Showing papers in "The Journal of Membrane Biology in 2006"

Structure of Fully Hydrated Fluid Phase Lipid Bilayers with Monounsaturated Chains

Abstract: Quantitative structures are obtained at 30 degrees C for the fully hydrated fluid phases of palmitoyloleoylphosphatidylcholine (POPC), with a double bond on the sn-2 hydrocarbon chain, and for dierucoylphosphatidylcholine (di22:1PC), with a double bond on each hydrocarbon chain. The form factors F(qz) for both lipids are obtained using a combination of three methods. (1) Volumetric measurements provide F(0). (2) X-ray scattering from extruded unilamellar vesicles provides /F(qz)/ for low q(z). (3) Diffuse X-ray scattering from oriented stacks of bilayers provides /F(qz)/ for high q(z). Also, data using method (2) are added to our recent data for dioleoylphosphatidylcholine (DOPC) using methods (1) and (3); the new DOPC data agree very well with the recent data and with (4) our older data obtained using a liquid crystallographic X-ray method. We used hybrid electron density models to obtain structural results from these form factors. The result for area per lipid (A) for DOPC 72.4 +/- 0.5 A(2) agrees well with our earlier publications, and we find A = 69.3 +/- 0.5 A2 for di22:1PC and A = 68.3 +/- 1.5 A2 for POPC. We obtain the values for five different average thicknesses: hydrophobic, steric, head-head, phosphate-phosphate and Luzzati. Comparison of the results for these three lipids and for our recent dimyristoylphosphatidylcholine (DMPC) determination provides quantitative measures of the effect of unsaturation on bilayer structure. Our results suggest that lipids with one monounsaturated chain have quantitative bilayer structures closer to lipids with two monounsaturated chains than to lipids with two completely saturated chains.

Volume-sensitive chloride channels involved in apoptotic volume decrease and cell death.

Abstract: Apoptosis is an essential process in organ development, tissue homeostasis, somatic cell turnover, and the pathogenesis of degenerative diseases. Apoptotic cell death occurs in response to a variety of stimuli in physiological and pathological circumstances. Efflux of K(+) and Cl(-) leads to apoptotic volume decrease (AVD) of the cell. Both mitochondrion-mediated intrinsic, and death receptor-mediated extrinsic, apoptotic stimuli have been reported to rapidly activate Cl(-) conductances in a large variety of cell types. In epithelial cells and cardiomyocytes, the AVD-inducing anion channel was recently determined to be the volume-sensitive outwardly rectifying (VSOR) Cl(-) channel which is usually activated by swelling under non-apoptotic conditions. Blocking the VSOR Cl(-) channel prevented cell death in not only epithelial and cardiac cells, but also other cell types, by inhibiting the induction of AVD and subsequent apoptotic events. Ischemia-reperfusion-induced apoptotic death in cardiomyocytes and brain neurons was also prevented by Cl(-) channel blockers. Furthermore, cancer cell apoptosis induced by the anti-cancer drug cisplatin was recently found to be associated with augmented activity of the VSOR Cl(-) channel and to be inhibited by a Cl(-) channel blocker. The apoptosis-inducing VSOR Cl(-) channel is distinct from ClC-3 and its molecular identity remains to be determined.

Structure and function of cationic amino acid transporters (CATs).

Abstract: The CAT proteins (CAT for cationic amino acid transporter) are amongst the first mammalian amino acid transporters identified on the molecular level and seem to be the major entry path for cationic amino acids in most cells. However, CAT proteins mediate also efflux of their substrates and thus may also deplete cells from cationic amino acids under certain circumstances. The CAT proteins form a subfamily of the solute carrier family 7 (SLC7) that consists of four confirmed transport proteins for cationic amino acids: CAT-1 (SLC7A1), CAT-2A (SLC7A2A), CAT-2B (SLC7A2B), and CAT-3 (SLC7A3). SLC7A4 and SLC7A14 are two related proteins with yet unknown function. One focus of this review lies on structural and functional differences between the different CAT isoforms. The expression of the CAT proteins is highly regulated on the level of transcription, mRNA stability, translation and subcellular localization. Recent advances toward a better understanding of these mechanisms provide a second focus of this review.

Structure and function of the hair cell ribbon synapse.

Abstract: Faithful information transfer at the hair cell afferent synapse requires synaptic transmission to be both reliable and temporally precise. The release of neurotransmitter must exhibit both rapid on and off kinetics to accurately follow acoustic stimuli with a periodicity of 1 ms or less. To ensure such remarkable temporal fidelity, the cochlear hair cell afferent synapse undoubtedly relies on unique cellular and molecular specializations. While the electron microscopy hallmark of the hair cell afferent synapse--the electron-dense synaptic ribbon or synaptic body--has been recognized for decades, dissection of the synapse's molecular make-up has only just begun. Recent cell physiology studies have added important insights into the synaptic mechanisms underlying fidelity and reliability of sound coding. The presence of the synaptic ribbon links afferent synapses of cochlear and vestibular hair cells to photoreceptors and bipolar neurons of the retina. This review focuses on major advances in understanding the hair cell afferent synapse molecular anatomy and function that have been achieved during the past years.

