Choline transport specificity in animal cells and tissues

1. Beta carbolines inhibit choline transport in rat brain. 2. The aziridinium ring on the nitrogen of mustard analogs of choline causes irreversible binding to the carrier in rat brain. 3. The uptake system in rat brain is stereoselective, requires a quaternary nitrogen, and prefers analogs with a nitrogen-oxygen distance of about 3.26 A. 4. In mouse brain troxonium derivatives inhibit choline transport. 5. In cuttlefish optic lobes and torpedo electric organ pyrene derivatives potently inhibit choline transport. 6. In guinea pig placenta, the affinity of the choline carrier remains high even when this molecule lacks one or two methyl groups.     

Carrier-mediated transport system for choline and its related quaternary ammonium compounds on rat intestinal brush-border membrane.

The characteristics of the intestinal transport system for choline were investigated using isolated brush-border membrane vesicles from rat small intestine. In spite of the diminutive lipid solubility, the uptake of choline by membrane vesicles reflected smooth permeation into intravesicular space rather than the binding to the membrane surface. Physiological conditions, present in the intact intestine, such as an inward-directed Na+ or H+ gradient and inside negative membrane potentials, didn't directly involve in choline transport across the brush-border membrane. Moreover, an outward-directed H+ gradient had no significant effect on the time course of choline transport. However, in the absence of a driving-force, the initial uptake of choline exhibited a saturable manner. A kinetic analysis of the initial uptake rate gave an apparent Km of 159 microM. Furthermore, unlabeled choline caused both cis-inhibition and trans-stimulation for labeled choline transport, suggesting the existence of a carrier-mediated transport system for choline, classified as so-called 'facilitated diffusion'. Since tetramethylammonium, acetylcholine, and N1-methylnicotinamide caused both cis-inhibition and trans-stimulation, they appear to be accepted as the substrate of choline carrier. On the other hand, quaternary ammonium compounds (QACs) such as those which possessed hydrophobic parts in their molecules exhibited only cis-inhibition. They also inhibited Na(+)-dependent D-glucose transport, indicating that they influenced various carrier-mediated transport systems non-specifically due to interaction with the membrane. These findings strongly suggest that the choline transport system on the brush-border membrane of rat intestine recognizes only small molecular QACs as its substrate.     

Salinity‐Gradient Power Generation with Ionized Wood Membranes

Reverse electrodialysis (RED) is known as an efficient way of converting the salinity gradient between river water and sea water into energy. However, the high cost and complex fabrication of the necessary ion exchange membranes greatly prohibit the development of the RED process. For the first time, an ionized wood membrane is demonstrated for this application, benefiting from the advantages of natural wood, which is abundant, low cost, sustainable, and easy to scale. The wood membrane maintains the aligned nanochannels of the cellulose nanofibers derived from the natural wood. The surface of the nanochannels can be functionalized to positively or negatively charged by in situ modifying the hydroxyl groups on the cellulose chains to quaternary ammonium or carboxyl groups, respectively. These charged aligned nanochannels serve as nanofluidic passages for selective ion transport with opposite polarity through the wood membrane, resulting in efficient charge separation and generating an electrochemical potential difference. The all‐wood RED device with 100 cells using a scalable stacking geometry generates an output voltage as high as 9.8 V at open circuit from a system of synthetic river water and sea water.     

Alterations of the carrier-mediated transport of an anionic solute,methotrexate, by charged liposomes in Ehrlich ascites tumor cells

