Anion channels from rat brain synaptosomal membranes incorporated into planar bilayers

Summary Synaptic membranes from rat brain were incorporated into planar lipid bilayers, and the characteristics of two types of anion-selective channels (type I and type II) were investigated. In asymmetric BaCl₂ buffers (cis, 100mm/trans, 25mm), single channel conductances at –40 mV were 70 pS (type I) and 120 pS (type II). Permeability ratios (P _Na:P _Ba:P _Cl) calculated from the Goldman-Hodgkin-Katz current equation for type I and type II channels were 0.230.041 and 0.050.031, respectively. Both channels exhibited characteristic voltage-dependent bursting activities. Open probability for type I channels had a maximum of 0.7 at about 0 mV and decreased to zero at greater transmembrane potentials of either polarity. Type II channels were relatively voltage independent at negative voltages and were inactivated at highly positive voltages. Type I channels showed spontaneous irreversible inactivation often preceded by sudden transition to subconducting states. DIDS blocked type I channels only from thecis side, while it blocked type II channels from either side.     

Protein transport to the epididymal lumen

Summary Experiments were performed to clarify the debate over the entry of circulating proteins into the epididymal lumen by use of the marker horseradish peroxidase (HRP). Epididymal tubules from the caput epididymidis of the rat were immersed in medium TC 199 containing HRP (3.5 mg/ ml) for 5 min to 3 h at 33° C. Sections were examined for the presence of tracer within the epithelial cells by electron microscopy. From 5 min to 3 h, vesicles containing peroxidase reaction products were found throughout the cytoplasm of the principal cells. Vesicles occurred close to both the basal and apical membranes, and many were found opening into the interstitial space and lumen, depending on the length of incubation. By 5 min labelled vesicles were infrequently found in the apical part of the cells. Reaction product was observed in the epididymal lumen adhering to the microvilli from 30 min of incubation onwards. At all periods of incubation peroxidase was present at the base of the epithelium and between the cells, but it was never found within the tight junctional complexes, and no reaction deposits were found within epithelial cells of tubules incubated in the absence of peroxidase. It is concluded that large molecules leaving the capillaries may enter the epididymal lumen in the caput by means of fluid-phase endocytosis.     

Anion transport in the colon

The principal anions transported by colonic epithelium are Cl-, HCO3- and organic anions (OA-), particularly acetate, butyrate and pyruvate, these last being formed by microbial degradation of carbohydrate. In the normal absorptive rat colon, Cl- is transported from lumen to plasma both by the transcellular and paracellular pathways. The transcellular route appears to depend on amiloride-insensitive coupling of Na+-Cl- at the mucosal (apical) membrane, the Na+ electrochemical gradient energizing Cl- uptake. Intraluminal [HCO3-] rises as Cl- as absorbed, and a mucosal Cl- -HCO3- exchange carrier has been postulated. In some species (and in distal colon of the rat when sodium-depleted), the putative Na+-Cl- carrier is absent so that Cl- absorption then depends largely on the paracellular electrochemical gradient. Absorption of OA- is independent of the transepithelial p.d., is associated with HCO3- secretion and is considerably reduced by acetazolamide. In the absence of Cl-, OA- supports Na+ absorption but does not depend on it continuing unchanged when the latter is blocked. Colonic epithelium can become secretory and an example of this state is congenital chloridorrhoea in which an elevated transepithelial p.d. is associated with excessive Cl- secretion. Here, it appears that the Na+-Cl- and Cl- -HCO3- carriers are lost and Cl- conductance of the mucosal membrane substantially increased. The transepithelial uphill movements of Cl- or HCO3- in the absorptive and secretory colon appear to depend on coupling to other ionic flows, and there seems to be no need to postulate active transport of these ions.     

Mechanism of phosphorylation in the lumen of the Golgi apparatus. Translocation of adenosine 5'-triphosphate into Golgi vesicles from rat liver and mammary gland

The occurrence of phosphorylated secretory proteins such as caseins and vitellogenin and the recent characterization of phosphorylated proteoglycans, in the xylose and protein core, has raised the question of where in the cell and how this phosphorylation occurs. Previous studies have described a casein kinase activity in the lumen of the Golgi apparatus and this organelle as the site of xylose addition to the protein core of proteoglycans. We now report the translocation in vitro of ATP into the lumen of rat liver and mammary gland Golgi vesicles which are sealed and have the same membrane topographical orientation as in vivo. The entire ATP molecule was translocated into the lumen of the Golgi vesicles; this was established by using ATP radiolabeled with tritium in the adenine and gamma-32P. Translocation was temperature dependent and saturable, with an apparent Km of 0.9 microM and Vmax of 58 pmol/mg protein/min. Preliminary evidence suggests that translocation of ATP into the vesicles' lumen is coupled to exit of AMP from the lumen. Following translocation of ATP into the lumen of the vesicles, proteins were phosphorylated.     