Mechanisms of Acid and Base Secretion by the Airway Epithelium

Abstract: One of the main functions of the airway epithelium is to inactivate and remove infectious particles from inhaled air and thereby prevent infection of the distal lung This function is achieved by mucociliary and cough clearance and by antimicrobial factors present in the airway surface liquid (ASL) There are indications that airway defenses are affected by the pH of the ASL and historically, acidification of the airway surfaces has been suggested as a measure of airway disease However, even in health, the ASL is slightly acidic, and this acidity might be part of normal airway defense Only recently research has focused on the mechanisms responsible for acid and base secretion into the ASL Advances resulted from research into the airway disease associated with cystic fibrosis (CF) after it was found that the CFTR Cl(-) channel conducts HCO (3) (-) and, therefore, may contribute to ASL pH However, the acidity of the ASL indicated parallel mechanisms for H(+) secretion Recent investigations identified several H(+) transporters in the apical membrane of the airway epithelium These include H(+) channels and ATP-driven H(+) pumps, including a non-gastric isoform of the H(+)-K(+) ATPase and a vacuolar-type H(+) ATPase Current knowledge of acid and base transporters and their potential roles in airway mucosal pH regulation is reviewed here

The role of TRP channels in oxidative stress-induced cell death.

Abstract: The transient receptor potential (TRP) protein superfamily is a diverse group of voltage-independent calcium-permeable cation channels expressed in mammalian cells. These channels have been divided into six subfamilies, and two of them, TRPC and TRPM, have members that are widely expressed and activated by oxidative stress. TRPC3 and TRPC4 are activated by oxidants, which induce Na+ and Ca2+ entry into cells through mechanisms that are dependent on phospholipase C. TRPM2 is activated by oxidative stress or TNFα, and the mechanism involves production of ADP-ribose, which binds to an ADP-ribose binding cleft in the TRPM2 C-terminus. Treatment of HEK 293T cells expressing TRPM2 with H2O2 resulted in Ca2+ influx and increased susceptibility to cell death, whereas coexpression of the dominant negative isoform TRPM2-S suppressed H2O2-induced Ca2+ influx, the increase in [Ca2+]i, and onset of apoptosis. U937-ecoR monocytic cells expressing increased levels of TRPM2 also exhibited significantly increased [Ca2+]i and increased apoptosis after treatment with H2O2 or TNFα. A dramatic increase in caspase 8, 9, 3, 7, and PARP cleavage was observed in TRPM2-expressing cells, demonstrating a downstream mechanism through which cell death is mediated. Inhibition of endogenous TRPM2 function through three approaches, depletion of TRPM2 by RNA interference, blockade of the increase in [Ca2+]i through TRPM2 by calcium chelation, or expression of the dominant negative splice variant TRPM2-S protected cell viability. H2O2 and amyloid β-peptide also induced cell death in primary cultures of rat striatal cells, which endogenously express TRPM2. TRPM7 is activated by reactive oxygen species/nitrogen species, resulting in cation conductance and anoxic neuronal cell death, which is rescued by suppression of TRPM7 expression. TRPM2 and TRPM7 channels are physiologically important in oxidative stress-induced cell death.

Gap junctions and cochlear homeostasis.

Abstract: Gap junctions play a critical role in hearing and mutations in connexin genes cause a high incidence of human deafness. Pathogenesis mainly occurs in the cochlea, where gap junctions form extensive networks between non-sensory cells that can be divided into two independent gap junction systems, the epithelial cell gap junction system and the connective tissue cell gap junction system. At least four different connexins have been reported to be present in the mammalian inner ear, and gap junctions are thought to provide a route for recycling potassium ions that pass through the sensory cells during the mechanosensory transduction process back to the endolymph. Here we review the cochlear gap junction networks and their hypothesized role in potassium ion recycling mechanism, pharmacological and physiological gating of cochlear connexins, animal models harboring connexin mutations and functional studies of mutant channels that cause human deafness. These studies elucidate gap junction functions in the cochlea and also provide insight for understanding the pathogenesis of this common hereditary deafness induced by connexin mutations.

K + Channels in Apoptosis

Abstract: A proper rate of programmed cell death or apoptosis is required to maintain normal tissue homeostasis. In disease states such as cancer and some forms of hypertension, apoptosis is blocked, resulting in hyperplasia. In neurodegenerative diseases, uncontrolled apoptosis leads to loss of brain tissue. The flow of ions in and out of the cell and its intracellular organelles is becoming increasingly linked to the generation of many of these diseased states. This review focuses on the transport of K+ across the cell membrane and that of the mitochondria via integral K+-permeable channels. We describe the different types of K+ channels that have been identified, and investigate the roles they play in controlling the different phases of apoptosis: early cell shrinkage, cytochrome c release, caspase activation, and DNA fragmentation. Attention is also given to K+ channels on the inner mitochondrial membrane, whose activity may underlie anti- or pro-apoptotic mechanisms in neurons and cardiomyocytes.

