的具体机制仍有待研究。本文旨在研究壳聚糖与细菌细胞膜相互作用的分子机制。方法 本研究利用全原子分子动力学模拟技术主要探究了完全脱乙酰化的不同聚合度壳聚糖（八聚糖、十二聚糖和十六聚糖）与革兰氏阴性菌外膜（outer membrane,OM）和革兰氏阳性菌质膜（cytoplasmic membrane,CM）相互作用的动态过程。结果 壳聚糖主要依靠其氨基、碳6位羟基和碳3位羟基与OM和CM的头部极性区发生快速结合。随后壳聚糖末端糖基单元倾向于插入OM内部，深度约1 nm，并与脂质分子脂肪酸链上的羰基形成稳定的氢键相互作用。与之相比，壳聚糖分子难以稳定地插入CM内部。壳聚糖结合对膜结构性质产生影响，主要表现在降低OM和CM的单分子脂质面积，显著减少OM和CM极性区的Ca2+和Na+数目，破坏阳离子介导的脂质间相互作用。结论 本研究证明，壳聚糖带正电的氨基基团是介导其与膜相互作用的关键，并破环脂质间的相互作...     

Endothelium,the dynamic interface in cardiac lipid transport

Vascular endothelium is the dynamic interface in transport of lipid from blood to myocytes in heart and arteries. The luminal surface of endothelium is the site of action of lipoprotein lipase on chylomicrons and VLDL and the site of uptake of fatty acids from albumin. Fatty acids and monoacylglycerols are transported from the lumen in an interfacial continuum of endothelial and myocyte membranes. Lipoprotein lipase is transferred from myocytes to the vascular lumen, and is anchored there, by proteoheparan sulfate in cell membranes. Insulin, needed for synthesis of lipoprotein lipase and esterfication of fatty acids, is captured from the blood stream and delivered to myocytes by endothelial insulin receptors. Fatty acids, monoacylglycerols, lipoprotein lipase and insulin are transported along the same route, but by different mechanisms. The route involves the plasma membrane of endothelium and myocytes, the membrane lining transendothelial channels, and intercellular contacts. (Mol Cell Biochem116: 181–191, 1992)     

From mosaic to patchwork: Matching lipids and proteins in membrane organization

Abstract

Biological membranes encompass and compartmentalize cells and organelles and are a prerequisite to life as we know it. One defining feature of membranes is an astonishing diversity of building blocks. The mechanisms and principles organizing the thousands of proteins and lipids that make up membrane bilayers in cells are still under debate. Many terms and mechanisms have been introduced over the years to account for certain phenomena and aspects of membrane organization and function. Recently, the different viewpoints – focusing on lipids vs. proteins or physical vs. molecular driving forces for membrane organization – are increasingly converging. Here we review the basic properties of biological membranes and the most common theories for lateral segregation of membrane components before discussing an emerging model of a self-organized, multi-domain membrane or ‘patchwork membrane'.     

Ion transport through dimethyl sulfoxide (DMSO) induced transient water pores in cell membranes

Abstract

It is well known that dimethyl sulphoxide (DMSO) increases membrane permeability, which makes it widely used as a vehicle to facilitate drug delivery across biological membranes. However, the mechanism of how DMSO increases membrane permeability has not been well understood. Recently, molecular dynamics simulations have demonstrated that DMSO can induce water pores in biological membranes, but no direct experimental evidence is so far available to prove the simulation result. Using FluxOR Tl⁺ influx assay and intracellular Ca²⁺ imaging technique, we studied the effect of DMSO on Tl⁺ and Ca²⁺ permeation across cell membranes. Upon application of DMSO on CHO-K1 cell line, Tl⁺ influx was transiently increased in a dose-dependent manner. The increase in Tl⁺ permeability induced by DMSO was not changed in the presence of blockers for K⁺ channel and Na⁺-K⁺ ATPase, suggesting that Tl⁺ permeates through transient water pores induced by DMSO to enter into the cell. In addition, Ca²⁺ permeability was significantly increased upon application of DMSO, indicating that the transient water pores induced by DMSO were non-selective pores. Furthermore, similar results could be obtained from RAW264.7 macrophage cell line. Therefore, this study provided experimental evidence to support the prediction that DMSO can induce transient water pores in cell membranes, which in turn facilitates the transport of active substances across membranes.     

