Proton-linked sugar transport systems in bacteria

The cell membranes of various bacteria contain proton-linked transport systems ford-xylose,l-arabinose,d-galactose,d-glucose,l-rhamnose,l-fucose, lactose, and melibiose. The melibiose transporter ofE. coli is linked to both Na⁺ and H⁺ translocation. The substrate and inhibitor specificities of the monosaccharide transporters are described. By locating, cloning, and sequencing the genes encoding the sugar/H⁺ transporters inE. coli, the primary sequences of the transport proteins have been deduced. Those for xylose/H⁺, arabinose/H⁺, and galactose/H⁺ transport are homologous to each other. Furthermore, they are just as similar to the primary sequences of the following: glucose transport proteins found in a Cyanobacterium, yeast, alga, rat, mouse, and man; proteins for transport of galactose, lactose, or maltose in species of yeast; and to a developmentally regulated protein of Leishmania for which a function is not yet established. Some of these proteins catalyze facilitated diffusion of the sugar without cation transport. From the alignments of the homologous amino acid sequences, predictions of common structural features can be made: there are likely to be twelve membrane-spanning -helices, possibly in two groups of six, there is a central hydrophilic region, probably comprised largely of -helix; the highly conserved amino acid residues (40–50 out of 472–522 total) form discrete patterns or motifs throughout the proteins that are presumably critical for substrate recognition and the molecular mechanism of transport. Some of these features are found also in other transport proteins for citrate, tetracycline, lactose, or melibiose, the primary sequences of which are not similar to each other or to the homologous series of transporters. The glucose/Na⁺ transporter of rabbit and man is different in primary sequence to all the other sugar transporters characterized, but it is homologous to the proline/Na⁺ transporter ofE. coli, and there is evidence for its structural similarity to glucose/H⁺ transporters in Plants.In vivo andin vitro mutagenesis of the lactose/H⁺ and melibiose/Na⁺ (H⁺) transporters ofE. coli has identified individual amino acid residues alterations of which affect sugar and/or cation recognition and parameters of transport. Most of the bacterial transport proteins have been identified and the lactose/H⁺ transporter has been purified. The directions of future investigations are discussed.     

Effects of membrane potential and surface potential on the kinetics of solute transport

A theoretical study has been made of the influence of the transmembrane potential difference and the surface potential of living cells on the kinetics of carried-mediated solute transport. It is assumed that the form of the free energy barrier within the membrane may be approximated by one dominant symmetrical peak, and that the electrical field is constant. Both single-ion transport kinetics and cotransport of an ion with a neutral solute are dealt with. Provided that the surface potential and the transmembrane potential are constant, the concentration dependence of the uptake rate is given by the Michaelis-Menten equation. The kinetic parameters, the maximal rate of uptake and the K_m, depend on both the surface potential and the membrane potential in a rather complex way. It is shown that the intuitive notion, that the maximal rate of cation uptake will increase when the cell membrane is hyperpolarized, is wrong in its generallity. Both an increase or a decrease may occur, depending on the characteristics of the transport system involved. If the magnitude of the membrane potential and the surface potential depends on the substrate concentration, marked deviations from Michaelis-Menten kinetics may come to the fore. This may result in either apparent positive or apparent negative homotrope cooperative effects. Enhancement of the uptake rate of the substrate ion may occur on adding another cation, despite the fact that the membrane will become depolarized. The same type of complex transport kinetics as found for Rb⁺ and Na⁺ uptake in yeast cells can be simulated by using a single-site transport model and including effects of the membrane potential.     

Proton-linked D-xylose transport in Escherichia coli.