WHAMMing into the Golgi

A new paper from Campellone et al. in a recent issue of Cell identifies WHAMM, a multifunctional protein that stimulates Arp2/3-mediated actin polymerization, binds and organizes microtubules, and influences the structure and efficiency of the Golgi complex. WHAMM's membrane localization at the entry face of the Golgi complex is novel for an actin nucleation-promoting factor, and highlights the importance of the cytoskeleton in organizing the secretory pathway.     

Anion channels in plant cells

Plant anion channels allow the efflux of anions from cells. They are involved in turgor pressure control, changes in membrane potential, organic acid excretion, tolerance to salinity and inorganic anion nutrition. The recent molecular identification of anion channel genes in guard cells and in roots allows a better understanding of their function and of the mechanisms that control their activation.     

Characteristic transport of lactoferrin from the intestinal lumen into the bile via the blood in piglets

Lactoferrin is a major iron-binding protein in milk from several species, such as humans, monkeys, mice and sows. Using neonatal and weaner piglets, the characteristic transfer of lactoferrin from intestinal lumen into bile via the circulation was investigated. Bovine lactoferrin (1 or 3 g/kg body weight) was infused into the stomach through a polyethylene tube or into the duodenum through a duodenal catheter over 5 min. Peripheral blood and bile samples were collected after the infusion. Lactoferrin absorbed into plasma and bile were assayed quantitatively by double-antibody enzyme-linked immunosorbent assay, and homogeneity of bovine lactoferrin in plasma and bile was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting methods. Morphological investigation was carried out according to the peroxidase anti-peroxidase method. Following oral administration in neonatal pigs, bovine lactoferrin appeared in the blood circulation and reached a peak level after 2 h. It was confirmed immunohistochemically that lactoferrin was transported by endocytosis via the epithelial cells. Lactoferrin absorbed into the blood was also detected in the bile and reached a peak value 12 h after oral administration. Transportation of lactoferrin from the intestinal lumen into the bile via the bloodstream was also observed in weaner piglets. Lactoferrin transported into plasma and bile was confirmed to be the same substance as administrated lactoferrin by electrophoresis and immunoblotting methods. Lactoferrin transported into bile was re-absorbed into the blood in neonatal pigs. These results demonstrate that lactoferrin contained in milk is transported into the circulation from the intestinal lumen and excreted into the bile, suggesting the possibility of entero-hepatic circulation of lactoferrin in neonatal pigs.     

Retrograde vesicle transport in the Golgi

The Golgi apparatus is the central sorting and biosynthesis hub of the secretory pathway, and uses vesicle transport for the recycling of its resident enzymes. This system must operate with high fidelity and efficiency for the correct modification of secretory glycoconjugates. In this review, we discuss recent advances on how coats, tethers, Rabs and SNAREs cooperate at the Golgi to achieve vesicle transport. We cover the well understood vesicle formation process orchestrated by the COPI coat, and the comprehensively documented fusion process governed by a set of Golgi localised SNAREs. Much less clear are the steps in-between formation and fusion of vesicles, and we therefore provide a much needed update of the latest findings about vesicle tethering. The interplay between Rab GTPases, golgin family coiled-coil tethers and the conserved oligomeric Golgi (COG) complex at the Golgi are thoroughly evaluated.     

Inhibition of CMP-sialic acid transport into Golgi vesicles by nucleoside monophosphates.

We examined the interactions of nucleotides with the CMP-sialic acid transporter in order to better understand which features play a role in binding and to investigate the relationship between binding and subsequent transport. With respect to the sugar, the transporter requires a complete ribose ring for tight binding, and the 2'-ara hydrogen makes an important contact. The enzyme exhibits little specificity with respect to the 2'- and 3'-hydroxyls, as it tolerated substitutions ranging from fluorine to an azido group. In the base, the C4 amine and C2 carbonyl groups make important contacts, while the N3 nitrogen does not. However, adding a methyl group to N3 dramatically reduced binding, indicating that mass at this position sterically hinders binding. Adding a group at C5 had either no effect or slightly enhanced binding. To determine if the transporter recognizes these CMP analogues as substrates, we assayed them for their ability to trans stimulate CMP-sialic acid import. These data suggest that the enzyme transports a wide variety of NMPs, and the rate of transport is inversely proportional to the K(I) of the analogue. The importance of our findings for understanding the specificities of the different nucleotide-sugar tranlocators and the design of novel glycosylation inhibitors are discussed.     