Solute Transport Process in Intestinal Epithelial Cells

In rat small intestine, the active transport of organic solutes results in significant depolarization of the membrane potential measured in an epithelial cell with respect to a grounded mucosal solution and in an increase in the transepithelial potential difference. According to the analysis with an equivalent circuit model for the epithelium, the changes in emf's of mucosal and serosal membranes induced by active solute transport were calculated using the measured conductive parameters. The result indicates that the mucosal cell membrane depolarizes while the serosal cell membrane remarkably hyperpolarizes on the active solute transport. Corresponding results are derived from the calculations of emf's in a variety of intestines, using the data that have hitherto been reported. The hyperpolarization of serosal membrane induced by the active solute transport might be ascribed to activation of the serosal electrogenic sodium pump. In an attempt to determine the causative factors in mucosal membrane depolarization during active solute transport, cell water contents and ion concentrations were measured. The cell water content remarkably increased and, at the same time, intracellular monovalent ion concentrations significantly decreased with glucose transport. Net gain of glucose within the cell was estimated from the restraint of osmotic balance between intracellular and extracellular fluids. In contrast to the apparent decreases in intracellular Na⁺ and K⁺ concentrations, significant gains of Na⁺ and K⁺ occurred with glucose transport. The quantitative relationships among net gains of Na⁺, K⁺ and glucose during active glucose transport suggest that the coupling ratio between glucose and Na⁺ entry by the carrier mechanism on the mucosal membrane is approximately 1:1 and the coupling ratio between Na⁺-efflux and K⁺-influx of the serosal electrogenic sodium pump is approximately 4:3 in rat small intestine. In addition to the electrogenic ternary complex inflow across the mucosal cell membrane, the decreases in intracellular monovalent ion concentrations, the temporary formation of an osmotic pressure gradient across the cell membrane and the streaming potential induced by water inflow through negatively charged pores of the cell membrane in the course of an active solute transport in intestinal epithelial cells are apparently all possible causes of mucosal membrane depolarization.     

Electrical properties of chloride transport across theNecturus proximal tubule

Summary The chloride conductance of the basolateral cell membrane of theNecturus proximal tubule was studied using conventional and chloride-sensitive liquid ion exchange microelectrodes. Individual apical and basolateral cell membrane and shunt resistances, transepithelial and basolateral, cell membrane potential differences, and electromotive forces were determined in control and after reductions in extracellular Cl^–. When extracellular Cl^– activity is reduced in both apical and basolateral solutions the resistance of the shunt increases about 2.8 times over control without any significant change in cell membrane resistances. This suggests a high Cl^– conductance of the paracellular shunt but a low Cl^– conductance of the cell membranes. Reduction of Cl^– in both bathing solutions or only on the basolateral side hyperpolarizes both the basolateral cell membrane potential difference and electromotive force. Hyperpolarization of the basolateral cell membrane potential difference after low Cl^– perfusion was abolished by exposure to HCO ₃ ^– -free solutions and SITS treatment. In control conditions, intracellular Cl^– activity was significantly higher than predicted from the equilibrium distribution across both the apical and basolateral cell membranes. Reducing Cl^– in only the basolateral solution caused a decrease in intracellular Cl^–. From an estimate of the net Cl^– flux across the basolateral cell membrane and the electrochemical driving force, a Cl^– conductance of the basolateral cell membrane was predicted and compared to measured values. It was concluded that the Cl^– conductance of the basolateral cell membrane was not large enough to account for the measured flux of Cl^– by electrodiffusion alone. Therefore these results suggest the presence of an electroneutral mechanism for Cl^– transport across the basolateral cell membrane of theNecturus proximal tubule cell.     

A study of dynamic membrane phenomena during the gastric secretory cycle: Fusion,retrieval and recycling of membranes

Summary The possibility of recycling, fusion and retrieval of membranes during the gastric secretory process was studied in isolated gastric mucosae of the toadBufo marinus. Incorporation and efflux of¹⁴C-inulin and horseradish peroxidase (HRP) into the tissue as well as transmission and freeze-fracture electron microscopic studies during the secretory cycle were done. HRP and¹⁴C-inulin were incorporated into the tubulovesicular membrane system during the secreting-resting transition. Upon restimulation, markers were released towards the lumen. Marker efflux preceded onset of H⁺ secretion. Morphological transformations in the oxyntic cell as evidenced from transmission and freeze-fracture electron microscopy preceded acid secretion coinciding with marker efflux. At this time, images that have been associated with membrane fusion were found in the apical membranes of oxyntic cells. The results are consistent with a model where membrane area increases by a fusion mechanism at the expense of the tubulovesicular system. This transformation precedes the onset of H⁺ secretion. Upon cessation of the stimulus or inhibition, membranes are retrieved and the tubulovesicular system reformed. Retrieved membranes could be then reutilized in the next secretory cycle.     