Summary Interaction of positively charged liposomes with Ehrlich ascites tumor cells increases the bidirectional transmembrane fluxes of the anionic folic acid analog, methotrexate. Negative liposomes reduce methotrexate influx. Stimulation of methotrexate influx by positively charged liposomes is time and concentration dependent, requiring at least a 5-min incubation with 2.5mm phosphatidylcholine containing 20% stearylamine for maximum effect. Stimulation is not appreciably reversed by washing the cells. Similar increases are observed for influx and efflux so that there is no change in the steady-state methotrexate electrochemical-potential difference across the cell membrane. The increase in influx appears to be a stimulation of the carrier-mediated transport process for methotrexate since both control and stimulated influx are abolished by the competitive inhibitor, 5-formyltetrahydrofolate or the sulfhydryl group inhibitor,p-chloromercuriphenylsulfonic acid and the Q₁₀ of the system remains unchanged. Influx of 5-methyltetrahydrofolate, which shares the same transport carrier as methotrexate, is also stimulated. However, the transport of folic acid, which is structurally similar to methotrexate but does not utilize the carrier, is unaffected. The kinetic change induced by positively charged liposomes is an increase in theV _ma ⁱⁿ , while theK _t ⁱⁿ remains unchanged. Trans-stimulation of methotrexate influx by 5-formyltetrahydrofolate occurs to the same extent in the presence or absence of positively charged liposomes. The liposomes have no apparent effect on the intracellular water, the extracellular space, or the chloride distribution ratio. The data suggest that interaction of positively charged liposomes with Ehrlich ascites tumor cells accelerates the rate of transposition of the membrane carrier system for methotrexate, altering the kinetics of transport without a change in transport thermodynamics.     

The binding and translocation steps in transport as related to substrate structure. A study of the choline carrier of erythrocytes

The relationships between structure, affinity and transport activity in the choline transport system of erythrocytes have been investigated in order to (i) explore the nature of the carrier site and its surroundings, and (ii) determine the dependence of the carrier reorientation process on binding energies and steric restraints due to the substrate molecule. Affinity constants and maximum transport rates for a series of trialkyl derivatives of ethanolamine were obtained by a method that involves measuring the trans effect of unlabeled analogs upon the movement of radioactive choline. The main conclusions are as follows: (1) An analysis of transport kinetics shows that the affinity constants determined experimentally differ from the actual dissociation constants in a predictable way. The better the substrate, the higher the apparent affinity relative to the true value, whereas the affinity of non-transported inhibitors is underestimated by a constant factor. (2) The carrier-choline complex undergoes far more rapid reorientation (translocation) than the free carrier. (3) The carrier imposes a strict upper limit upon the size of a substrate molecule that can participate in the carrier reorientation process; this limit corresponds to the choline structure. A smaller substrate such as tetramethylammonium, despite relatively weak binding forces, is unhindered in its translocation, suggesting that a carrier conformational change, dependent upon substrate binding energy, is not required for transport. (4) Small increases in the size of the quaternary ammonium head, as in triethylcholine, sharply lower affinity, consistent with a high degree of specificity for the trimethylammonium group. (5) Lengthening the alkyl substituent in derivatives of dimethyl- and diethylaminoethanol causes a regular increase in affinity, suggestive of unspecific hydrophobic bonding in a region very near the substrate site.     

The binding and translocation steps in transport as related to substrate structure. A study of the choline carrier of erythrocytes.

The relationships between structure, affinity and transport activity in the choline transport system of erythrocytes have been investigated in order to (i) explore the nature of the carrier site and its surroundings, and (ii) determine the dependence of the carrier reorientation process on binding energies and steric restraints due to the substrate molecule. Affinity constants and maximum transport rates for a series of trialkyl derivatives of ethanolamine were obtained by a method that involves measuring the trans effect of unlabeled analogs upon the movement of radioactive choline. The main conclusions are as follows: (1) An analysis of transport kinetics shows that the affinity constants determined experimentally differ from the actual dissociation constants in a predictable way. The better the substrate, the higher the apparent affinity relative to the true value, whereas the affinity of non-transported inhibitiors is underestimated by a constant factor. (2) The carrier-choline complex undergoes far more rapid reorientation (translocation) than the free carrier. (3) The carrier imposes a strict upper limit upon the size of a substrate molecule that can participate in the carrier reorientation process; this limit corresponds to the choline structure. A smaller substrate such as tetramethylammonium, despite relatively weak binding forces , is unhindered in its translocation, suggesting that a carrier conformational change, dependent upon substrate binding energy, is not required for transport. (4) Small increases in the size of the quaternary ammonium head, as in triethylcholine, sharply lower affinity, consistent with a high degree of specificity for the trimethylammonium group. (5) Lengthening the alkyl substituent in derivatives of dimethyl- and diethylaminoethanol causes a regular increase in affinity, suggestive of unspecific hydrophobic bonding in a region very near the substrate site.     