Anion transport and membrane morphology

Summary Freeze-fracture electronmicroscopy has been used to examine the membrane ultrastructure of human red blood cells in the presence of inhibitors of chloride exchange. The extent of inhibition was correlated with a decrease of intramembrane particle density on the B-fracture face. Dimethylsulfoxide (DMSO) and glycerol, which markedly and reversibly reduced the intramembrane particle density, were shown to drastically and reversibly inhibit chloride self-exchange. DMSO was shown to be a noncompetitive inhibitor of chloride flux.     

Cell-to-cell transport via the lumen of the endoplasmic reticulum

Plasmodesmata are plasma membrane‐lined channels through which cytoplasmic molecules move from cell‐to‐cell in plants. Most plasmodesmata contain a desmotubule, a central tube of endoplasmic reticulum (ER), that connects the ER of adjacent cells. Here we demonstrate that molecules of up to 10.4 kDa in size can move between the ER lumen of neighbouring leaf trichome or epidermal cells via the desmotubule lumen. Fluorescent molecules of up to 10 kDa, microinjected into the ER of Nicotiana trichome cells, consistently moved into the ER and nuclei of neighbouring trichome cells. This movement occurred more rapidly than movement via the cytoplasmic pathway. A fluorescent 3‐kDa dextran microinjected into the ER of a basal trichome cell moved into the ER and nuclei of epidermal cells across a barrier to cytoplasmic movement. We constructed a 10.4‐kDa recombinant ER‐lumenal reporter protein (LRP) from a fragment of the endogenous ER‐lumenal binding protein AtBIP1. Following transient expression of the LRP in the ER of Tradescantia leaf epidermal cells, it often moved into the nuclear envelopes of neighbouring cells. However, green fluorescent protein targeted to the ER lumen (ER‐GFP) did not move from cell to cell. We propose that the ER lumen of plant cells is continuous with that of their neighbours, and allows movement of small ER‐lumenal molecules between cells.     

The UDPase activity of the Kluyveromyces lactis Golgi GDPase has a role in uridine nucleotide sugar transport into Golgi vesicles.

In Saccharomyces cerevisiae a Golgi lumenal GDPase (ScGda1p) generates GMP, the antiporter required for entry of GDP-mannose, from the cytosol, into the Golgi lumen. Scgda1 deletion strains have severe defects in N- and O-mannosylation of proteins and glycosphingolipids. ScGda1p has also significant UDPase activity even though S. cerevisiae does not utilize uridine nucleotide sugars in its Golgi lumen. Kluyveromyces lactis, a species closely related to S. cerevisiae, transports UDP-N-acetylglucosamine into its Golgi lumen, where it is the sugar donor for terminal N-acetylglucosamine of the mannan chains. We have identified and cloned a K. lactis orthologue of ScGda1p. KlGda1p is 65% identical to ScGda1p and shares four apyrase conserved regions with other nucleoside diphosphatases. KlGda1p has UDPase activity as ScGda1p. Transport of both GDP-mannose, and UDP-GlcNAc was decreased into Golgi vesicles from Klgda1 null mutants, demonstrating that KlGda1p generates both GMP and UMP required as antiporters for guanosine and uridine nucleotide sugar transport into the Golgi lumen. Membranes from Klgda1 null mutants showed inhibition of glycosyltransferases utilizing uridine- and guanosine-nucleotide sugars, presumably due to accumulation of nucleoside diphosphates because the inhibition could be relieved by addition of apyrase to the incubations. KlGDA1 and ScGDA1 restore the wild-type phenotype of the other yeast gda1 deletion mutant. Surprisingly, KlGDA1 has only a role in O-glycosylation in K. lactis but also complements N-glycosylation defects in S. cerevisiae. Deletion mutants of both genes have altered cell wall stability and composition, demonstrating a broader role for the above enzymes.     

Non-mitochondrial ATP transport

Exchange of organelle ATP with cytosolic ADP through the ADP/ATP carrier is a well-characterized feature of mitochondrial metabolism. Obligate intracellular bacteria, such as Rickettsia prowazekii, and higher-plant plastids possess another type of adenylate transporter, which exchanges bacterial or plastidic ADP for ATP from the eukaryotic (host cell) cytoplasm. The bacterial and plastidic transporters are similar but do not share significant sequence similarities with the mitochondrial carrier. Recent molecular and biochemical studies are providing deeper insight into the functional and evolutionary relationships between the bacterial and the plant transport proteins.     