Organic anion transporting polypeptides of the OATP/SLCO superfamily: identification of new members in nonmammalian species, comparative modeling and a potential transport mode.

Abstract: Organic anion-transporting polypeptides (human, OATPs; other animals, Oatps; gene symbol, SLCO/Slco) form a transport protein superfamily that mediates the translocation of amphipathic substrates across the plasma membrane of animal cells. So far, OATPs/Oatps have been identified in human, rat and mouse tissues. In this study, we used bioinformatic tools to detect new members of the OATP/SLCO superfamily in nonmammalian species and to build models for the three-dimensional structure of OATPs/Oatps. New OATP/SLCO superfamily members, some of which form distinct novel families, were identified in chicken, zebrafish, frog, fruit fly and worm species. The lack of OATP/SLCO superfamily members in plants, yeast and bacteria suggests the emergence of an ancient Oatp protein in an early ancestor of the animal kingdom. Structural models were generated for the representative members OATP1B3 and OATP2B1 based on the known structures of the major facilitator superfamily of transport proteins. A model was also built for the large extracellular region between transmembrane helices 9 and 10, following the identification of a novel homology with the Kazal-type serine protease inhibitors. Along with the electrostatic potential and the conservation of key amino acid residues, we propose a common transport mechanism for all OATPs/Oatps, whereby substrates are translocated through a central, positively charged pore in a rocker-switch type of mechanism. Several amino acid residues were identified that may play crucial roles in the proposed transport mechanism.

Protein secretion and membrane insertion systems in gram-negative bacteria

Abstract: In contrast to other organisms, gram-negative bacteria have evolved numerous systems for protein export. Eight types are known that mediate export across or insertion into the cytoplasmic membrane, while eight specifically mediate export across or insertion into the outer membrane. Three of the former secretory pathway (SP) systems, type I SP (ISP, ABC), IIISP (Fla/Path) and IVSP (Conj/Vir), can export proteins across both membranes in a single energy-coupled step. A fourth generalized mechanism for exporting proteins across the two-membrane envelope in two distinct steps (which we here refer to as type II secretory pathways [IISP]) utilizes either the general secretory pathway (GSP or Sec) or the twin-arginine targeting translocase for translocation across the inner membrane, and either the main terminal branch or one of several protein-specific export systems for translocation across the outer membrane. We here survey the various well-characterized protein translocation systems found in living organisms and then focus on the systems present in gram-negative bacteria. Comparisons between these systems suggest specific biogenic, mechanistic and evolutionary similarities as well as major differences.

COPI-mediated Transport

Abstract: COPI-coated vesicles are protein and liquid carriers that mediate transport within the early secretory pathway. In this Topical Review, we present their main protein components and discuss current models for cargo sorting. Finally, we describe the striking similarities that exist between the COPI system and the two other characterized types of vesicular carriers: COPII- and clathrin-coated vesicles.

Molecular Mechanisms of Urea Transport in Plants

Abstract: 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.

Ca2+ -permeable acid-sensing ion channels and ischemic brain injury.

Abstract: Acidosis is a common feature of brain in acute neurological injury, particularly in ischemia where low pH has been assumed to play an important role in the pathological process. However, the cellular and molecular mechanisms underlying acidosis-induced injury remain unclear. Recent studies have demonstrated that activation of Ca2+-permeable acid-sensing ion channels (ASIC1a) is largely responsible for acidosis-mediated, glutamate receptor-independent, neuronal injury. In cultured mouse cortical neurons, lowering extracellular pH to the level commonly seen in ischemic brain activates amiloride-sensitive ASIC currents. In the majority of these neurons, ASICs are permeable to Ca2+, and an activation of these channels induces increases in the concentration of intracellular Ca2+ ([Ca2+]i). Activation of ASICs with resultant [Ca2+]i loading induces time-dependent neuronal injury occurring in the presence of the blockers for voltage-gated Ca2+ channels and the glutamate receptors. This acid-induced injury is, however, inhibited by the blockers of ASICs, and by reducing [Ca2+]o. In focal ischemia, intracerebroventricular administration of ASIC1a blockers, or knockout of the ASIC1a gene protects brain from injury and does so more potently than glutamate antagonism. Furthermore, pharmacological blockade of ASICs has up to a 5 h therapeutic time window, far beyond that of glutamate antagonists. Thus, targeting the Ca2+-permeable acid-sensing ion channels may prove to be a novel neuroprotective strategy for stroke patients.

Membrane reserves and hypotonic cell swelling.

