Effects of spatial variation in membrane diffusibility and solubility on the lateral transport of membrane components.

There exist many examples of membrane components (e.g. receptors) accumulating in special domains of cell membranes. We analyze how certain variations in lateral diffusibility and solubility of the membrane would increase the efficiency of transport to these regions. A theorem is derived to show that the mean-time-of capture, tc, for particles diffusing to a trap from an annular region surrounding it, is intermediate to the tc values that correspond to the minimum and maximum diffusion coefficients that obtain in this region. An analytical solution for tc as a function of the gradient of diffusivity surrounding a trap is derived for circular geometry. Since local diffusion coefficients can be increased dramatically by reducing the concentration of intra-membrane particles and/or allowing them to form aggregates, such mechanisms could greatly enhance the diffusion-limited transport of particular membrane components to a trap (e.g. coated pit). If the trap is surrounded by an annular region in which the probe particles' partition function is increased, say, by the local segregation of certain phospholipids, tc is shown to vary inversely with the logarithm of the relative partition function. We provide some conjectural examples to illustrate the magnitude of the effects which heterogeneities in diffusibility and solubility may have in biological membranes.     

Modulation of lateral diffusion in the plasma membrane by protein density

The rate of lateral diffusion of proteins over micron-scale distances in the plasma membrane (PM) of mammalian cells is much slower than in artificial membranes [1, 2]. Different models have been advanced to account for this discrepancy. They invoke either effects on the apparent viscosity of cell membranes through, for example, protein crowding [3, 4], or a role for cortical factors such as actin or spectrin filaments [1]. Here, we use photobleaching to test specific predictions of these models [5]. Neither loss of detectable cortical actin nor knockdown of spectrin expression has any effect on diffusion. Disruption of the PM by formation of ventral membrane sheets or permeabilization induces aggregation of membrane proteins, with a concomitant increase in rates of diffusion for the nonaggregated fraction. In addition, procedures that directly increase or decrease the total protein content of the PM in live cells cause reciprocal changes in lateral diffusion rates. Our data imply that slow diffusion over micron-scale distances is an intrinsic property of the membrane itself and that the density of proteins within the membrane is a significant parameter in determining rates of lateral diffusion.     

Nucleotide binding by membrane components of bacterial periplasmic binding protein-dependent transport systems.

Bacterial periplasmic binding protein-dependent transport systems require the function of a specific substrate-binding protein, located in the periplasm, and several membrane-bound components. We present evidence for a nucleotide-binding site on one of the membrane components from each of three independent transport systems, the hisP, malK and oppD proteins of the histidine, maltose and oligopeptide permeases, respectively. The amino acid sequence of the oppD protein has been determined and this protein is shown to share extensive homology with the hisP and malK proteins. Three lines of evidence lead us to propose the existence of a nucleotide-binding site on each of these proteins. A consensus nucleotide-binding sequence can be identified in the same relative position in each of the three proteins. The oppD protein binds to a Cibacron Blue affinity column and can be eluted by ATP but not by CTP or NADH. The oppD protein is labelled specifically by the nucleotide affinity analogue 5'-p-fluorosulphonylbenzoyladenosine. The identification of a nucleotide-binding site provides strong evidence that transport by periplasmic binding protein-dependent systems is energized directly by the hydrolysis of ATP or a closely related nucleotide. The hisP, malK and oppD proteins are thus responsible for energy-coupling to their respective transport systems.     

Modulation of glycosylation and transport of viral membrane glycoproteins by a sodium ionophore