Anion channels in the sea urchin sperm plasma membrane

Ionic fluxes in sea urchin sperm plasma membrane regulate cell motility and the acrosome reaction (AR). Although cationic channels mediate some of the ionic movements, little is known about anion channels in these cells. The fusion of sperm plasma membranes into lipid bilayers allowed identification of a 150 pS anion channel. This anion channel was enriched from detergent-solubilized sperm plasma membranes using a wheat germ agglutinin Sepharose column. Vesicles formed from this preparation were fused into black lipid membranes (BLM), yielding single channel anion-selective activity with the properties of those found in the sperm membranes. The following anion selectivity sequence was found: NO₃^? > CNS^? > Br^? > CI^?. This anion channel has a high open probability at the holding potentials tested, it is partially blocked by 4,4′-diisothiocyano-2,2′ -stilbendisulfonic acid (DIDS), and it often displays substates. The sperm AR was also inhibited by DIDS. © 1993 Wiley-Liss, Inc.     

ATP release through pannexon channels

Extracellular adenosine triphosphate (ATP) serves as a signal for diverse physiological functions, including spread of calcium waves between astrocytes, control of vascular oxygen supply and control of ciliary beat in the airways. ATP can be released from cells by various mechanisms. This review focuses on channel-mediated ATP release and its main enabler, Pannexin1 (Panx1). Six subunits of Panx1 form a plasma membrane channel termed ‘pannexon’. Depending on the mode of stimulation, the pannexon has large conductance (500 pS) and unselective permeability to molecules less than 1.5 kD or is a small (50 pS), chloride-selective channel. Most physiological and pathological stimuli induce the large channel conformation, whereas the small conformation so far has only been observed with exclusive voltage activation of the channel. The interaction between pannexons and ATP is intimate. The pannexon is not only the conduit for ATP, permitting ATP efflux from cells down its concentration gradient, but the pannexon is also modulated by ATP. The channel can be activated by ATP through both ionotropic P2X as well as metabotropic P2Y purinergic receptors. In the absence of a control mechanism, this positive feedback loop would lead to cell death owing to the linkage of purinergic receptors with apoptotic processes. A control mechanism preventing excessive activation of the purinergic receptors is provided by ATP binding (with low affinity) to the Panx1 protein and gating the channel shut.     

ATP release via anion channels

ATP serves not only as an energy source for all cell types but as an ‘extracellular messenger’ for autocrine and paracrine signalling. It is released from the cell via several different purinergic signal efflux pathways. ATP and its Mg²⁺ and/or H⁺ salts exist in anionic forms at physiological pH and may exit cells via some anion channel if the pore physically permits this. In this review we survey experimental data providing evidence for and against the release of ATP through anion channels. CFTR has long been considered a probable pathway for ATP release in airway epithelium and other types of cells expressing this protein, although non-CFTR ATP currents have also been observed. Volume-sensitive outwardly rectifying (VSOR) chloride channels are found in virtually all cell types and can physically accommodate or even permeate ATP⁴⁻ in certain experimental conditions. However, pharmacological studies are controversial and argue against the actual involvement of the VSOR channel in significant release of ATP. A large-conductance anion channel whose open probability exhibits a bell-shaped voltage dependence is also ubiquitously expressed and represents a putative pathway for ATP release. This channel, called a maxi-anion channel, has a wide nanoscopic pore suitable for nucleotide transport and possesses an ATP-binding site in the middle of the pore lumen to facilitate the passage of the nucleotide. The maxi-anion channel conducts ATP and displays a pharmacological profile similar to that of ATP release in response to osmotic, ischemic, hypoxic and salt stresses. The relation of some other channels and transporters to the regulated release of ATP is also discussed.     

Anion permeation in human ClC-4 channels

ClC-4 and ClC-5 are mammalian ClC isoforms with unique ion conduction and gating properties. Macroscopic current recordings in heterologous expression systems revealed very small currents at negative potentials, whereas a substantially larger instantaneous current amplitude and a subsequent activation were observed upon depolarization. Neither the functional basis nor the physiological impact of these channel features are currently understood. Here, we used whole-cell recordings to study pore properties of human ClC-4 channels heterologously expressed in tsA201 or HEK293 cells. Variance analysis demonstrated that the prominent rectification of the instantaneous macroscopic current amplitude is due to a voltage-dependent unitary current conductance. The single channel amplitudes are very small, i.e., 0.10 +/- 0.02 pA at +140 mV for external Cl(-) and internal I(-). Conductivity and permeability sequences were determined for various external and internal anions, and both values increase for anions with lower dehydration energies. ClC-4 exhibits pore properties that are distinct from other ClC isoforms. These differences can be explained by assuming differences in the size of the pore narrowing and the electrostatic potentials within the ion conduction pathways.