The addition of xylose to energy-depleted cells of Escherichia coli elicited an alkaline pH change which failed to appear in the presence of uncoupling agents. Accumulation of [14C]xylose by energy-replete cells was also inhibited by uncoupling agents, but not by fluoride or arsenate. Subcellular vesicles of E. coli accumulated [14C]xylose provided that ascorbate plus phenazine methosulfate were present for respiration, and this accumulation was inhibited by uncoupling agents or valinomycin. Therefore, the transport of xylose into E. coli appears to be energized by a proton-motive force, rather than by a phosphotransferase or directly energized mechanism. Its specificity for xylose as inducer and substrate and the genetic location of a xylose-H+ transport-negative mutation near mtl showed that the xylose-H+ system is distinct from other proton-linked sugar transport systems of E. coli.     

Proton-linked L-fucose transport in Escherichia coli.

1. Addition of L-fucose to energy-depleted anaerobic suspensions of Escherichia coli elicited an uncoupler-sensitive alkaline pH change diagnostic of L-fucose/H+ symport activity. 2. L-Galactose or D-arabinose were also substrates, but not inducers, for the L-fucose/H+ symporter. 3. L-Fucose transport into subcellular vesicles was dependent upon respiration, displayed a pH optimum of about 5.5, and was inhibited by protonophores and ionophores. 4. These results showed that L-fucose transport into E. coli was energized by the transmembrane electrochemical gradient of protons. 5. Neither steady state kinetic measurements nor assays of L-fucose binding to periplasmic proteins revealed the existence of a second L-fucose transport system.     

Cevadine-induced changes of membrane potential and sodium transport of muscle membrane in a chloride-free solution

Cevadine-induced changes in membrane potential, sodium transport, intracellular Na, K, and water content were investigated in sartorius muscles incubated in chloride-free (glutamate) Ringer. Cevadine sensitivity of muscles incubated in glutamate Ringer was about five times greater than that of muscles incubated in normal Ringer. Therefore, even 0.005 mmol/l cevadine could induce depolarization and membrane potential oscillations. The membrane potential oscillations were recorded much longer from muscles incubated in chloride-free Ringer (even in the 15th hour of treatment) than in normal Ringer. Depolarization and membrane potential oscillations reversed more slowly in cevadine-free glutamate Ringer than in alkaloid-free normal Ringer. The rhythmic activity could be recorded even in the 10th-15th hour of incubation in cevadine-free glutamate Ringer. Cevadine increased the 24Na uptake of muscles incubated in glutamate Ringer by an average of 230%. In comparison, the cevadine-induced increase of 24Na uptake of muscles incubated in normal Ringer was approximately 350%. In the presence of cevadine the 24Na loss of muscles incubated either in glutamate or in normal Ringer increased to the same degree, i.e. three times. The increase of 24Na loss developed faster in glutamate Ringer than in the presence of chloride. The water content of muscles incubated in cevadine containing, chloride-free (glutamate) Ringer did not increase significantly. Muscles incubated in normal Ringer with cevadine showed a 42.7% increase of water content in 2 hours. Intracellular Na content and Na concentration increased by about 60% during a 2-hour-treatment with cevadine in a chloride-free environment. At the same time, cevadine treatment increased the intracellular Na content and Na concentration of muscles incubated in normal Ringer by about 160% and 80%, respectively. The cevadine-induced decrease of intracellular K content and concentration of muscles incubated in glutamate Ringer was 5% and 10%, respectively, in 2 hours. On the other hand, the decrease of intracellular K concentration in muscles incubated in cevadine-containing normal Ringer occasionally reached 30% due to the increase of water content of the muscles. The cevadine-induced increase of the wet weight of muscles incubated in normal Ringer was practically irreversible. It was not possible to eliminate the increase of wet weight even by washout lasting for 10-15 hours.     

Persistent directional cell migration requires ion transport proteins as direction sensors and membrane potential differences in order to maintain directedness

Background  

Ion transport proteins generate small electric fields that can induce directional cell motility; however, little is known about their mechanisms that lead to directedness. We investigated Na, K-ATPase (NaKA) and Na+/H+ exchanger isoforms (NHE1 and 3) in SaOS-2 and Calvarial osteoblasts, which present anode- and cathode- directed motility, during electrotaxis.     