Choline transport into rat liver mitochondria. Characterization and kinetics of a specific transporter.

Rat liver mitochondria possess a specific choline transporter in the inner membrane. The transporter shows saturable kinetics at high membrane potential with a Km of 220 microM and a Vmax of 0.4 nmol/mg of protein/min at pH 7.0 and 25 degrees C. At physiological concentrations of choline, the rate of choline uptake by the transporter shows a linear dependence on membrane potential; uptake is distinct from the nonspecific cation diffusion process. Hemicholinium-3, hemicholinium-15, quinine, and quinidine, all analogues of choline, are high affinity competitive inhibitors of choline transport with Ki values of 17, 55, 15, and 127 microM, respectively. The choline transporter is distinct from other known mitochondrial transporters. Rat heart mitochondria do not appear to possess a choline transporter. Evidence suggests that the transporter is an electrophoretic uniporter. Analogue studies have shown that the hydroxyl and the quaternary ammonium groups of choline are necessary for binding to the transporter. A comparison of molecular models of choline and the high affinity inhibitors has provided evidence for the preferred conformation of choline for binding to the transporter. The presence of a choline transporter in the mitochondrial inner membrane provides a potential site for control of choline oxidation and hence supply of endogenous betaine.     

Looking for probes of gated channels: studies of the inhibition of glucose and choline transport in erythrocytes

Two seemingly contradictory sets of observations have been made in studies of biological transport, which are essential for our understanding of the transport mechanism: carriers are integral membrane proteins, which span the membrane and are not free to rotate across the membrane; carriers appear to function like a ferryboat, with a substrate binding site moving back and forth from one side of the membrane to the other. To reconcile these facts, it is necessary to postulated gated channels connecting the substrate site with the two membrane surfaces: the channels are arranged so that as one opens the other closes, with the result that the substrate site is alternately accessible from opposite sides of the membrane. Based on these properties, the following distinguishing features of molecules specifically bound in the channels may be predicted: if sufficiently bulky, they inhibit transport; they bind outside the substrate site (though adjacent to it), they bind asymmetrically either to the outward-facing carrier and on the outer surface of the membrane, or to the inward-facing carrier and on the inner surface of the membrane. The asymmetrical inhibition of the glucose and choline transport systems of erythrocytes by various inhibitors is examined, and the behavior in every case is found to conform with these criteria. From the results it may be concluded that the glucose carrier binds cytochalasin B in the inner gated channel and phloretin and tetrathionate in the outer gated channel.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Kinetic Data on the Inhibition of High-affinity Choline Transport into Rat Forebrain Synaptosomes by Choline-like Compounds and Nitrogen Mustard Analogues