Abstract: To accommodate expanding volume (V) during hyposmotic swelling, animal cells change their shape and increase surface area (SA) by drawing extra membrane from surface and intracellular reserves. The relative contributions of these processes, sources and extent of membrane reserves are not well defined. In this study, the SA and V of single substrate-attached A549, 16HBE14o−, CHO and NIH 3T3 cells were evaluated by reconstructing cell three-dimensional topology based on conventional light microscopic images acquired simultaneously from two perpendicular directions. The size of SA reserves was determined by swelling cells in extreme 98% hypotonic (∼6 mOsm) solution until membrane rupture; all cell types examined demonstrated surprisingly large membrane reserves and could increase their SA 3.6 ± 0.2-fold and V 10.7 ± 1.5-fold. Blocking exocytosis (by N-ethylmaleimide or 10°C) reduced SA and V increases of A549 cells to 1.7 ± 0.3-fold and 4.4 ± 0.9-fold, respectively. Interestingly, blocking exocytosis did not affect SA and V changes during moderate swelling in 50% hypotonicity. Thus, mammalian cells accommodate moderate (<2-fold) V increases mainly by shape changes and by drawing membrane from preexisting surface reserves, while significant endomembrane insertion is observed only during extreme swelling. Large membrane reserves may provide a simple mechanism to maintain membrane tension below the lytic level during various cellular processes or acute mechanical perturbations and may explain the difficulty in activating mechanogated channels in mammalian cells.

Properties of shaker-type potassium channels in higher plants.

Abstract: Potassium (K+), the most abundant cation in biological organisms, plays a crucial role in the survival and development of plant cells, modulation of basic mechanisms such as enzyme activity, electrical membrane potentials, plant turgor and cellular homeostasis. Due to the absence of a Na+/K+ exchanger, which widely exists in animal cells, K+ channels and some type of K+ transporters function as K+ uptake systems in plants. Plant voltage-dependent K+ channels, which display striking topological and functional similarities with the voltage-dependent six-transmembrane segment animal Shaker-type K+ channels, have been found to play an important role in the plasma membrane of a variety of tissues and organs in higher plants. Outward-rectifying, inward-rectifying and weakly-rectifying K+ channels have been identified and play a crucial role in K+ homeostasis in plant cells. To adapt to the environmental conditions, plants must take advantage of the large variety of Shaker-type K+ channels naturally present in the plant kingdom. This review summarizes the extensive data on the structure, function, membrane topogenesis, heteromerization, expression, localization, physiological roles and modulation of Shaker-type K+ channels from various plant species. The accumulated results also help in understanding the similarities and differences in the properties of Shaker-type K+ channels in plants in comparison to those of Shaker channels in animals and bacteria.

Structure and Function of Sodium-coupled GABA and Glutamate Transporters

Abstract: Neurotransmitter transporters are key elements in the termination of the synaptic actions of the neurotransmitters. They use the energy stored in the electrochemical ion gradients across the plasma membrane of neurons and glial cells for uphill transport of the transmitters into the cells surrounding the synapse. Therefore specific transporter inhibitors can potentially be used as novel drugs for neurological disease. Sodium-coupled neurotransmitter transporters belong to either of two distinct families. The glutamate transporters belong to the SLC1 family, whereas the transporters of the other neurotransmitters belong to the SLC6 family. An exciting and recent development is the emergence of the first high-resolution structures of archeal and bacterial members belonging to these two families. In this review the functional results on prototypes of the two families, the GABA transporter GAT-1 and the glutamate transporters GLT-1 and EAAC1, are described and discussed within the perspective provided by the novel structures.

Potential roles of electrogenic ion transport and plasma membrane depolarization in apoptosis

Abstract: Apoptosis is characterized by the programmed activation of specific biochemical pathways leading to the organized demise of cells. To date, aspects of the intracellular signaling machinery involved in this phenomenon have been extensively dissected and characterized. However, recent studies have elucidated a novel role for changes in the intracellular milieu of the cells as important modulators of the cell death program. Specially, intracellular ionic homeostasis has been reported to be a determinant in both the activation and progression of the apoptotic cascade. Several apoptotic insults trigger specific changes in ionic gradients across the plasma membrane leading to depolarization of the plasma membrane potential (PMP). These changes lead to ionic imbalance early during apoptosis. Several studies have also suggested the activation and/or modulation of specific ionic transport mechanisms including ion channels, transporters and ATPases, as mediators of altered intracellular ionic homeostasis leading to PMP depolarization during apoptosis. However, the role of PMP depolarization and of the changes in ionic homeostasis during the progression of apoptosis are still unclear. This review summarizes the current knowledge regarding the causes and consequences of PMP depolarization during apoptosis. We also review the potential electrogenic ion transport mechanisms associated with this event, including the net influx/efflux of cations and anions. An understanding of these mechamisms could lead to the generation of new therapeutic approaches for a variety of diseases involving apoptosis.