Genetically encoded fluorescent sensors of membrane potential

Imaging activity of neurons in intact brain tissue was conceived several decades ago and, after many years of development, voltage-sensitive dyes now offer the highest spatial and temporal resolution for imaging neuronal functions in the living brain. Further progress in this field is expected from the emergent development of genetically encoded fluorescent sensors of membrane potential. These fluorescent protein (FP) voltage sensors overcome the drawbacks of organic voltage sensitive dyes such as non-specificity of cell staining and the low accessibility of the dye to some cell types. In a transgenic animal, a genetically encoded sensor could in principle be expressed specifically in any cell type and would have the advantage of staining only the cell population determined by the specificity of the promoter used to drive expression. Here we critically review the current status of these developments.     

Proton transport via the membrane surface

Some proton pumps, such as cytochrome c oxidase (C(c)O), translocate protons across biological membranes at a rate that considerably exceeds the rate of proton transport to the entrance of the proton-conducting channel via bulk diffusion. This effect is usually ascribed to a proton-collecting antenna surrounding the channel entrance. In this paper, we consider a realistic phenomenological model of such an antenna. In our model, a homogeneous membrane surface, which can mediate proton diffusion toward the channel entrance, is populated with protolytic groups that are in dynamic equilibrium with the solution. Equations that describe coupled surface-bulk proton diffusion are derived and analyzed. A general expression for the rate constant of proton transport via such a coupled surface-bulk diffusion mechanism is obtained. A rigorous criterion is formulated of when proton diffusion along the surface enhances the transport. The enhancement factor is found to depend on the ratio of the surface and bulk diffusional constants, pK(a) values of surface protolytic groups, and their concentration. A capture radius for a proton on the surface and an effective size of the antenna are found. The theory also predicts the effective distance that a proton can migrate on the membrane surface between a source (such as CcO) and a sink (such as ATP synthase) without fully equilibrating with the bulk. In pure aqueous solutions, protons can travel over long distances (microns). In buffered solutions, the travel distance is much shorter (nanometers); still the enhancement effect of the surface diffusion on the proton flow to a target on the surface can be tens to hundreds at physiological buffer concentrations. These results are discussed in a general context of chemiosmotic theory.     

Measurement of membrane potential in polymorphonuclear leukocytes and its changes during surface stimulation

The membrane potential of guinea pig polymorphonuclear leukocytes has been assessed with two indirect probes, tetraphenylphosphonium (TPP⁺) and 3,3′-dipropylthiadicarbocyanine (diS-C₃-(5)). The change in TPP⁺ concentration in the medium was measured with a TPP⁺-selective electrode. By monitoring differences in accumulation of TPP⁺ in media containing low and high potassium concentrations, a resting potential of −58.3 mV was calculated. This potential is composed of a diffusion potential due to the gradient of potassium, established by the Na⁺, K⁺ pump, and an electrogenic potential. The chemotactic peptide fMet-Leu-Phe elicits a rapid efflux of accumulated TPP⁺ (indicative of depolarization) followed by its reaccumulation (indicative of repolarization). In contrast, stimulation with concanavalin A results in a rapid and sustained depolarization without a subsequent repolarization. The results obtained with TPP⁺ and diS-C₃-(5) were comparable. Such changes in membrane potential were observed in the absence of extracellular sodium, indicating that an inward movement of sodium is not responsible for the depolarization. Increasing potassium levels, which lead to membrane depolarization, had no effect on the oxidative metabolism in nonstimulated or in fMet-Leu-Phe-stimulated cells. Therefore, it seems unlikely that membrane depolarization per se is the immediate stimulus for the respiratory burst.     