Analysis of viral glycoprotein expression on surfaces of monensin- treated cells using a fluorescence-activated cell sorter (FACS) demonstrated that the sodium ionophore completely inhibited the appearance of the vesicular stomatitis virus (VSV) G protein on (Madin- Darby canine kidney) MDCK cell surfaces. In contrast, the expression of the influenza virus hemagglutinin (HA) glycoprotein on the surfaces of MDCK cells was observed to occur at high levels, and the time course of its appearance was not altered by the ionophore. Viral protein synthesis was not inhibited by monensin in either VSV- or influenza virus-infected cells. However, the electrophoretic mobilities of viral glycoproteins were altered, and analysis of pronase-derived glycopeptides by gel filtration indicated that the addition of sialic acid residues to the VSV G protein was impaired in monensin-treated cells. Reduced incorporation of fucose and galactose into influenza virus HA was observed in the presence of the ionophore, but the incompletely processed HA protein was cleaved, transported to the cell surface, and incorporated into budding virus particles. In contrast to the differential effects of monensin on VSV and influenza virus replication previously observed in monolayer cultures of MDCK cells, yields of both viruses were found to be significantly reduced by high concentrations of monensin in suspension cultures, indicating that cellular architecture may play a role in determining the sensitivity of virus replication to the drug. Nigericin, an ionophore that facilitates transport of potassium ions across membranes, blocked the replication of both influenza virus and VSV in MDCK cell monolayers, indicating that the ion specificity of ionophores influences their effect on the replication of enveloped viruses.     

Modulation of membrane function by cholesterol.

The molecular basis for the essential role of cholesterol in mammalian (and other cholesterol-requiring) cells has long been the object of intense interest. Cholesterol has been found to modulate the function of membrane proteins critical to cellular function. Current literature supports two mechanisms for this modulation. In one mechanism, the requirement of 'free volume' by integral membrane proteins for conformational changes as part of their functional cycle is antagonized by the presence of high levels of cholesterol in the membrane. In the other mechanism, the sterol modulates membrane protein function through direct sterol-protein interactions. This mechanism provides an explanation for the stimulation of the activity of important membrane proteins and for the essential requirement of a structurally-specific sterol for cell viability. In some cases, these latter membrane proteins exhibit little or no activity in the absence of the specific sterol required for growth of that cell type. The specific sterol required varies from one cell type to another and is unrelated to the ability of that sterol to affect the bulk properties of the membrane.     

Modulation of P-glycoprotein-mediated drug transport by alterations in lipid fluidity of rat liver canalicular membrane vesicles.

P-glycoprotein (P-gp) is believed to function as an ATP-dependent efflux pump for natural product anti-cancer drugs in multidrug-resistant (MDR) tumor cells and in certain normal tissues. P-gp has been localized to the apical plasma membrane of the bile canaliculus where it has been shown to transport [3H]daunomycin. In this study, we investigated whether alterations in membrane lipid fluidity of canalicular membrane vesicles (CMV) could modulate the P-gp-mediated accumulation of [3H]daunomycin and [3H]vinblastine. Accumulation of both cytotoxic agents was stimulated by ATP, exhibited temperature dependence and osmotic sensitivity, and followed Michaelis-Menten kinetics. Alterations in CMV lipid fluidity were induced by the known fluidizers, 2-(2-methoxyethoxy)ethyl 8-(cis-2-n-octylcyclopropyl)octanoate (A2C) and benzyl alcohol, and were assessed by fluorescence polarization techniques using the fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene (DPH). Both A2C (2.5-5.0 microM) and benzyl alcohol (10-20 mM) produced a dose-dependent increase in CMV lipid fluidity. Moreover, both fluidizers, at the above doses, significantly inhibited (p < 0.05) the ATP-dependent accumulation of [3H]daunomycin. [3H]Vinblastine accumulation was also inhibited by A2C (p < 0.05). Lower doses of A2C (0.6 microM) and benzyl alcohol (1 mM) failed to influence either lipid fluidity or P-gp-mediated drug accumulation. Kinetic analysis revealed that A2C (5.0 microM) noncompetitively inhibited [3H]daunomycin accumulation and uncompetitively inhibited [3H]vinblastine accumulation with apparent Ki values of approximately 1.5 and approximately 1.2 microM, respectively. Verapamil competitively inhibited P-gp-mediated accumulation of [3H]daunomycin but failed to alter the fluidity of CMV. Taken together, the present results demonstrate that while increases in membrane fluidity of CMV are not necessarily required to inhibit P-gp-mediated drug accumulation, they can inhibit these processes, at least in CMV. Alterations in the physical state of CMV, therefore, appear to be at least one important modulator of P-gp function.     