Serotonin Transporters – Structure and Function

Abstract: Serotonin transporter (SERT) selectively transports 5-hydroxytryptamine (5-HT) into nerve cells together with Na and Cl and, in the same reaction, transports a K ion out of the cell. SERT is a member of the SLC6 gene family designated the neurotransmitter sodium symporter (NSS) family 2.A.22 by Saier (1999). Also in this family are many other transporters responsible for reuptake of small neurotransmitters, including glycine, c-aminobutyric acid (GABA), dopamine (DA), norepinephrine (NE) and 5-HT across the plasma membrane of neurons and glia. The neurotransmitter transporters are plasma membrane proteins that take up extracellular neurotransmitters after release and thereby terminate the transmitters action at extracellular receptor sites. They represent the first step in the process of transmitter recycling. These plasma membrane neurotransmitter transporters use transmembrane ion gradients of Na, Cl and K and an internal negative membrane potential for transport of their substrate neurotransmitters (Rudnick, 2002; Rudnick & Clark, 1993). SERT is of interest also as a drug target. A variety of compounds that are used to treat clinical depression, including fluoxetine (Prozac), sertraline (Zoloft), paroxetine (Paxil) and citalopram (Celexa), are inhibitors of SERT. In addition to drugs that specifically target SERT, this transporter is also affected by cocaine and amphetamines, psychostimulant drugs that are widely abused. Cocaine acts as a simple inhibitor of SERT and also of the closely related NSS transporters for NE and DA (NET and DAT, respectively) (Gu, Wall & Rudnick, 1994).

Hair Cells–Beyond the Transducer

Abstract: This review considers the “tween twixt and twain” of hair cell physiology, specifically the signaling elements and membrane conductances which underpin forward and reverse transduction at the input stage of hair cell function and neurotransmitter release at the output stage Other sections of this review series outline the advances which have been made in understanding the molecular physiology of mechanoelectrical transduction and outer hair cell electromotility Here we outline the contributions of a considerable array of ion channels and receptor signaling pathways that define the biophysical status of the sensory hair cells, contributing to hair cell development and subsequently defining the operational condition of the hair cells across the broad dynamic range of physiological function

Intracellular Monovalent Ions as Second Messengers

Abstract: It is generally accepted that electrochemical gradients of monovalent ions across the plasma membrane, created by the coupled function of pumps, carriers and channels, are involved in the maintenance of resting and action membrane potential, cell volume adjustment, intracellular Ca2+ handling and accumulation of glucose, amino acids, nucleotides and other precursors of macromolecular synthesis. In the present review, we summarize data showing that side-by-side with these classic functions, modulation of the intracellular concentration of monovalent ions in a physiologically reasonable range is sufficient to trigger numerous cellular responses, including changes in enzyme activity, gene expression, protein synthesis, cell proliferation and death. Importantly, the engagement of monovalent ions in regulation of the above-listed cellular responses occurs at steps upstream of Ca2+ i and other key intermediates of intracellular signaling, which allows them to be considered as second messengers. With the exception of HCO 3 − -sensitive soluble adenylyl cyclase, the molecular origin of sensors involved in the function of monovalent ions as second messengers remains unknown.

The Role of the Tight Junction in Paracellular Fluid Transport across Corneal Endothelium. Electro-osmosis as a Driving Force

Abstract: The mechanism of epithelial fluid transport is controversial and remains unsolved. Experimental difficulties pose obstacles for work on a complex phenomenon in delicate tissues. However, the corneal endothelium is a relatively simple system to which powerful experimental tools can be applied. In recent years our laboratory has developed experimental evidence and theoretical insights that illuminate the mechanism of fluid transport across this leaky epithelium. Our evidence points to fluid being transported via the paracellular route by a mechanism requiring junctional integrity, which we attribute to electro-osmotic coupling at the junctions. Fluid movements can be produced by electrical currents. The direction of the movement can be reversed by current reversal or by changing junctional electrical charges by polylysine. Aquaporin 1 (AQP1) is the only AQP present in these cells, and its deletion in AQP1 null mice significantly affects cell osmotic permeability but not fluid transport, which militates against the presence of sizable water movements across the cell. By contrast, AQP1 null mice cells have reduced regulatory volume decrease (only 60% of control), which suggests a possible involvement of AQP1 in either the function or the expression of volume-sensitive membrane channels/transporters. A mathematical model of corneal endothelium predicts experimental results only when based on paracellular electro-osmosis, and not when transcellular local osmosis is assumed instead. Our experimental findings in corneal endothelium have allowed us to develop a novel paradigm for this preparation that includes: (1) paracellular fluid flow; (2) a crucial role for the junctions; (3) hypotonicity of the primary secretion; (4) an AQP role in regulation and not as a significant water pathway. These elements are remarkably similar to those proposed by the Hill laboratory for leaky epithelia.