Modifications in choline transport activity as a function of membrane potential and the sodium gradient

Advantage was taken of a preparation of proteoliposomes made using Torpedo presynaptic membranes in which both the internal and external media can be controlled to investigate the effects of membrane potential and the Na+ gradient on choline transport activity. Under control conditions, Na+ outside and K+ inside, choline was concentrated by proteoliposomes and this phenomenon was sensitive to hemicholinium-3 and high levels of external choline. While proteoliposomes showed no permeability towards K+ spontaneously, in the presence of valinomycin a transmembrane potential was developed. The rate of transport was higher, the greater the inside negative potential. Both the affinity and the maximal velocity of high affinity transport rose in the presence of a potential. Likewise, the affinity and velocity of this transporter increased with increasing external Na+. Increasing internal Na+, on the other hand, caused a decrease in affinity and had little effect on the maximal velocity. The low affinity component was much less, if at all, affected by these changes. These results are consistent with a model of high affinity choline transport in which Na+ binds before choline and the carrier-Na+-choline complex is positively charged. However, these results do not provide a direct explanation for choline transport activation by nerve activity, underlining the need to study the effects of parameters other than membrane potential and the Na+ gradient on choline transport activity.     

Sodium-induced conformation changes in membrane transport proteins

In the presence of KCl, tryptic digestion of vesicles derived from pigeon erythrocyte membranes inactivates sodium-dependent uptake of alanine by the vesicles, whereas digestion in the presence of NaCl does not. Extensive degradation of vesicle proteins occurs under both conditions. Similarly, the extent of inhibition by N-ethylmaleimide of the sodium-dependent influxes of both glycine and alanine into human erythrocytes is greater when the cells are exposed to the thiol reagent in the presence of KCl than when NaCl is used. These observations are interpreted as providing evidence for sodium-induced conformation changes in these transport proteins.     

Intracellular membrane transport systems in Trypanosoma brucei

Trypanosomes belong to the order kinetoplastida, an early diverging group of organisms in the eukaryotic lineage. The principal reasons for interest in these organisms are twofold; they provide a superb distant triangulation point from which to assess global features of eukaryotic biology and, more importantly, they are representative of a number of pathogenic parasitic protozoa with a huge public health impact --Trypanosoma brucei, T. cruzi and Leishmania spp. Recent advances in the study of intracellular transport in T. brucei have been considerable, and a fuller picture of the complexity, function and role that the endomembrane system plays in trypanosomes is finally emerging.     

Light-induced changes in membrane potential in Spirogyra

Spirogyra cells exhibited changes in membrane potential whenthey were exposed to light. Cells made chloroplast-free didnot show any light-induced potential change (LPC) upon illuminationwith white light and also monochromatic red (680 nm) and farred (720 nm) light. LPC was observed when the cell containedonly a small fragment of chloroplast, whether the cell had anucleus or not. The magnitude of LPC depended on the amountof chloroplast in the cell. DCMU at 10^–5 M, CCCP at 10^–5 M and DNP at 10^–4M at pH 5.5 suppressed LPC, while CCCP at 1–5 ? 10^–6M, NH₄Cl at 5 ? 10^–2 M and DNP at 10^–4 M at pH 7.0stimulated LPC. PMS at 10^–4 M stimulated LPC and couldinduce LPC which was completely inhibited by DCMU. These factssuggest that LPC is related to noncyclic and cyclic electronflows. The influences of light and dark conditions and various metabolicinhibitors (DCMU, DNP, CCCP, NH₄Cl) on ATP level have been investigated.No significant difference in the ATP level was observed betweencells in the light and dark. DNP at 10^–4 M (pH 5.5) andCCCP at 5 ? 10^–6 M decreased the ATP level significantly,while DCMU and NH₄Cl only slightly. Good correlation was notfound between the total ATP level and LPC in Spirogyra. LPC occurred even when the external medium contained only asingle salt such as KCl, NaCl or CaSO₄. LPC was also recorded in chloroplasts in situ and in vitro.The mode of LPC of chloroplasts was quite different from thatof the cell. On illumination, the chloroplast potential changedvery rapidly and transiently in the positive direction thenrecovered spontaneously to almost the original potential level. Possible causes of LPC are discussed in relation to the electrogenicion pump. ¹ Present address: Department of Botany, Faculty of Science,University of Tokyo, Hongo, Bunkyo, Tokyo 113, Japan. (Received November 9, 1977; )     

Electromobile surface charge alters membrane potential changes induced by applied electric fields.

