Short-range intracellular trafficking of small molecules across endoplasmic reticulum junctions

Intracellular trafficking is not mediated exclusively by vesicles. Additional, non-vesicular mechanisms transport material, in particular small molecules such as lipids and Ca(2+) ions, from one organelle to another. This transport occurs at narrow cytoplasmic gaps called membrane contact sites (MCSs), at which two organelles come into close apposition. Despite the conservation of these structures throughout evolution, little is known about this transport, largely because of a lack of knowledge of almost all molecular components of MCSs. Recently, this situation has started to change because the structural proteins that bridge an MCS are now known in a single case, and proteins implicated in lipid trafficking have been localized to MCSs. In the light of these advances, I hypothesize that the endoplasmic reticulum has a central role in the trafficking of lipids and ions by forming a network of MCSs with most other intracellular organelles.     

Nuclear magnetic resonance studies of cation transport across vesicle bilayer membranes.

We analyze an increasingly popular NMR method analogous to the black lipid membrane (BLM) isotopic tracer experiment for the study of mediated cation transport but involving the preparation of vesicles with an environment asymmetric in that paramagnetic metal ions are present only outside the vesicles. This asymmetry is manifest in the NMR spectrum as two distinct resonances for magnetic nuclei in outside and inside lipid headgroups. As mediated transport begins and for the paramagnetic metal ions enter the vesicles, the inner headgroup resonance line shifts and changes shape with a time course containing much information on the actual ion transport mechanism. Processes by which the ions enter the vesicles one or a few at a time (such as via a diffusive carrier) are easily distinguishable from those by which the ions enter in large bursts (such as by pore activation). The limiting case where intervesicular mediator exchange is slow relative to cation transport (the situation for integral membrane proteins) is treated analytically. Computer simulated curves indicate conditions necessary for certain changes in the line shape which are analogous to the "current jumps" observed in BLM conductance studies. The theory derived allows estimates of the average number of ions entering the first few bursts, how often the bursts occur, and how they depend on the concentration of the mediating species in the vesicular membrane. Preliminary experimental spectra illustrating some of the various possible line shape behaviors are presented.     

Evaluations of a mechanistic hypothesis for the influence of extracellular ions on electroporation due to high-intensity,nanosecond pulsing

The effect of ions present in the extracellular medium on electroporation by high-intensity, short-duration pulsing is studied through molecular dynamic simulations. Our simulation results indicate that mobile ions in the medium might play a role in creating stronger local electric fields across membranes that then reinforce and strengthen electroporation. Much faster pore formation is predicted in higher conductivity media. However, the impact of extracellular conductivity on cellular inflows, which depend on transport processes such as electrophoresis, could be different as discussed here. Our simulation results also show that interactions between cations (Na⁺ in this case) and the carbonyl oxygen of the lipid headgroups could impede pore resealing.     

Metal ions in the structure and function of RNA

It is becoming increasingly clear that RNA is more than a passive carrier of genetic information. Folded RNA molecules play key roles in almost every aspect of cellular metabolism, including protein transport, RNA splicing, peptide bond formation, and translational regulation. This is facilitated by the multifunctional nature of RNA biopolymers which can serve as rigid structural scaffolds, conformational switches, and catalysts for chemical reactions. In all cases, metal ions play a crucial role in RNA function. For folded RNA molecules, the pathway for adopting proper tertiary structure, and the stabilization of that structure, depends on specific and nonspecific interactions with certain classes of metal ions. There is a rapidly expanding repertoire of RNA structural motifs that typically sequester metal ions, and these are being studied using new spectroscopic and chemical methodologies. Many ribozymes (catalytic RNA molecules) depend on metal ions as cofactors that are explicitly involved in the chemical mechanism of catalysis. All of these functions are exemplified by recent studies of group II introns, which are among the largest ribozymes found in Nature. In this case, there are specific roles for metal ions in the folding pathway, the tertiary structure and the chemical mechanism.     

Copper-albumin: What is its functional role?

The small copper fraction in animal blood that is bound to albumin protein is generally considered to have a transport role for the metal. However, several studies have concluded that copper ion incorporation into copper-enzymes requires caeruloplasmin to act as the transport form. The kinetic rates of Cu(albumin) reactions are also not in keeping with a general transport function. Only in the portal bloodstream does the Cu(albumin) appear to have a well-established transport role, i.e. in delivering the metal ions from the intestine to the liver. Thereafter the evidence as to its function is less certain; it could act as a storage form of the metal or have no role at all. Similar considerations apply to other metal-albumin fractions e.g. those of zinc and nickel.     