Tetrahymena pyriformis as a Model System for Membrane Studies*†

SYNOPSIS. Tetrahymena pyriformis is an exceptionally useful subject for studying metabolic interrelationships among intracellular membranes. Its advantages include the striking differences in lipid composition among the cell's various functionally distinct membrane systems, indicating a pronounced lipid specificity at the membrane sites. The magnitude of these differences permits analysis of the mechanisms underlying the specificity. Even more valuable is the unique physical isolation of ciliate surface membranes from the cytoplasm of the cell. In contrast to the almost immediate equilibration of newly made lipids with preexisting lipids found in most cells, Tetrahymena surface membranes have a lipid turnover slow enough to be conveniently analyzed. Finally, the well-studied responses of Tetrahymena to such physiological stresses as heat and starvation may be used to evaluate the effects of environmental factors on membrane formation.     

Topology of amino-phospholipids in the red cell membrane

The red cell membrane has an asymmetric arrangement of phospholipids. The amino-phospholipids are localized primarily on the inner surface of the membrane and the choline phospholipids are localized to a large extent on the outer surface of the membrane. Evidence is presented based on the use of covalent chemical probes in sequence that the red cell membrane contains heterogeneous domains of PE and PS and that the transport systems for Pi and K⁺ are asymmetrically arranged. Certain amino groups of PE, PS, and/or protein localized on the outer membrane surface are involved in Pi transport and certain amino groups of PE, PS, and/or protein localized on the inner surface of the membrane are involved in K⁺ transport. Cross-linking studies with DFDNB show that the cross-linked PE-PE molecules are rich in plasmalogens. This suggests that clusters of plasmalogen forms of PE occur in the membrane. Both PE and PS are cross-linked to membrane protein. These PE and PS molecules contain 24–28% 16:0 and 18:0 fatty acids and 12% fatty aldehydes. PE and PS molecules are cross-linked to a spectrin-rich fraction. It is proposed that the binding of spectrin to membrane PE and PS may help anchor spectrin to the inner surface of the membrane and regulate shape changes in the cell. K⁺-valinomycin forms a complex with TNBS and converts it from a non-penetrating proble to a penetrating probe. Valinomycin enhances K⁺ leak and Pi leak in the red cells. SITS inhibits completely the valinomycin-induced Pi leak and inhibits partially the valinomycin induced K⁺ leak. Valinomycin and IAA have additive effects on Pi leak. Ouabin has no effect on basal or valino-mycin-induced Pi leak. These data suggest that Pi leak and K⁺ leak occur by separate transport systems. In summary, the amino-phospholipids in the red cell membrane are asymmetrically arranged; some occur in clusters and some are closely associated with membrane proteins. Amino-phospholipids also are believed to bind spectrin to the inner surface of the membrane and also may play a role in cation and anion leak.     

The amino acids upstream of NH(2)-terminal dileucine motif play a role in regulating the intracellular sorting of the Class III transporters GLUT8 and GLUT12

Abstract

The transport of glucose across cell membranes is mediated by a family of facilitative glucose transporters (GLUTs). The class III glucose transporters GLUT8 and GLUT12 both contain a similar [DE]XXXL[LI] dileucine sorting signal in their amino terminus. This type of dileucine motif facilitates protein trafficking to various organelles or to the plasma membrane via interactions with adaptor protein (AP) complexes. The [DE]XXXL[LI] motif in GLUT8 is thought to direct it to late endosomal/lysosomal compartments via its interactions with AP1 and AP2. Unlike GLUT8, the [DE]XXXL[LI] motif does not direct GLUT12 to a lysosomal compartment. Rather, GLUT12 resides in the Golgi network and at the plasma membrane. In a previous study, we found that exchanging the XXX (TQP) residues in GLUT8 with the corresponding residues in GLUT12 (GPN) resulted in a dramatic missorting of GLUT8 to the cell surface. We postulated that the XXX amino acids upstream of the dileucine motif in GLUT8 influence the degree of interaction between the [DE]XXXL[LI] motif and adaptor proteins. To further explore its trafficking mechanisms, we created mutant constructs to identify the role that each of the individual XXX amino acids has for regulating the intracellular sorting of GLUT8. Here we find that the XXX amino acids, specifically the position of a proline -2 from the dileucine residues, influence the affinity of APs for GLUT8 and GLUT12.     