Modulation of erythrocyte membrane proteins by membrane cholesterol and lipid fluidity.

Human erythrocyte membranes were enriched or depleted of cholesterol and effects on membrane proteins assessed with a membrane-impermeant sulfhydryl reagent, [35S]glutathione-maleimide. Reaction of the probe with intact cells quantifies exofacial sulfhydryl groups and reaction with leaky ghost membranes permits quantification of endofacial sulfhydryl groups. The mean endofacial sulfhydryl titer of cholesterol-enriched membranes exceeded that of cholesterol-depleted membrane by approximately 45 nmol/mg of protein or 64%. The corresponding exofacial titer of cholesterol-enriched cells was less than that of cholesterol-depleted cells by approximately 0.4 nmol/mg of protein, or 14%. Labeled membranes were examined by autoradiography of sodium dodecyl sulfate-polyacrylamide gel electropherograms to determine the labeling patterns of individual protein bands. Cholesterol enrichment enhanced the surface labeling of Coomassie brilliant blue stained bands 1,2,3, and 5, decreased the labeling of band 6, and did not change significantly that of band 4. The results demonstrate that changes in membrane cholesterol which influence lipid fluidity can alter the surface labeling of both intrinsic and extrinsic membrane proteins.     

ATP-dependent calcium transport in rat parotid basolateral membrane vesicles. Modulation by agents which elevate cyclic AMP

ATP-dependent Ca2+ transport was studied in basolateral membrane vesicles prepared from rat parotid gland slices incubated without or with agents which increase cyclic AMP. Isoproterenol (10(-5) M), forskolin (2 X 10(-6) M) and 8-bromocyclic AMP (2 X 10(-3) M) all increased ATP-dependent 45Ca2+ uptake 1.5- to 3-fold. The effect of isoproterenol was concentration-dependent and blocked by the beta-adrenergic antagonist propranolol. Enhanced uptake did not appear an artifact of vesicle preparation as apparent vesicle sidedness, 45Ca2+ efflux rates, specific activity of marker enzymes and equilibrium Ca2+ content were identical in vesicle preparations from control and 8-bromocyclic AMP-treated slices. Kinetic studies showed the ATP-dependent Ca2+ transport system in vesicles from 8-bromocyclic AMP-treated slices displayed a approximately 50% increase in Vmax and in Km Ca2+, compared to controls. The data suggest that physiological secretory stimuli to rat parotid acinar cells, which involve cyclic AMP, result in a readjustment of the basolateral membrane ATP-dependent Ca2+ pump.     

Modulation of membrane fusion by calcium-binding proteins.

The effects of several Ca2+-binding proteins (calmodulin, prothrombin, and synexin) on the kinetics of Ca2+-induced membrane fusion were examined. Membrane fusion was assayed by following the mixing of aqueous contents of phospholipid vesicles. Calmodulin inhibited slightly the fusion of phospholipid vesicles. Bovine prothrombin and its proteolytic fragment 1 had a strong inhibitory effect on fusion. Depending on the phospholipid composition, synexin could either facilitate or inhibit Ca2+-induced fusion of vesicles. The effects of synexin were Ca2+ specific. 10 microM Ca2+ was sufficient to induce fusion of vesicles composed of phosphatidic acid/phosphatidylethanolamine (1:3) in the presence of synexin and 1 mM Mg2+. We propose that synexin may be involved in intracellular membrane fusion events mediated by Ca2+, such as exocytosis, and discuss possible mechanisms facilitating fusion.     