The physiology and evolution of urea transport in fishes

Abstract: This review summarizes what is currently known about urea transporters in fishes in the context of their physiology and evolution within the vertebrates. The existence of urea transporters has been investigated in red blood cells and hepatocytes of fish as well as in renal and branchial cells. Little is known about urea transport in red blood cells and hepatocytes, in fact, urea transporters are not believed to be present in the erythrocytes of elasmobranchs nor in teleost fish. What little physiological evidence there is for urea transport across fish hepatocytes is not supported by molecular evidence and could be explained by other transporters. In contrast, early findings on elasmobranch renal urea transporters were the impetus for research in other organisms. Urea transport in both the elasmobranch kidney and gill functions to retain urea within the animal against a massive concentration gradient with the environment. Information on branchial and renal urea transporters in teleost fish is recent in comparison but in teleosts urea transporters appear to function for excretion and not retention as in elasmobranchs. The presence of urea transporters in fish that produce a copious amount of urea, such as elasmobranchs and ureotelic teleosts, is reasonable. However, the existence of urea transporters in ammoniotelic fish is curious and could likely be due to their ability to manufacture urea early in life as a means to avoid ammonia toxicity. It is believed that the facilitated diffusion urea transporter (UT) gene family has undergone major evolutionary changes, likely in association with the role of urea transport in the evolution of terrestriality in the vertebrates.

Molecular Modeling of PepT1 — Towards a Structure

Abstract: The proton-coupled uptake of di- and tri-peptides is the major route of dietary nitrogen absorption in the intestine and of reabsorption of filtered protein in the kidney In addition, the transporters involved, PepT1 (SLC15a1) and PepT2 (SLC15a2), are responsible for the uptake and tissue distribution of a wide range of pharmaceutically important compounds, including β-lactam antibiotics, angiotensin-converting enzyme inhibitors, anti-cancer and anti-viral drugs PepT1 and PepT2 are large proteins, with over 700 amino acids, and to date there are no reports of their crystal structures, nor of those of related proteins from lower organisms Therefore there is virtually no information about the protein 3-D structure, although computer-based approaches have been used to both model the transmembrane domain (TM) layout and to produce a substrate binding template These models will be discussed, and a new one proposed from homology modeling rabbit PepT1 to the recently crystallized bacterial transporters LacY and GlpT Understanding the mechanism by which PepT1 and PepT2 bind and transport their substrates is of great interest to researchers, both in academia and in the pharmaceutical industries

Urea Transport in Bacteria: Acid Acclimation by Gastric Helicobacter spp

Abstract: Urea transporters in bacteria are relatively rare. There are three classes, the ABC transporters such as those expressed by cyanobacteria and Corynebacterium glutamicum, the Yut protein expressed by Yersinia spp and the UreI expressed by gastric Helicobacter spp. This review focuses largely on the UreI proton-gated channel that is part of the acid acclimation mechanism essential for gastric colonization by the latter. UreI is a six-transmembrane polytopic integral membrane protein, N and C termini periplasmic, and is expressed in all gastric Helicobacter spp that have been studied but also in Helicobacter hepaticus and Streptococcus salivarius. The first two are proton-gated, the latter is pH insensitive. Site-directed mutagenesis and chimeric constructs have identified histidines and dicarboxylic amino acids in the second periplasmic loop of H. pylori and the first loop of H. hepaticus UreI and the C terminus of both as involved in a hydrogen-bonding dependence of proton gating, with the membrane domain in these but not in the UreI of S. salivarius responding to the periplasmic conformational changes. UreI and urease are essential for gastric colonization and urease associates with UreI during acid exposure, facilitating activation of the UreA and UreB apoenzyme complex by Ni2+ insertion by the UreF-UreH and UreE-UreG assembly proteins. Transcriptome analysis of acid responses of H. pylori also identified a cytoplasmic and periplasmic carbonic anhydrase as responding specifically to changes in periplasmic pH and these have been shown to be essential also for acid acclimation. The finding also of upregulation of the two-component histidine kinase HP0165 and its response element HP0166, illustrates the complexity of the acid acclimation processes involved in gastric colonization by this pathogen.

Aquaporins and CFTR in ocular epithelial fluid transport.

Abstract: Aquaporins (AQPs) and the cystic fibrosis transmembrane conductance regulator (CFTR) provide the molecular routes for transport of water and chloride, respectively, through many epithelial tissues. In ocular epithelia, fluid transport generally involves secondary active chloride transport, which creates the osmotic gradient to drive transepithelial water transport. This review is focused on the role of AQPs and CFTR in water and ion transport across corneal/conjunctival epithelia, corneal endothelium, ciliary epithelium, and retinal pigment epithelium. The potential relevance of water and chloride transport to common disorders of ocular fluid balance is also considered. Recent data suggest AQPs and CFTR as attractive targets for drug development for therapy of keratoconjunctivitis sicca, recurrent corneal erosions, corneal edema, glaucoma, retinal detachment, and retinal ischemia.