Abstract: A series of choline analogues and nitrogen mustard derivatives were evaluated as inhibitors of high-affinity transport of choline in rat forebrain synaptosomes. When synaptosomes were preincubated for 10 min with choline mustard aziridinium ion, monoethylcholine and monoethylcholine mustard aziridinium ion, the agents appeared to be equipotent as inhibitors of high-affinity uptake (K_i=2.63, 3.15 and 2.72 μm , respectively). Acetylcholine mustard aziridinium ion was less potent than these compounds (K_i= 27.8 μm ), but it was more potent than ethoxycholine and ethoxycholine mustard aziridinium ion (K_i= 500 and 403 μm ) as a blocker of choline transport. From study with these compounds it was concluded that the high-affinity choline transport mechanism shows specificity for hydroxylated compounds over those in which the same hydroxyl has been acetylated (10-fold) and that the carbonyl oxygen of the acetylated analogues is important, as its removal (to form the ethylether derivative) decreased affinity another 20-fold. The presence of an aziridinium ring on the quaternary nitrogen in place of two methyl groups did not affect the blocking of transport at 10 min of inhibitor preincubation and replacement of a methyl group on the nitrogen by an ethyl group did not alter affinity for the high-affinity carrier. The aziridinium ring on the nitrogen of the mustard analogues was important, however, in determining the extent of reversibility of the binding of these agents to the carrier protein. Choline transport was not restored by washing synaptosomes that were incubated with choline mustard aziridinium ion or monoethylcholine mustard aziridinium ion, but was readily obtained in washed synaptosomes preincubated with monoethylcholine, hemicholinium-3, or pyrrolcholine. The results indicate that the mustard analogues may be potent alkylators of the high-affinity choline carrier and thus, useful agents in monitoring acetylcholine turnover in systems where the carrier is blocked.     

The choline transport system of erythrocytes. Distribution of the free carrier in the membrane

A method is described, based on the kinetics of transport, for determining the equilibrium distribution of the carrier site on the inner and outer surfaces of the cell membrane, and this method is applied to the choline carrier of human erythrocytes. This method depends on measurement of flux ratios for both entry and exit, i.e., the transport rates of a low concentration of labeled substrate into a solution which contains either no substrate or a saturating concentration of unlabeled substrate. The concentrations of inward-facing and outward-facing carrier are found to be nearly equal, and therefore the 5-fold difference in choline affinity on the inner and outer surfaces of the membrane cannot be explained by an unequal carrier distribution. It is also shown that both reorientation and dissociation of the carrier-substrate complex are far more rapid than reorientation of the free carrier.     

Characterization of the quaternary amine transporters of Rhizobium leguminosarum bv. viciae 3841

Rhizobium leguminosarum bv. viciae 3841 contains six putative quaternary ammonium transporters (Qat), of the ABC family. Qat6 was strongly induced by hyperosmosis although the solute transported was not identified. All six systems were induced by the quaternary amines choline and glycine betaine. It was confirmed by microarray analysis of the genome that pRL100079-83 (qat6) is the most strongly upregulated transport system under osmotic stress, although other transporters and 104 genes are more than threefold upregulated. A range of quaternary ammonium compounds were tested but all failed to improve growth of strain 3841 under hyperosmotic stress. One Qat system (gbcXWV) was induced 20-fold by glycine betaine and choline and a Tn5::gbcW mutant was severely impaired for both transport and growth on these compounds, demonstrating that it is the principal system for their use as carbon and nitrogen sources. It transports glycine betaine and choline with a high affinity (apparent K(m), 168 and 294 nM, respectively).     

Choline mustard: an irreversible ligand for use in studies of choline transport mechanisms at the cholinergic nerve terminal

It has been shown in our laboratory that choline mustard aziridinium ion is a potent and irreversible inhibitor of choline transport into rat brain synaptosomes; this compound showed selectivity for the sodium-dependent, high affinity carrier in that it was 30 times more potent as an inhibitor when compared with the effect on sodium-independent, low affinity choline uptake. In the present study, this mustard analogue did not inhibit synaptosomal uptake of 5-hydroxytryptamine, noradrenaline, or gamma-aminobutyric acid, thereby confirming further the specificity of this compound for the choline carrier. Studies of the effect of depolarization of the nerve terminals on the inactivation of choline carriers by choline mustard were performed. It was determined that alkylation of the carrier was significantly increased in nerve endings previously depolarized. The enhancing effect of depolarization on choline transport velocity and on the alkylation of choline carriers by choline mustard was dependent upon the presence of sodium in the external medium. Possible mechanisms for the enhanced inactivation of choline carriers by choline mustard aziridinium ion are proposed, and kinetic interactions of choline mustard with the high affinity choline carrier and with choline acetyltransferase are reviewed and discussed.     