Pathways for alanine transport in intestinal basal lateral membrane vesicles

Summary Membrane vesicles obtained from the basal lateral membranes of the rat intestinal epithelium were used to study the pathways for neutral amino acid transport.In the absence of sodium there was a stereospecific uptake ofl-alanine which exhibited saturation kinetics (K _m 0.73mm andV _max 5.3 nmol/mg min at 22°C). The activation energy for this process was 8.1 kcal/mole between 5 and 25°C. Preloading the vesicles with alanine increased the unidirectional influx of alanine into the vesicle. Competition experiments indicated that the affinity of the sodium-independent transport system was glutamine > threonine > alanine > phenylalanine > valine > methionine > glycine > histidine > proline, N-MeAIB. These are the characteristics of the classical L transport system.External sodium increased the rate of the stereospecificl-alanine uptake. The Na-dependent flux had aK _m of 0.04mm and aV _max of 0.26 nmol/mg min at 22°, and an activation energy of 9.1 kcal/mole between 5 and 25°C. Competition experiments suggest the existence of three separate pathways for alanine transport in the presence of sodium. A major pathway is shared by all other amino acids tested (i.e., threonine, glutamine, methionine, phenylalanine, valine, proline and N-MeAIB). This resembles the classical A system. A second pathway is unavailable to either phenylalanine or N-MeAIB; this is reminiscent of the classical ASC system; and the third is a novel pathway which is shared by N-MeAIB but not phenylalanine.The sodium-independent and the sodium-dependent transport ofl-alanine was blocked by PCMBS and significantly inhibited by DTP and NEM. It is concluded that the sodium-independent system (the L-like system) accounts for the efflux of neutral amino acids from the epithelium to the blood during the absorption of amino acids from the gut, and that the sodium-dependent transport processes may play an important role in the supply of amino acids to the epithelium in the absence of amino acids from the gut lumen.     

Transient alterations in the lateral mobility of erythrocyte membrane components during Sendai virus-mediated fusion.

Destabilization of the target membrane structure by fusion-promoting viral glycoproteins is assumed to be an essential part of the fusion mechanism. To explore this possibility, we employed fluorescence photobleaching recovery to investigate changes in the lateral mobility of native membrane constituents in human red blood cells (RBCs) during the course of Sendai virus-mediated fusion. The mobile fraction of RBC membrane proteins labeled with 5-(4,6-dichloro-5-triazin-2-yl)aminofluorescein increased significantly in the course of fusion, relaxing back to the original values upon completion of the fusion process. A different effect was observed on the lateral mobility of a fluorescent lipid probe, N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine, incorporated initially into the external monolayer. In this case, the lateral diffusion coefficient (rather than the mobile fraction) increased during fusion; this increase was permanent in the absence of Mg-ATP and transient in its presence. An active viral fusion protein was required to mediate the effects on both protein and lipid mobility. These effects, which take place on the same time scale as that of the fusion process, suggest that the organization of the RBC membrane is perturbed during fusion and that the observed changes may be related to the fusion mechanism.     

Modulation of brain mitochondrial membrane permeability and synaptosomal Ca2+ transport by dopamine oxidation

Effects of dopamine on the membrane permeability transition, thioredoxin reductase activity, production of free radicals and oxidation of sulfhydryl groups in brain mitochondria and the Ca²⁺ uptake by Na⁺-Ca²⁺ exchange and sulfhydryl oxidation in brain synaptosomes were examined. The brain mitochondrial swelling and the fall of transmembrane potential were altered by pretreatment of dopamine in a dose dependent manner. Depressive effect of dopamine on mitochondrial swelling was reversed by 10 g/ml catalase, and 10 mM DMSO. The activities of thioredoxin reductase in intact or disrupted mitochondria were decreased by dopamine (1-100 M), 25 M Zn²⁺ and 50 M Mn²⁺. Dopamine-inhibited enzyme activity was reversed by 10 g/ml SOD and 10 g/ml catalase. Pretreatment of dopamine decreased Ca²⁺ transport in synaptosomes, which was restored by 10 g/ml SOD and 10 mM DMSO. Dopamine (1-100 M) in the medium containing mitochondria produced superoxide anion and hydrogen peroxide, while its effect on nitrite production was very weak. The oxidation of sulfhydryl groups in mitochondria and synaptosomes were enhanced by dopamine with increasing incubation times. Results suggest that dopamine could modulate membrane permeability in mitochondria and calcium transport at nerve terminals, which may be ascribed to the action of free radicals and the loss of reduced sulfhydryl groups.     