Electrogenic Kinetics of a Mammalian Intestinal Type IIb Na + /P i Cotransporter

Abstract: The kinetics of a type IIb Na+-coupled inorganic phosphate (Pi) cotransporter (NaPi-IIb) cloned from mouse small intestine were studied using the two-electrode voltage clamp applied to Xenopus oocytes. In the steady state, mouse NaPi-IIb showed a curvilinear I-V relationship, with rate-limiting behavior only for depolarizing potentials. The Pi dose dependence was Michaelian, with an apparent affinity constant for Pi (\( {K_{\rm m}}^{\rm P_i} \)) of 10 ± 1 μM at −60 mV. Unlike for rat NaPi-IIa, \( {K_{\rm m}}^{\rm P_i} \) increased with membrane hyperpolarization, as reported for human NaPi-IIa, flounder NaPi-IIb and zebrafish NaPi-IIb2. The apparent affinity constant for Na+ (\( {K_{\rm m}}^{\rm Na} \)) was 23 ± 1 mM at −60 mV, and the Na+ activation was cooperative with a Hill coefficient of approximately 2. Pre-steady-state currents were documented in the absence of Pi and showed a strong dependence on external Na+. The hyperpolarizing shift of the charge distribution midpoint potential was 65 mV/log[Na]. Approximately half the moveable charge was attributable to the empty carrier. A comparison of the voltage dependence of steady-state Pi-induced current and pre-steady-state charge movement indicated that for −120 mV ≤ V ≤ 0 mV the voltage dependence of the empty carrier was the main determinant of the curvilinear steady-state cotransport characteristic. External protons partially inhibited NaPi-IIb steady-state activity, independent of the titration of mono- and divalent Pi, and immobilized pre-steady-state charge movements associated with the first Na+ binding step.

Control of Volume and Turgor in Stomatal Guard Cells

Abstract: Water loss from plants is determined by the aperture of stomatal pores in the leaf epidermis, set by the level of vacuolar accumulation of potassium salt, and hence volume and turgor, of a pair of guard cells. Regulation of ion fluxes across the tonoplast, the key to regulation of stomatal aperture, can only be studied by tracer flux measurements. There are two transport systems in the tonoplast. The first is a Ca(2+)-activated channel, inhibited by phenylarsine oxide (PAO), responsible for the release of vacuolar K(+)(Rb(+)) in response to the "drought" hormone, abscisic acid (ABA). This channel is sensitive to pressure, down-regulated at low turgor and up-regulated at high turgor, providing a system for turgor regulation. ABA induces a transient stimulation of vacuolar ion efflux, during which the flux tracks the ion content (volume, turgor), suggesting ABA reduces the set-point of a control system. The second system, which is PAO-insensitive, is responsible for an ion flux from vacuole to cytoplasm associated with inward water flow following a hypo-osmotic transfer. It is suggested that this involves an aquaporin as sensor, and perhaps also as responder; deformation of the aquaporin may render it ion-permeable, or, alternatively, the deformed aquaporin may signal to an associated ion channel, activating it. Treatment with inhibitors of aquaporins, HgCl(2) or silver sulfadiazine, produces a large transient increase in ion release from the vacuole, also PAO-insensitive. It is suggested that this involves the same aquaporin, either rendered directly ion-permeable, or signalling to activate an associated ion channel.

Tuning in to the amazing outer hair cell: membrane wizardry with a twist and shout.