Apparent cooperativity of amino acid transport in Halobacterium halobium: Effect of electrical potential

Active serine accumulation in cell envelope vesicles from Halobacterium halobium proceeds by co-transport with Na⁺ and can be induced by either transmembrane electrical potential or transmembrane Na⁺ concentration difference. It was shown earlier that in the former case the initial transport rate is a fourth-power function of the magnitude of the electrochemical potential difference of sodium ions, and in the latter, a second-power function. A possible interpretation of this finding is cooperativity of sodium-transporting sites in the transport carrier. When both kinds of driving force are imposed simultaneously on the vesicles, fourth-power dependence on the total potential difference of sodium ions is obtained, suggesting that the transport carrier is regulated by the electrical potential. Heat treatment of the vesicles at 48 ° partially inactivates transport and abolishes this effect of the electrical potential.     

Model of active transport of ions in cardiac cell

A model of the active transport of ions in a cardiac muscle cell, which takes into account the active transport of Na⁺, K⁺, Ca²⁺, Mg²⁺, HCO₃⁻ and Cl⁻ ions, has been constructed. The model allows independent calculations of the resting potential at the biomembrane and concentrations of basic ions (sodium, potassium, chlorine, magnesium and calcium) in a cell. For the analysis of transport processes in cardiac cell hierarchical algorithm “one ion-one transport system” was offered. The dependence of the resting potential on concentrations of the ions outside a cell has been established. It was shown, that ions of calcium and magnesium, despite their rather small concentration, play an essential role in maintenance of resting potential in cardiac cell. The calculated internal concentrations of ions are in good agreement with the corresponding experimental values.     

Characterization of orthophosphate-induced active cation transport in isolated liver mitochondria

The kinetics of the P_i-induced active transport of ions by isolated liver mitochondria were studied by monitoring photometrically mitochondrial volume changes. In a previous communication, these volume changes were shown to correlate quantitatively with the net uptake or release of ions. In the present study the specificity of the P_i role was further characterized. The data support the contention that cations are actively transported. Anions follow the transfer of cations to maintain electrical neutrality. The relationship of the transport system to oxidative phosphorylation was investigated by simultaneously monitoring both processes under different experimental conditions. The results of the experiments are quantitatively consistent with a model proposed for the P_i-induced active transport in isolated rat liver mitochondria. The model includes the following features. 1. P_i induces an inwardly directed, carrier-mediated active transport of cations. 2. The transport is coupled to the energy-conserving reactions of the cytochrome chain. 3. The efflux of ions accumulated in the presence of low P_i concentrations occurs by passive diffusion. 4. Net accumulation ceases when the rates of active transport and passive diffusion become equal. 5. The active transport competes with oxidative phosphorylation for a common, nonphosphorylated, high-energy intermediate.     

Anion inhibitor-sensitive unidirectional sodium movements in the human erythrocyte

The increased unidirectional sodium influx found when human erythrocytes are suspended in isotonic salt solutions containing bicarbonate ions as a replacement for chloride ions was examined. The increased sodium movement appears to have the transport characteristics of anion movement. Inhibitors of anion transport such as furosemide, fluorodinitrobenzene (FDNB), and 4-acetamido-4'-isothiocyano-stilbene-2- 2'-disulfonic acid (SITS) drastically inhibit these augmented sodium movements. An ion-pair mechanism appears to phenomenologically describe much of the data. A possible role for carbamino groups is considered. Such a model, however, required additional assumptions to explain the selectivity and the anion inhibitor effects.     

The effect of potassium diffusion through the Schwann cell layer on potassium conductance of the squid axon

Summary The accumulation of K⁺ ions in the intermembranous spaces of the Schwann cell layer during K⁺ ion current flow may lead to appreciable changes of the K⁺ equilibrium potential. Thus, for an evaluation of the K⁺ conductance of the axolemma, the transport of K⁺ ions through the Schwann cell layer has to be characterized quantitatively. In the present work this is done for a simplified model of the geometrical arrangement of the slit-like channels traversing the Schwann cell region.The K⁺ transport through the slits is treated for two cases: (a) Assuming that electro-kinetic volume flow does not affect K⁺ transport. In this case, pure diffusion of K⁺ ions accounts for their removal from the intermembranous spaces. Estimates on electro-kinetic volume flow show that this case applies to axons ofLoligo forbesi in voltage clamps of fairly small depolarizations. (b) For the case of appreciable electro-kinetic volume flow, evidence is adduced that its main effect is a widening of the slits through the Schwann cell layer. This physical situation could be treated only for the steady-state of convective diffusion of K⁺ ions in the slits.This case is applied to experiments on large depolarizing voltage clamps forLoligo pealii axons. It is shown that a widening of the slits to up to eight times the resting width is to be expected.In both cases (a) and (b), marked deviations of the K⁺ conductance of the axolemma from the Hodgkin-Huxley conductance result. The series resistance of the Schwann cell layer and the decay of after-effects of trains of action potentials are described by the theory.     