Uptake of Some Quaternary Ammonium Ions by Human Erythrocytes

In many biophysical studies on erythrocytes some quaternary ammonium ions are used as replacements for Na⁺ and K⁺ of the physiological solutions. The object of this work was to study the possible uptake of quaternary ammonium ions by erythrocytes. Uptake of C¹⁴–choline chloride and C¹⁴–tetramethylammonium chloride by human erythrocytes was proved. It was shown that the compounds were neither incorporated into phospholipids of the cell nor converted to any other metabolites. Studies of uptake as a function of time, at several external concentrations of choline and tetramethylammonium, showed that within the first 4 hours uptake was a linear function of time regardless of the external concentration of the quaternary ammonium ions. The effects of various external concentrations of choline and tetramethylammonium ions on the rate of uptake by the cells were studied. The results showed the presence of two distinct mechanisms for the uptake of choline: one, a facilitated uptake mechanism which becomes saturated at low external concentrations of the ion; the other, a simple diffusion mechanism in which the rate of uptake is proportional to concentration. For the facilitated part of the uptake the external choline concentration at which half-maximum rate was obtained was found to be 0.02 mm. Although the kinetic studies with tetramethylammonium ion were not as extensive as those with choline, they did suggest the presence of similar mechanisms for the uptake of both ions. Tetramethylammonium and tetraethylammonium ions were shown to be competitive inhibitors of the facilitated choline uptake.     

Lysine excretion by Corynebacterium glutamicum. 2. Energetics and mechanism of the transport system.

Lysine excretion in Corynebacterium glutamicum was characterized as secondary transport process. It is modulated by three forces: the membrane potential, the chemical potential of lysine, and the proton gradient. The ATP content of the cells did not correlate with the export activity. Lysine is excreted in symport with presumably two OH- ions which is not distinguishable experimentally from an antiport mechanism against two protons. The substrate-loaded carrier is uncharged. When the external substrate concentration is low and no proton gradient present, reorientation of the positively charged, unloaded carrier is rate-limiting. Export then depends on the membrane potential. When the external substrate is high, translocation of the loaded, uncharged carrier is rate-limiting, and export is not modulated by the membrane potential. The lysine secretion system in C. glutamicum is shown to be well adapted to the requirements of metabolite export.     

Effects of atropine on the melanophores and light-reflecting chromatophores of some teleost fishes

1. The mechanism of the action of atropine, which is known to accelerate the dispersion response of fish melanophores, was examined by use of various receptor antagonists.2. The effects of atropine were found to be independent of adenosine receptors, beta-adrenoceptors and MSH receptors on the melanophore membrane.3. Analogs of atropine, such as scopolamine, also had a potent pigment-dispersing effect on melanophores, whereas the quaternary ammonium derivatives, which are positively charged molecules, had only a small effect.4. These results suggest that the possible site of atropine action is within the chromatophores themselves.5. In addition to the melanosome-dispersing effect, atropine caused a shift in the spectral peak of reflected light toward shorter wavelengths and the dispersion of leucosomes in the motile iridophores of the blue damselfish and in the leucophores of the medaka, respectively.     

The transport of potassium through lipid bilayer membranes by the neutral carriers valinomycin and monactin

Summary Stationary conductance experiments on neutral and negatively charged bilayer membranes in the presence of valinomycin or monactin agree with a recently proposed carrier transport model, which is common to both carrier types. This model assumes an interface reaction between a cation from the aqueous solution and a carrier molecule from the membrane phase to establish charge transport across the interface. The transport across the membrane interior is described by some kind of Eyring model. The discussion of the current-voltage characteristic, the dependence of membrane conductance on the carrier and K⁺ concentrations, and the comparison with appropriate experiments allow correlation of the different rate constants of the transport model. The results show that the rate constants partly depend on the surface charge of the membranes. This dependency can be described by introducing the Gouy-Chapman theory for charged surfaces into the transport model.It was found that the carrier molecules could be added either to the aqueous phase or to the membrane-forming solution. The quantitative treatment of this phenomenon gives an evaluation of the partition coefficient of the carrier molecules between the membrane bulk phase and water.     

