Regulation of PTEN in neurons by myosin-based transport mechanisms

Our understanding of the function and regulation of the PTEN phosphatase now extends far beyond its initial characterization as a tumor suppressor with an important role in nervous system development and function. A number of developmental neurological disorders, which harbor mutations in PTEN or the PI3K pathway, are associated with increases in neuronal cell size (i.e., specific forms of autism or tuberous sclerosis). In light of this, the discovery of a myosin-based transport mechanism offers a new outlook in exploring further the underlying mechanisms of cell size control and its consequences to nervous system function. Overall, dysregulation of the PTEN:MyosinV interaction may provide an additional signaling intersection which increases the growth potential of a number of cell types. Indeed, members of the MyosinV family are widely expressed in different combinations in tissues (Rodriguez and Cheney, 2002), and have been shown to be differentially regulated in various cancers.     

Physico-chemical mechanisms of organic acid transport in the apical membrane cells of the proximal kidney tubules in rats. I. Kinetic transport parameters and effect of the lipid phasic state on transport

The kinetics of the transport of 3H-para-aminohippuric acid (PAH) and the influence of the temperature on the initial rate of transport were studied on the vesicles of a purified fraction of the apical membrane isolated from cells of kidney proximal tubules. The PAH transport is accomplished owing to the facilitate diffusion mechanism. The apparent Michaelis constant at 36 degrees C was equal to 7.0 + 1.0 mM, the maximum rate was 15 nmol/min on 1 mg of protein, the inhibition constant for the PAH transport by probenecid being 0.5 mM. At 22 degrees C the apparent Michaelis constant was drastically increased. When the temperature dependence of the initial rate of PAH transport into vesicles was replotted in the form of the Arrhenius plot, there was a turning-point of the line at 28-30 degrees C. The same turning-point is shown on the Arrhenius plot for temperature dependence of alkaline phosphatase activity (a marker enzyme for the apical membrane). The electron paramagnetic resonance spectra analysis of 5-doxylstearate-labeled apical membrane preparation reveals a thermotropic transition near 21-29 degrees C. It is concluded that the function of the carrier and the activity of alkaline phosphatase depend on the phasic state of membrane lipids; the normal function of membrane proteins is possible under the liquid-crystalline state of the lipid bilayer.     

Ionic transport mechanisms underlying fluid secretion by the pancreas

The pancreas is a 'leaky' epithelium and secretes a juice in which sodium and potassium have concentrations similar to those of plasma. The characteristic features of the secretion are its isosmolality and its high bicarbonate concentration. It is the latter that has attracted considerable attention. Secretion in the isolated cat pancreas is directly proportional to the bicarbonate concentration in the nutrient fluid. The ability of the gland to secrete weak acids has led to the view that because of the very different chemical nature of the anions, it is most likely that it is a component common to all buffers, the proton, that is subject to active transport. This is supported by the decrease in pH and the increase in rho CO2 of the venous effluent when secretion occurs and the sensitivity of secretion to the pH of the nutritional extracellular fluid. It is proposed that the cellular mechanisms are as follows: CO2 diffuses into the cell and is hydrated to carbonic acid under the influence of carbonic anhydrase. The bicarbonate ion so formed diffused into the ductular lumen and the proton is transported backwards through the epithelium with a proton pump (Mg2+ -ATPase) provisionally located in the luminal membrane and a hydrogen-sodium exchange carrier located in the basolateral membrane. Energy for the latter process is derived from the sodium gradient between extracellular fluid and cell. This gradient is maintained by a (Na+ + K+)-ATPase also located in the basolateral membrane. Chloride appears to be transported partly through a chloride-bicarbonate exchange mechanism but largely passively together with a large sodium and potassium component through the paracellular pathway. Osmotic equilibrium is likely to occur in the small ductules.     

PfMDR1: mechanisms of transport modulation by functional polymorphisms

ATP-Binding Cassette (ABC) transporters are efflux pumps frequently associated with multidrug resistance in many biological systems, including malaria. Antimalarial drug-resistance involves an ABC transporter, PfMDR1, a homologue of P-glycoprotein in humans. Twenty years of research have shown that several single nucleotide polymorphisms in pfmdr1 modulate in vivo and/or in vitro drug susceptibility. The underlying physiological mechanism of the effect of these mutations remains unclear. Here we develop structural models for PfMDR1 in different predicted conformations, enabling the study of transporter motion. Such analysis of functional polymorphisms allows determination of their potential role in transport and resistance. The bacterial MsbA ABC pump is a PfMDR1 homologue. MsbA crystals in different conformations were used to create PfMDR1 models with Modeller software. Sequences were aligned with ClustalW and analysed by Ali2D revealing a high level of secondary structure conservation. To validate a potential drug binding pocket we performed antimalarial docking simulations. Using aminoquinoline as probe drugs in PfMDR1 mutated parasites we evaluated the physiology underlying the mechanisms of resistance mediated by PfMDR1 polymorphisms. We focused on the analysis of well known functional polymorphisms in PfMDR1 amino acid residues 86, 184, 1034, 1042 and 1246. Our structural analysis suggested the existence of two different biophysical mechanisms of PfMDR1 drug resistance modulation. Polymorphisms in residues 86/184/1246 act by internal allosteric modulation and residues 1034 and 1042 interact directly in a drug pocket. Parasites containing mutated PfMDR1 variants had a significant altered aminoquinoline susceptibility that appears to be dependent on the aminoquinoline lipophobicity characteristics as well as vacuolar efflux by PfCRT. We previously described the in vivo selection of PfMDR1 polymorphisms under antimalarial drug pressure. Now, together with recent PfMDR1 functional reports, we contribute to the understanding of the specific structural role of these polymorphisms in parasite antimalarial drug response.     