Mechanism of transport and storage of neurotransmitters

This review will focus on the bioenergetics, mechanism, and molecular basis of neurotransmitter transport. As indicated in the next section, these processes play an important role in the overall process of synaptic transmission. During the last few years, direct evidence has been obtained that these processes are coupled chemiosmotically, i.e., the accumulation of neurotransmitters is driven by ion gradients. Two types of neurotransmitter transport systems have been identified: sodium-coupled systems located in the synaptic plasma membrane of nerves (and sometimes in the plasma membrane of glial cells) and proton-coupled systems which are part of the membrane of intracellular storage organelles. From a bioenergetic point of view, the sodium-coupled systems are especially interesting, since it has recently been discovered that many systems require other ions in addition to sodium. It has now been demonstrated in several cases that, besides sodium ions, these additional ions, such as chloride and potassium, serve as additional coupling ions. These systems will be reviewed here in considerable detail with emphasis on the role of the additional ions. In the second part of the review we shall focus on neurotransmitter transport into storage organelles. Although both sodium and proton coupled systems have been reviewed in the past, there has been a shift from a kinetic and thermodynamic to a biochemical approach. In fact, a few transporters have been identified and functionally reconstituted. These developments have of course been incorporated in this review.     

NAD(P)H oxidase and renal epithelial ion transport

A fundamental requirement for cellular vitality is the maintenance of plasma ion concentration within strict ranges. It is the function of the kidney to match urinary excretion of ions with daily ion intake and nonrenal losses to maintain a stable ionic milieu. NADPH oxidase is a source of reactive oxygen species (ROS) within many cell types, including the transporting renal epithelia. The focus of this review is to describe the role of NADPH oxidase-derived ROS toward local renal tubular ion transport in each nephron segment and to discuss how NADPH oxidase-derived ROS signaling within the nephron may mediate ion homeostasis. In each case, we will attempt to identify the various subunits of NADPH oxidase and reactive oxygen species involved and the ion transporters, which these affect. We will first review the role of NADPH oxidase on renal Na(+) and K(+) transport. Finally, we will review the relationship between tubular H(+) efflux and NADPH oxidase activity.     

Divalent cations and nifedipine action on Ca(2+) transport into myometrial plasmalemma vesicles

While using 45Ca2+ on the model of "outside-out configuration" vesicules of the myometrium cells sarcolemma an investigation of Cd2+, Zn2+, Co2+ and niphedipin on Ca2+ transport into the vesicules in the conditions of protons gradient transmembrane dissipation has been conducted. The above listed substances blocking effect corresponds to their physicochemical properties. Cadmium and zinc ions are considerably more effective in suppressing Ca2+ transport into the vesicules under the dissipation of delta pH on the membrane if compare with the case of delta pH = 0. In the case of niphedipin inhibiting action an opposite result is observed. The hypothesis has been made, that dissipation of delta pH on the sarcolemma is capable to strengthen the transmembrane Ca2+ transport by means of changing the channel structures conformation.     

Certain aspects of the mechanism of intestinal transport of sodium, potassium, calcium and magnesium in rats

Intestinal absorption of sodium, potassium, calcium and magnesium was studied in rats by the method of intestinal perfusion using ouabain as an inhibitor of sodium-potassium dependent ATPase. At the same time the activity of ATPase and phosphatase were determined in homogenates of intestinal mucosa. A significant effect on the concentration of the determined ions was demonstrated in the transport of these ions, and also an unquestionable participation of intestinal ATPase in the direction and intensity of this transport. It was found that the multidirectional effect of ouabain on the transport of cations depended on their concentration. In the case of concentrations of cations similar to those in the mean food rations it has been demonstrated that ouabain increased the absorption of sodium, potassium and calcium and inhibited the absorption of magnesium. With a threefold higher ions concentration the absorption of potassium and magnesium was inhibited, without changing the transport of sodium and calcium. The possible explanation of the mechanism of these effects is discussed.     

Transport numbers in transdermal iontophoresis

Parameters determining ionic transport numbers in transdermal iontophoresis have been characterized. The transport number of an ion (its ability to carry charge) is key to its iontophoretic delivery or extraction across the skin. Using small inorganic ions, the roles of molar fraction and mobility of the co- and counterions present have been demonstrated. A direct, constant current was applied across mammalian skin in vitro. Cations were anodally delivered from either simple M(+)Cl(-) solutions (single-ion case, M(+) = sodium, lithium, ammonium, potassium), or binary and quaternary mixtures thereof. Transport numbers were deduced from ion fluxes. In the single-ion case, maximum cationic fluxes directly related to the corresponding ionic aqueous mobilities were found. Addition of co-ions decreased the transport numbers of all cations relative to the single-ion case, the degree of effect depending upon the molar fraction and mobility of the species involved. With chloride as the principal counterion competing to carry current across the skin (the in vivo situation), a maximum limit on the single or collective cation transport number was 0.6-0.8. Overall, these results demonstrate how current flowing across the skin during transdermal iontophoresis is distributed between competing ions, and establish simple rules with which to optimize transdermal iontophoretic transport.     