Abstract: of 28rd Meeting of the Assoc. Res. Otolaryngol. New Orleans, LA Gulley, R.S., Reese, T.S. 1977. Regional specialization of the hair cell plasmalemma in the organ of Corti. Anat. Rec. 189:109–124 Hall, A. 1998. Rho GTPases and the actin cytoskeleton. Science 279:509–514 Hallworth, R. 1995. Passive compliance and active force generation in the guinea pig outer hair cell. J. Neurophysiol. 74:2319–2328 Hallworth, R. 1997. Modulation of outer hair cell compliance and force by agents that affect hearing. Hear. Res. 114:204–212 Hallworth, R., Evans, B.N., Dallos, P. 1993. The location and mechanism of electromotility in guinea pig outer hair cells. J. Neurophysiol. 70:549–558 Halter, J.A., Kruger, R.P., Yium, M.J., Brownell, W.E. 1997. The influence of the subsurface cisterna on the electrical properties of the outer hair cell. Neuroreport 8:2517–2521 Hammarlund, M., Davis, W.S., Jorgensen, E.M. 2000. Mutations in b-spectrin disrupt axon outgrowth and sarcomere structure. J. Cell Biol. 149:931–942 He, D.Z.Z. 1997. Relationship between the development of outer hair cell electromotility and efferent innervation: a study in cultured organ of Corti of neonatal gerbils. J. Neurosci. 17:3634–3643 He, D.Z., Dallos, P. 1999. Somatic stiffness of cochlear outer hair cells is voltage-dependent. Proc. Natl. Acad. Sci. USA 96:8223– 8228 He, D.Z., Dallos, P. 2000. Properties of voltage-dependent somatic stiffness of cochlear outer hair cells. J. Assoc. Res. Otolaryngol. 1:64–81 He, D.Z., Evans, B.N., Dallos, P. 1994. First appearance and development of electromotility in neonatal gerbil outer hair cells. Hear. Res. 78:77–90 He, D.Z., Jia, S., Dallos, P. 2003. Prestin and the dynamic stiffness of cochlear outer hair cells. J. Neurosci. 23:9089–9096 Holley MC.1996. Outer hair cell motility. In: The Cochlea. Dallos, P., Popper, A., Fay, R., editors pp. 386–434, Springer Verlag, New York Holley, M.C., Ashmore, J.F. 1988a. A cytoskeletal spring in cochlear outer hair cells. Nature 335:635–637 Holley, M.C., Ashmore, J.F. 1988b. On the mechanism of a high-frequency force generator in outer hair cells isolated from the guinea pig cochlea. Proc. R. Soc. Lond. B. 232:413– 429 Holley, M.C., Ashmore, J.F. 1990a. A cytoskeletal spring for the control of cell shape in outer hair cells isolated from the guinea pig cochlea. Eur. Arch. Otorhinolaryngol. 247:4–7 Holley, M.C., Ashmore, J.F. 1990b. Spectrin, actin and the structure of the cortical lattice in mammalian cochlear outer hair cells. J. Cell Sci. 96:283–291 Holley, M.C., Kalinec, F., Kachar, B. 1992. Structure of the cortical cytoskeleton in mammalian outer hair cells. J. Cell Sci. 102:569–580 Huang, G., Santos-Sacchi, J. 1993. Mapping the distribution of the outer hair cell motility voltage sensor by electrical amputation. Biophys. J. 65:2228–2236 Huang, G., Santo-Sacchi, J. 1994. Motility Voltage Sensor of the outer hair cell resides within the lateral plasma membrane. Proc. Natl. Acad. Sci. USA 91:12268–12272 Iida, K., Konno, K., Oshima, T., Tsumoto, K., Ikeda, K., Kumagai, I., Kobayashi, T., Wada, H. 2003. Stable expression of the motor protein prestin in Chinese hamster ovary cells. Jsme International Journal Series C-Mechanical Systems Machine Elements and Manufacturing 46:1266–1274 Iwasa, K.H. 1993. Effect of stress on the membrane capacitance of the auditory outer hair cell. Biophys. J. 65:492–498 Iwasa, K.H. 1994. A membrane motor model for the fast motility of the outer hair cell. J. Acoust. Soc. Am. 96:2216– 2224 Iwasa, K.H. 2000. Effect of membrane motor on the axial stiffness of the cochlear outer hair cell. J. Acoust Soc. Am. 107:2764–

BKCa channels activating at resting potential without calcium in LNCaP prostate cancer cells.

Abstract: Large-conductance Ca2+-dependent K+ (BK(Ca)) channels are activated by intracellular Ca2+ and membrane depolarization in an allosteric manner. We investigated the pharmacological and biophysical characteristics of a BK(Ca)-type K+ channel in androgen-dependent LNCaP (lymph node carcinoma of the prostate) cells with novel functional properties, here termed BK(L). K+ selectivity, high conductance, activation by Mg2+ or NS1619, and inhibition by paxilline and penitrem A largely resembled the properties of recombinant BK(Ca) channels. However, unlike conventional BK(Ca) channels, BK(L) channels activated in the absence of free cytosolic Ca2+ at physiological membrane potentials; the half-maximal activation voltage was shifted by about -100 mV compared with BK(Ca) channels. Half-maximal Ca2+-dependent activation was observed at 0.4 microM: for BK(L) (at -20 mV) and at 4.1 microM: for BK(Ca) channels (at +50 mV). Heterologous expression of hSlo1 in LNCaP cells increased the BK(L) conductance. Expression of hSlo-beta1 in LNCaP cells shifted voltage-dependent activation to values between that of BK(L) and BK(Ca) channels and reduced the slope of the P (open) (open probability)-voltage curve. We propose that LNCaP cells harbor a so far unknown type of BK(Ca) subunit, which is responsible for the BK(L) phenotype in a dominant manner. BK(L)-like channels are also expressed in the human breast cancer cell line T47D. In addition, functional expression of BK(L) in LNCaP cells is regulated by serum-derived factors, however not by androgens.

AQP and the Control of Fluid Transport in a Salivary Gland

Abstract: Experiments were performed with the perfused rat submandibular gland in vitro to investigate the nature of the coupling between transported salt and water by varying the osmolarity of the source bath and observing the changes in secretory volume flow. Glands were submitted to hypertonic step changes by changing the saline perfusate to one containing different levels of sucrose. The flow rate responded by falling to a lower value, establishing a new steady-state flow. The rate changes did not correspond to those expected from a system in which fluid production is due to simple osmotic equilibration, but were much larger. The changes were fitted to a model in which fluid production is largely paracellular, the rate of which is controlled by an osmosensor system in the basal membrane. The same experiments were done with glands from rats that had been bred to have very low levels of AQP5 (the principal aquaporin of the salivary acinar cell) in which little AQP5 is expressed at the basal membrane. In these rats, salivary secretion rates after hypertonic challenges were small and best modelled by simple osmotic equilibration. In rats which had intermediate AQP5 levels the changes in flow rate were similar to those of normal rats although their AQP5 levels were reduced.