Chloride transport by lobster hepatopancreas is facilitated by several anion antiport mechanisms

Three anion antiporters have previously been demonstrated in lobster hepatopancreatic basolateral membrane vesicles (BLMV) to perform vital physiological functions in the crustacean. Cl(-) was shown to be transported by all three of the documented antiporters. The stilbene, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid, also known as SITS, strongly inhibited Cl(-)/SO(4)(2-), Cl(-)/oxalate(2-) and Cl(-)/HCO(3)(-) exchange. It was concluded that Cl(-) could be transported by different modes of the documented existing anion antiporters in the lobster hepatopancreatic BLMV.     

Sulfate transport mechanisms in epithelial systems

A novel invertebrate gastrointestinal transport mechanism has been shown to couple chloride-sulfate exchange in an electrogenic fashion. In the lobster, Homarus americanus, the hepatopancreas, or digestive gland, exists as an outpocketing of the digestive tract, representing a single cell layer separating the gut lumen and an open circulatory system composed of hemolymph. Investigations utilizing independently prepared brush border and basolateral membrane vesicles revealed discrete antiport systems which possess the capacity to bring about a transcellular secretion of sulfate. The luminal antiport system functions as a high-affinity, one-to-one chloride-sulfate exchanger that is stimulated by an increase in luminal hydrogen ion concentration. Such a system would take advantage of the high chloride concentration of ingested seawater as well as the high proton concentrations generated during digestion, which further suggests a potential regulation by resident sodium-proton exchangers. Exchange of one chloride for one divalent sulfate ion provides the driving force for electrogenic vectorial translocation. The basolateral antiport system was found to be electroneutral in nature, responsive to gradients of the dicarboxylic anion oxalate while lacking in proton stimulation. No evidence of sodium-sulfate co-transport, commonly reported for the brush border of vertebrate renal and intestinal epithelia, was observed in either membrane preparation. The two antiporters together can account for the low hemolymph to seawater sulfate levels previously described in decapod crustaceans. A secretory pathway for sulfate based upon electrogenic chloride-antiport may appear among invertebrates partly in response to digestion taking place in a seawater environment. J. Exp. Zool. 289:245-253, 2001.     

Physico-chemical characterization of proteins by capillary electrophoresis

The electrophoretic mobility of proteins was successfully determined by means of capillary electrophoresis (CE) with various background electrolytes (BGEs). The objective was focused on the variation in BGE physico-chemical composition and the consequential impact on the observed protein charge. Experimental and calculated mobilities, according to Henry's equation, versus ionic strength have been compared. For positively-charged lysozyme, a good agreement between observed and calculated mobilities was observed using triethanolamine chloride at pH 7.0 as the BGE. Mobility close to zero was shown using borate (pH 8.0) and phosphate (pH 7.0) at a low ionic strength of about 20 mmol l⁻¹, and as a consequence, specific adsorption of oxyanions was evidenced. Lysozyme retention in the case of reversed-phase high-performance liquid chromatography (RP-HPLC) was decreased by the presence of phosphate ions. CE and HPLC are complementary tools for characterizing the behaviour of lysozyme. On the other hand, the mobility of the negatively-charged α-lactalbumin remained constant as regards phosphate at pH 7.0 in the 20–200 mmol l⁻¹ range, contrary to the decrease that had been expected with the increasing ionic strength. β-Lactoglobulin exhibited increasingly lower mobilities than those expected of boric acid/borate at pH 7.0 and 8.0 (I=20 mmol l⁻¹).     

Nonparallel transport and mechanisms of secretion.

After many years of controversy, it is now clear that at least some cells and tissues that secrete more than one product can vary the composition of the secreted mixture as the result of the differential transport of various substances out of the cells that secrete them. In this article we discuss this phenomenon, non-parallel transport or secretion, and how it has and continues to inform us about how cells release the products they manufacture. We focus on expression of the phenomenon in the secretion of digestive enzymes by the exocrine pancreas, where it has been studied most extensively.     

Molecular mechanisms of phosphate transport in plants

Membrane-spanning transport proteins are responsible for the selective passage of most mineral nutrients and metabolites across cellular and intracellular membranes. This review's focus is on summarising the current state of research covering the molecular regulation and biochemical mechanisms involved in the transport of phosphorus, an often growth-limiting nutrient, in vascular plants. Physiological data illustrating the tight control of Pi homeostasis on the cellular as well as on the organism's level are discussed together with the recent results on molecular transport mechanisms.     