The inhibitory effect of choline and other quaternary ammonium compounds on thiamine transport in isolated rat hepatocytes

Quaternary ammonium compounds, such as choline and acetylcholine significantly inhibited thiamine uptake in isolated rat hepatocytes. Kinetic analysis using Lineweaver-Burk and Dixon plots of inhibition experiments revealed that choline and acetylcholine were purely competitive inhibitors for thiamine uptake with Ki values of 0.61 mM and 0.31 mM, respectively. Among quaternary ammonium compounds, hemicholinium-3 and curare were the strongest inhibitors, and kinetic studies showed that these compounds were also purely competitive inhibitors with Ki values of 12.5 microM and 4.3 microM, respectively. These results indicate that choline, acetylcholine and their structural analogs share a common binding site with thiamine in isolated rat hepatocytes. On the other hand, choline uptake by isolated rat hepatocytes occurred by a saturable mechanism with a Kt of 162 +/- 3.85 microM and Vmax of 80.1 +/- 1.30 pmol/10(5) cells per min as well as by a nonsaturable mechanism. Thiamine, pyrithiamine, oxythiamine, chloroethylthiamine and dimethialium inhibited choline uptake, while thiamine phosphates such as thiamine monophosphate and thiamine pyrophosphate insignificantly inhibited uptake. Although a Lineweaver-Burk plot of choline uptake in the presence of thiamine showed that thiamine also competitively inhibited choline uptake, a Dixon plot of the inhibition experiment was hyperbolic and indicated that the inhibition of choline uptake by thiamine was 'pseudo-competitive'. On the basis of these results, it is suggested that in isolated rat hepatocytes thiamine and choline do not share common transport sites.     

Carrier-mediated inhibition of choline acetyltransferase

Incubation of rat forebrain synaptosomes with choline mustard aziridinium ion in a sodium-rich medium caused a time-dependent inhibition of the high-affinity transport of choline, as well as a significant decrease in intrasynaptosomal choline acetyltransferase activity. In the absence of added sodium choline uptake by a sodium-independent mechanism was also blocked in a time-dependent manner but intrasynaptosomal choline acetyl-transferase activity was unaltered. Neither monoethylcholine nor hemicholinium-3 changed intrasynaptosomal choline acetyl-transferase activity but competitively inhibited the transport of choline. The results indicate that there may be a fraction of choline acetyltransferase that is closely associated with the sodium-dependent high-affinity choline transport system and that this fraction can be irreversibly inhibited by choline mustard aziridinium ion, perhaps indirectly mediated by alkylation of the carrier.     

Effects on transport of rapidly penetrating,competing substrates: Activation and inhibition of the choline carrier in erythrocytes by imidazole

Summary The properties of the choline transport system are fundamentally altered in saline solution containing 5mm imidazole buffer instead of 5mm phosphate: (i) The system no longer exhibits accelerated exchange. (ii) Choline in the external compartment fails to increase the rate of inactivation of the carrier by N-ethylmaleimide. (iii) Depending on the relative concentrations of choline and imidazole, transport may be activated or inhibited. The maximum rates are increased more than fivefold by imidazole, but at moderate substrate concentrations activation is observed with low concentrations of imidazole and inhibition with high concentrations. (iv) The imidazole effect is asymmetric, there being a greater tendency to activate exit than entry. All this behavior is predicted by the carrier model if imidazole is a substrate of the choline carrier having a high maximum transport rate but a relatively low affinity, and if imidazole rapidly enters the cell by simple diffusion, so that it can add to carrier sites on both sides of the membrane. Addition at thecis side inhibits, and at thetrans side activates. According to the carrier model, asymmetry is a necessary consequence of the potassium ion gradient in red cells, potassium ion being another substrate of the choline system.