The relation between extracellular electric fields and changes in membrane potential that such fields directly induce has previously been described both theoretically and experimentally. It is clearly established that extracellular electric-field-induced membrane potential changes are well described by Poisson's equation of electrostatics. A modification of this simple theory to include effects of the electric-field-induced redistribution of charged cell surface components is introduced and is shown to produce major alterations in calculated membrane potential changes over times of the order of minutes to hours. Implications for biological systems which respond to extracellular electric fields are discussed.     

Anion transport systems in the plasma membrane of vertebrate cells

In the case of the red blood cell, anion transport is a highly specific one-for-one exchange catalyzed by a major membrane protein known as band 3 or as capnophorin. This red cell anion-exchange system mediates the Cl-(-)HCO3- exchange responsible for most of the bicarbonate transport capacity of the blood. The rapidly expanding knowledge of the molecular biology and the transport kinetics of this specialized transport system is very briefly reviewed in Section III. Exchange diffusion mechanisms for anions are found in many cells other than erythrocytes. The exchange diffusion system in Ehrlich cells has several similarities to that in red cells. In several cell types (subsection IV-B), there is evidence that intracellular pH regulation depends on Cl-(-)HCO3- exchange processes. Anion exchange in other single cells is described in Section IV, and its role in pH regulation is described in Section VII. Anion exchange mechanism operating in parallel with, and only functionally linked to Na+-H+ or K+-H+ exchange mechanisms can also play a role in cell volume regulation as described in Section VII. In the Ehrlich ascites cell and other vertebrate cells, electroneutral anion transfer has been found to occur also by a cotransport system for cations and chloride operating in parallel with the exchange diffusion system. The cotransport system is capable of mediating secondary active chloride influx. In avian red cells, the cotransport system has been shown to be activated by adrenergic agonists and by cyclic AMP, suggesting that the cotransport is involved in regulatory processes (see subsection V-A.). In several cell types, cotransport systems are activated and play a role during volume regulation, as described in Section V and in Section VII. It is also likely that this secondary active cotransport of chloride plays a significant role for the apparently active extrusion of acid equivalents from certain cells. If a continuous influx of chloride against an electrochemical gradient is maintained by a cotransport system, the chloride disequilibrium can drive an influx of bicarbonate through the anion exchange mechanism, as described in Section VII. Finally, even the electrodiffusion of anions is shown to be regulated, and in Ehrlich cells and human lymphocytes an activation of the anion diffusion pathway plays a major role in cell volume regulation as described in Section VI and subsection VII-B.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interplay between the electrostatic membrane potential and conformational changes in membrane proteins

Transmembrane electrostatic membrane potential is a major energy source of the cell. Importantly, it determines the structure as well as function of charge‐carrying membrane proteins. Here, we discuss the relationship between membrane potential and membrane proteins, in particular whether the conformation of these proteins is integrally connected to the membrane potential. Together, these concepts provide a framework for rationalizing the types of conformational changes that have been observed in membrane proteins and for better understanding the electrostatic effects of the membrane potential on both reversible as well as unidirectional dynamic processes of membrane proteins.     

Substrate-induced membrane potential changes in the perfused rat liver

In the hemoglobin-free perfused liver, administration of pyruvate, lactate fructose, alanine and palmitate elicited a sustained hyperpolarization of the cell membrane. In contrast, glucose, galactose, lysine, acetate or α-aminoisobutyric acid had no effect on the membrane potential. The pattern of the substrate induced hyperpolarization was different from glucagon- or cyclic AMP-induced hyperpolarization in the onset and duration of the response and ouabain sensitivity. The effect of cyclic AMP (5 · 10^-4 M) on membrane potential was additive to the effect of the hyperpolarizing substrates and seems to involve a mechanism different from the substrate-induced potential changes.