Calcium transport mechanisms of PC12 cells

Many studies of Ca2+ signaling use PC12 cells, yet the balance of Ca2+ clearance mechanisms in these cells is unknown. We used pharmacological inhibition of Ca2+ transporters to characterize Ca2+ clearance after depolarizations in both undifferentiated and nerve growth factor-differentiated PC12 cells. Sarco-endoplasmic reticulum Ca2+ ATPase (SERCA), plasma membrane Ca2+ ATPase (PMCA), and Na+/Ca2+ exchanger (NCX) account for almost all Ca2+ clearance in both cell states, with NCX and PMCA making the greatest contributions. Any contribution of mitochondrial uniporters is small. The ATP pool in differentiated cells was much more labile than that of undifferentiated cells in the presence of agents that dissipated mitochondrial proton gradients. Differentiated PC12 cells have a small component of Ca2+ clearance possessing pharmacological characteristics consistent with secretory pathway Ca2+ ATPase (SPCA), potentially residing on Golgi and/or secretory granules. Undifferentiated and differentiated cells are similar in overall Ca2+ transport and in the small transport due to SERCA, but they differ in the fraction of transport by PMCA and NCX. Transport in neurites of differentiated PC12 cells was qualitatively similar to that in the somata, except that the ER stores in neurites sometimes released Ca2+ instead of clearing it after depolarization. We formulated a mathematical model to simulate the observed Ca2+ clearance and to describe the differences between these undifferentiated and NGF-differentiated states quantitatively. The model required a value for the endogenous Ca2+ binding ratio of PC12 cell cytoplasm, which we measured to be 268 +/- 85. Our results indicate that Ca2+ transport in undifferentiated PC12 cells is quite unlike transport in adrenal chromaffin cells, for which they often are considered models. Transport in both cell states more closely resembles that of sympathetic neurons, for which differentiated PC12 cells often are considered models. Comparison with other cell types shows that different cells emphasize different Ca2+ transport mechanisms.     

Molecular mechanisms of urea transport in plants

Urea is a soil nitrogen form available to plant roots and a secondary nitrogen metabolite liberated in plant cells. Based on growth complementation of yeast mutants and “in-silico analysis”, two plant families have been identified and partially characterized that mediate membrane transport of urea in heterologous expression systems. AtDUR3 is a single Arabidopsis gene belonging to the sodium solute symporter family that cotransports urea with protons at high affinity, while members of the tonoplast intrinsic protein (TIP) subfamily of aquaporins transport urea in a channel-like manner. The following review summarizes current knowledge on the membrane localization, energetization and regulation of these two types of urea transporters and discusses their possible physiological roles in planta.     

Molecular mechanisms and regulation of ceramide transport

De novo biosynthesis of sphingolipids begins in the endoplasmic reticulum (ER) and continues in the Golgi apparatus and plasma membrane. A crucial step in sphingolipid biosynthesis is the transport of ceramide by vesicular and non-vesicular mechanisms from its site of synthesis in the ER to the Golgi apparatus. The recent discovery of the ceramide transport protein CERT has revealed a novel pathway for the delivery of ceramide to the Golgi apparatus for sphingomyelin (SM) synthesis. In addition to a ceramide-binding START domain, CERT has FFAT (referring to two phenylalanines [FF] in an acidic tract) and pleckstrin homology (PH) domains that recognize the ER integral membrane protein VAMP-associated protein (VAP) and Golgi-associated PtdIns 4-phosphate, respectively. Mechanisms for vectorial transport involving dual-organellar targeting and sites of deposition of ceramide in the Golgi apparatus are proposed. Similar Golgi-ER targeting motifs are also present in the oxysterol-binding protein (OSBP), which regulates ceramide transport and SM synthesis in an oxysterol-dependent manner. Consequently, this emerges as a potential mechanism for integration of sphingolipid and cholesterol metabolism. The identification of organellar targeting motifs in other related lipid-binding/transport proteins indicate that concepts learned from the study of ceramide transport can be applied to other lipid transport processes.     

Cellular mechanisms of potassium transport in plants

Potassium (K⁺) is the most abundant ion in the plant cell and is required for a wide array of functions, ranging from the maintenance of electrical potential gradients across cell membranes, to the generation of turgor, to the activation of numerous enzymes. The majority of these functions depend more or less directly upon the activities and regulation of membrane-bound K⁺ transport proteins, operating over a wide range of K⁺ concentrations. Here, we review the physiological aspects of potassium transport systems in the plasma membrane, re-examining fundamental problems in the field such as the distinctions between high- and low-affinity transport systems, the interactions between K⁺ and other ions such as NH₄⁺ and Na⁺, the regulation of cellular K⁺ pools, the generation of electrical potentials and the problems involved in measurement of unidirectional K⁺ fluxes. We place these discussions in the context of recent discoveries in the molecular biology of K⁺ acquisition and produce an overview of gene families encoding K⁺ transporters.