Solvent-tolerant bacteria in biocatalysis

The toxicity of fine chemicals to the producer organism is a problem in several biotechnological production processes. In several instances, an organic phase can be used to extract the toxic product from the aqueous phase during a fermentation. With the discovery of solvent-tolerant bacteria, more solvents can now be used in such two-liquid water–solvent systems. We are gaining new insights into the mechanisms of bacterial solvent tolerance, such as the active efflux of solvents from the cytoplasmic membrane and solvent-impermeable outer membranes.     

Ruthenium ammine complexes as electron acceptors for growth stimulation by plasma membrane electron transport

Ammineruthenium(III) complexes have been found to act as electron acceptors for the transplasmalemma electron transport system of animal cells. The active complexes hexaammineruthenium(III), pyridine pentaammineruthenium(III), and chloropentaammineruthenium(III) range in redox potential (E ₀) from 305 to –42 mV. These compounds also act as electron acceptors for the NADH dehydrogenase of isolated plasma membranes. Stimulation of HeLa cell growth, in the absence of calf serum, by these compounds provides evidence that growth stimulation by the transplasma membrane electron transport system is not entirely based on reduction and uptake of iron.     

Model membrane platforms to study protein-membrane interactions

Abstract

Constituting functional interactions between proteins and lipid membranes is one of the essential features of cellular membranes. The major challenge of quantitatively studying these interactions in living cells is the multitude of involved components that are difficult, if not impossible, to simultaneously control. Therefore, there is great need for simplified but still sufficiently detailed model systems to investigate the key constituents of biological processes. To specifically focus on interactions between membrane proteins and lipids, several membrane models have been introduced which recapitulate to varying degrees the complexity and physicochemical nature of biological membranes. Here, we summarize the presently most widely used minimal model membrane systems, namely Supported Lipid Bilayers (SLBs), Giant Unilamellar Vesicles (GUVs) and Giant Plasma Membrane Vesicles (GPMVs) and their applications for protein-membrane interactions.     

Integrity under stress: Host membrane remodelling and damage by fungal pathogens

Membrane bilayers of eukaryotic cells are an amalgam of lipids and proteins that distinguish organelles and compartmentalise cellular functions. The mammalian cell has evolved mechanisms to sense membrane tension or damage and respond as needed. In the case of the plasma membrane and phagosomal membrane, these bilayers act as a barrier to microorganisms and are a conduit by which the host interacts with pathogens, including fungi such as Candida, Cryptococcus, Aspergillus, or Histoplasma species. Due to their size, morphological flexibility, ability to produce long filaments, secrete pathogenicity factors, and their potential to replicate within the phagosome, fungi can assault host membranes in a variety of physical and biochemical ways. In addition, the recent discovery of a fungal pore‐forming peptide toxin further highlights the importance of membrane biology in the outcomes between host and fungal cells. In this review, we discuss the apparent “stretching” of membranes as a sophisticated biological response and the role of vesicular transport in combating membrane stress and damage. We also review the known pathogenicity factors and physical properties of fungal pathogens in the context of host membranes and discuss how this may contribute to pathogenic interactions between fungal and host cells.     