Modulation of basal and peptide hormone-stimulated Na transport by membrane cholesterol content in the A6 epithelial cell line.

These studies examined the effect of altering plasma membrane cholesterol on basal Na+ flux as well as on the natriferic responses to the peptide hormones, insulin and anti-diuretic hormone (ADH) in the A6 model renal cell line. Membrane cholesterol concentrations were depleted or enriched using methyl-beta-cyclodextrin (MbetaCD) or a MbetaCD/cholesterol inclusion complex respectively. Effects of changes in the apical and basolateral plasma membranes were examined independently. Apical membrane cholesterol removal or supplementation had no effect on the basal Na+ transport rate. Short-term apical membrane cholesterol supplementation also had no effect on insulin-stimulated Na+ transport or on the initial phase of the ADH response. Interestingly, the additional apical membrane cholesterol had an inhibitory effect on the ADH response after 30 minutes. Apical membrane cholesterol depletion partially inhibited the responses to both insulin and ADH. Conversely, supplementation of basolateral cholesterol caused a significant increase in basal Na+ flux. Removal of cholesterol from the basolateral plasma membrane caused a decrease in basal Na+ flux with a time course analogous to channel turnover and completely inhibited peptide hormone responses. None of the changes in membrane cholesterol content decreased transcellular resistance. These results indicate an important role for membrane cholesterol content in the regulation of ENaC-mediated Na+ uptake.     

1,25-Dihydroxycholecalciferol-related Na+/D-glucose transport in brush-border membrane vesicles from embryonic chick jejunum. Modulation by triiodothyronine

1,25-Dihydroxycholecalciferol, when present at and above 10 nM in an organ-culture system of embryonic chick jejunum, approximately doubled the rate of Na(+)-gradient-driven D-glucose uptake by brush-border membrane vesicles, but had no effect on Na(+)-independent D-glucose transfer. The sterol also had no effect on Na+ influx along an outside/inside Na+ gradient ([Na+]o = 100 mM; [Na+]i = 0 mM). This renders it unlikely that in embryonic intestine, calcitriol raises Na(+)-dependent D-glucose transport through changes in the electrochemical Na+ gradient. D-[U-14C]Glucose tracer exchange, measured under voltage-clamp condition at Na+/D-glucose equilibrium, revealed that addition of calcitriol to the culture medium approximately doubled the activity of the Na+/D-glucose transporter in the brush-border membrane. This was also reflected by an corresponding increase in the maximal velocity of the transfer process. Increased [3H]phlorizin binding after calcitriol treatment suggests that the steroid hormone activates Na+/D-glucose transport through increasing the number of carrier molecules in the brush-border membrane. 10 nM triiodothyronine, which by itself has no effect on Na(+)-dependent D-glucose transport, potentiated the effect of 1,25-dihydroxycholecalciferol such that in the presence of both hormones, Na+/D-glucose-carrier activity was increased fourfold above control levels.     

Brownian dynamics simulation of the lateral distribution of charged membrane components

Brownian dynamics simulations were performed to study the contribution of electric interactions between charged membrane components to their lateral distribution in a two-dimensional viscous liquid (bilayer lipid membrane). The electrostatic interaction potential was derived from an analytical solution of the linearized Poisson-Boltzmann equation for point charges in an electrolyte solution — membrane — electrolyte solution system. Equilibrium as well as dynamic quantities were investigated. The lateral organization of membrane particles, modelled by mobile cylinders in a homogeneous membrane separating two electrolyte solutions was described by spatial distribution functions, diffusion coefficients and cluster statistics. Disorder, local order and crystal-like arrangements were observed as a function of the particle charge, the closest possible distances between the charges and the particle density. The simulations revealed that the system is very sensitive to the position of the charges with respect to the electrolyte solution — membrane interface. Electrostatic interactions of charges placed directly on the membrane surface were almost negligible, whereas deeper charges demonstrated pronounced interaction. Biologically relevant parameters corresponded at most to local and transient ordering. It was found that lateral electric forces can give rise to a preferred formation of clusters with an even number of constituents provided that the closest possible charge-charge distances are small. It is concluded that lateral electrostatic interactions can account for local particle aggregations, but their impact on the global arrangement and movement of membrane components is limited. Correspondence to: D. Walther     