The identity of the current carriers in canine lingual epithelium in vitro

Ion transport across the lingual epithelium has been implicated as an early event in gustatory transduction. The fluxes of isotopically labelled Na+ and Cl- were measured across isolated canine dorsal lingual epithelium under short-circuit conditions. The epithelium actively absorbs Na+ and to a lesser extent actively secretes Cl-. Under symmetrical conditions with Krebs-Henseleit buffer on both sides, (1) Na+ absorption accounts for 46% of the short-circuit current (Isc); (2) there are two transcellular Na+ pathways, one amiloride-sensitive and one amiloride-insensitive; (3) ouabain, added to the serosal solution, inhibits both Isc and active Na+ absorption. When hyperosmotic (0.25 M) NaCl is placed in the mucosal bath, both Isc and Na+ absorption increase; net Na+ absorption is at least as much as Isc. Ion substitution studies indicate that the tissue may transport a variety of larger ions, though not as effectively as Na+ and Cl-. Thus we have shown that the lingual epithelium, like other epithelia of the gastrointestinal tract, actively transports ions. However, it is unusual both in its response to hyperosmotic solutions and in the variety of ions that support a transepithelial short-circuit current. Since sodium ion transport under hyperosmotic conditions has been shown to correlate well with the gustatory neural response, the variety of ions transported may likewise indicate a wider role for transport in taste transduction.     

Ion transport across transmembrane pores

To study the pore-mediated transport of ionic species across a lipid membrane, a series of molecular dynamics simulations have been performed of a dipalmitoyl-phosphatidyl-choline bilayer containing a preformed water pore in the presence of sodium and chloride ions. It is found that the stability of the transient water pores is greatly reduced in the presence of the ions. Specifically, the binding of sodium cations at the lipid/water interface increases the pore line tension, resulting in a destabilization of the pore. However, the application of mechanical stress opposes this effect. The flux of ions through these mechanically stabilized pores has been analyzed. Simulations indicate that the transport of the ions through the pores depends strongly on the size of the water channel. In the presence of small pores (radius <1.5 nm) permeation is slow, with both sodium and chloride permeating at similar rates. In the case in which the pores are larger (radius >1.5 nm), a crossover is observed to a regime where the anion flux is greatly enhanced. Based on these observations, a mechanism for the basal membrane permeability of ions is discussed.     

Physics of Ion Channels

We review the basic physics involved in transport of ions across membrane channels in cells. Electrochemical forces that control the diffusion of ions are discussed both from microscopic and macroscopic perspectives. A case is made for use of Brownian dynamics as the minimal phenomenological model that provides a bridge between experiments and more fundamental theoretical approaches. Application of Brownian and molecular dynamics methods to channels with known molecular structures is discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Structure and function of ATP7A and ATP7B proteins--Cu-transporting ATPases

Living organisms have developed refined and geneticaly controlled mechanisms of the copper metabolism and transport. ATP7A and ATP7B proteins play the key role in copper homeostasis in the organism. Both proteins are P-type Cu-transporting ATPases and use the energy of ATP hydrolysis to transfer the copper ions across the cellular membranes. Both proteins are localised in Golgi aparatus and involved in regulation of overall copper status in the body and their function is the export of excess copper from the cells and delivery of copper ions to Cu-dependent enzymes. Moreover in organism Cu-transporting ATPases are involved in absorption of dietary copper, Cu removal with the bile, placental copper transport and its secretion to the milk during lactation. Moreover it is known that Cu-transporting ATPases play a role in generation of anti-cancer drug resistance. Disturbances of ATP7A and ATP7B function caused by mutations lead to severe metabolic diseases Menkes and Wilson diseases, respectively.     

Water and potassium dynamics inside the KcsA K(+) channel

Molecular dynamics simulations and electrostatic modeling are used to investigate structural and dynamical properties of the potassium ions and of water molecules inside the KcsA channel immersed in a membrane-mimetic environment. Two potassium ions, initially located in the selectivity filter binding sites, maintain their position during 2 ns of dynamics. A third potassium ion is very mobile in the water-filled cavity. The protein appears engineered so as to polarize water molecules inside the channel cavity. The resulting water induced dipole and the positively charged potassium ion within the cavity are the key ingredients for stabilizing the two K(+) ions in the binding sites. These two ions experience single file movements upon removal of the potassium in the cavity, confirming the role of the latter in ion transport through the channel.