Electrical properties of the cellular transepithelial pathway inNecturus gallbladder: III. Ionic permeability of the basolateral cell membrane

Summary The ionic permeability of the basolateral membrane ofNecturus gallbladder epithelium was studied with intracellular microelectrode techniques. After removal of most of the subepithelial tissue (to reduce unstirred layer thickness), impalements were performed from the serosal side, and ionic substitutions were made in the serosal solution while a microelectrode was kept in a cell. Thus, it was possible to obtain continuous (and reversible) records of transepithelial and cell membrane potentials and to measure intermittently the transepithelial resistance and the ratio of cell membrane resistances. From these data and the mean value of the equivalent resistance of the cell membranes in parallel (obtained from cable analysis in a different group of tissues), absolute cell membrane and shunt resistances and equivalent electromotive forces (emf's) were calculated. From the changes of basolateral membrane emf (E _b ) produced by the substitutions, the conductance (G) and permeability (P) of the membrane for K, Cl and Na were estimated. Potassium-for-sodium substitutions produced large reductions of both cell membrane potentials, ofE _b , and of the resistance of the basolateral membrane (R _b ), indicating highG _K andP _K . Chloride substitution with isethionate or sulfate resulted in smaller changes of cell membrane potentials andE _b and in no significant change ofR _b , indicating small but measurable values ofG _Cl andP _Cl . Sodium substitutions with N-methyl-d-glucamine (NMDG) resulted in cell potential changes entirely attributable to the biionic potential produced in the shunt pathway (P _Na >P _NMDG ), and in no significant changes ofP _b orE _b , indicating thatG _Na andP _Na are undetectable. The question of the mechanism of Cl transport across the basolateral membrane was addressed by comparing the mean rate of transepithelial Cl transport (J _Cl ^net ) and the predicted passive Cl flux across the basolateral membrane (from the membrane Cl conductance, potential, and Cl equilibrium potential). The conclusion is that only a very small fraction of the Cl flux across the basolateral membrane can be electrodiffusional. Since the paracellular Cl conductance is also too low to account forJ _Cl ^net , these results suggest the presence of a neutral mechanism of Cl extrusion from the cells. This could be a NaCl pump, a downhill KCl transport mechanism, or a Cl–HCO₃ exchange mechanism.     

Effect of solution environment on the permeability of red blood cells

The cell membrane permeability governs the rate of solute transport into and out of the cell, significantly affecting the cell's metabolic processes, viability, and potential usefulness in both biotechnological applications and physiological systems. Most previous studies of the cell membrane permeability have neglected the possible effects of suspending medium on membrane transport, even though there is extensive experimental evidence that suspending phase composition can significantly affect other properties related to the cell membrane (e.g., cell deformability, fragility, and aggregation rate). This study examined the effects of suspending phase composition (both proteins and electrolytes) on the permeability of human red blood cells to the metabolites creatinine and uric acid. Data were obtained using a stirred ultrafiltration device with direct cell- and proteinfree sampling through a semipermeable membrane. Both the uric acid and creatinine permeabilities were strongly affected by the suspending phase composition, with the permeabilities in different buffer solutions varying by as much as a factor of three. The predominant factors affecting the permeability were the presence (or absence) of chloride, phosphate/adenine, and proteins, although the magnitude and even the direction of these effects were significantly different for creatinine and uric acid transport. The dramatic differences in behavior for uric acid and creatinine reflect the different transport mechanisms for these solutes, with uric acid transported by a carrier-mediated mechanism and creatinine transported by passive diffusion through the lipid bilayer. These results provide important insights into the effects of solution environment on cell membrane transport and other cell membrane-mediated properties. (c) 1994 John Wiley & Sons, Inc.     

Lipid analysis of mitochondrial membranes from the yeast Pichia pastoris

Here we describe for the first time isolation and biochemical characterization of highly purified mitochondrial inner and outer membranes from Pichia pastoris and systematic lipid analysis of submitochondrial fractions. Mitochondria of this yeast are best developed during growth on glycerol or sorbitol, but also on methanol or fatty acids. To obtain organelle membranes at high quality, methods of isolation and subfractionation of mitochondria originally developed for Saccharomyces cerevisiae were adapted and employed. A characteristic feature of the outer mitochondrial membrane of P. pastoris is the higher phospholipid to protein ratio and the lower ergosterol to phospholipid ratio compared to the inner membrane. Another marked difference between the two mitochondrial membranes is the phospholipid composition. Phosphatidylcholine and phosphatidylethanolamine are major phospholipids of both membranes, but the inner membrane is enriched in cardiolipin, whereas the outer membrane contains a high amount of phosphatidylinositol. The fatty acid composition of both mitochondrial membranes is similar. Variation of the carbon source, however, leads to marked changes of the fatty acid pattern both in total and mitochondrial membranes. In summary, our data are the first step to understand the P. pastoris lipidome which will be prerequisite to manipulate membrane components of this yeast for biotechnological purposes.