Arabidopsis plasma membrane proteomics identifies components of transport, signal transduction and membrane trafficking

In order to identify integral proteins and peripheral proteins associated with the plasma membrane, highly purified Arabidopsis plasma membranes from green tissue (leaves and petioles) were analyzed by mass spectrometry. Plasma membranes were isolated by aqueous two-phase partitioning, which yields plasma membrane vesicles with a cytoplasmic-side-in orientation and with a purity of 95%. These vesicles were turned inside-out by treatment with Brij 58 to remove soluble contaminating proteins enclosed in the vesicles and to remove loosely bound contaminating proteins. In total, 238 putative plasma membrane proteins were identified, of which 114 are predicted to have transmembrane domains or to be glycosyl phosphatidylinositol anchored. About two-thirds of the identified integral proteins have not previously been shown to be plasma membrane proteins. Of the 238 identified proteins, 76% could be classified according to function. Major classes are proteins involved in transport (17%), signal transduction (16%), membrane trafficking (9%) and stress responses (9%). Almost a quarter of the proteins identified in the present study are functionally unclassified and more than half of these are predicted to be integral.     

Modulation of thymocyte membrane potential by concanavalin A.

Binding of convanavalin A, a potent mitogenic lectin, to thymocyte surface membrane causes depolarization of membrane potential. The effect is suppressed by alpha-methyl-D-glucoside, a haptenic inhibitor of this lectin, or by low temperature. Colchicine and cytochalasin B aslso suppress the change. These data indicate that perturbation of thymocyte surface membrane receptors induced by concanavalin A might be linked to change in the functional state of cellular cytoskeletal systems in turn causing depolarization of thymocyte surface membrane. The initial event generated by receptor-ligand interaction on the outer surface could be translated into cellular interior action only under highly fluid conditions of membrane lipid. Depolarization of thymocyte plasma membrane may be involved in the triggering mechanism of metabolic burst associated with blastoid transformation.     

Modulation of P-glycoprotein phosphorylation and drug transport by sodium butyrate.

P-Glycoprotein (Pgp) expression in cell lines derived from tumors arising from cells which normally express Pgp can be increased by sodium butyrate and other differentiating agents. Although the Pgp level increased 25-fold after sodium butyrate treatment in SW620 human colon carcinoma cells, the intracellular accumulation of vinblastine, adriamycin, and actinomycin D increased rather than decreased. In contrast, colchicine showed the expected decrease in accumulation, as a result of increased efflux. Likewise, treatment of a Pgp-expressing multidrug-resistant SW620 subline with sodium butyrate resulted in active interference with Pgp function. This effect was partially reversed by phorbol esters with a resulting decrease in the accumulation of vinblastine, adriamycin, and actinomycin D. Sodium butyrate, while increasing Pgp levels, inhibited the phosphorylation of Pgp. Time course studies revealed a tight relationship between decreased Pgp phosphorylation and increased vinblastine accumulation after sodium butyrate treatment. Either treatment with phorbol esters or withdrawal of sodium butyrate increased Pgp phosphorylation while decreasing vinblastine accumulation. These studies suggest that the specificity of Pgp transport can be modulated by phosphorylation and that vinblastine, adriamycin, or actinomycin D transport, but not that of colchicine, is diminished after dephosphorylation by sodium butyrate.     

Modulation of membrane dynamics and cell motility by membrane tension

The plasma membrane of most cells is drawn tightly over the cytoskeleton of the cell, resulting in a significant tension being developed in the membrane. The tension in the membrane can be calculated from the force required to separate it from the cytoskeleton; and the force itself can be measured rapidly by using laser tweezers. Recent observations indicate that decreasing membrane tension stimulates endocytosis and increasing tension stimulates secretion. Thus, membrane tension provides a simple physical mechanism to control the area of the plasma membrane. Here, we speculate that tension is a global parameter that the cell uses to control physically plasma membrane dynamics, cell shape and cell motility.