Multidrug Transport Protein NorM from Vibrio cholerae Simultaneously Couples to Sodium- and Proton-Motive Force

Membrane transporters belonging to the multidrug and toxic compound extrusion family mediate the efflux of unrelated pharmaceuticals from the interior of the cell in organisms ranging from bacteria to human. These proteins are thought to fall into two classes that couple substrate efflux to the influx of either Na⁺ or H⁺. We studied the energetics of drug extrusion by NorM from Vibrio cholerae in proteoliposomes in which purified NorM protein was functionally reconstituted in an inside-out orientation. We establish that NorM simultaneously couples to the sodium-motive force and proton-motive force, and biochemically identify protein regions and residues that play important roles in Na⁺ or H⁺ binding. As the positions of protons are not available in current medium and high-resolution crystal structures of multidrug and toxic compound extrusion transporters, our findings add a previously unrecognized parameter to mechanistic models based of these structures.     

Reconstitution of cAMP-Dependent protein kinase regulated renal Na+−H+ exchanger

Summary Studies were performed to determine if the Na⁺–H⁺ exchanger, solubilized from renal brush border membranes from the rabbit and assayed in reconstituted artificial proteoliposomes, could be regulated by cAMP-dependent protein kinase. Octyl glucoside solubilized renal apical membrane proteins from the rabbit kidney were phosphorylated by incubation with ATP and highly purified catalytic subunit of cAMP-dependent kinase.²²Na⁺ uptake was determined subsequently after reconstitution of the proteins into proteoliposomes. cAMP-dependent protein kinase resulted in sustained protein phosphorylation and a concentration-dependent decrease in the amiloride-sensitive component of pH gradient-stimulated sodium uptake. The inhibitory effect of cAMP-dependent protein kinase demonstrated an absolute requirement for ATP and was blocked by the specific protein inhibitor of this kinase. cAMP-dependent protein kinase also inhibited²²Na⁺ uptake in the absence of a pH gradient (pH_in 6.0. pH_out 6.0) and the inhibitory effect was blocked by the specific inhibitor of the kinase. Solubilized membrane proteins exhibited little endogenous protein kinase or protein phosphatase activity.These studies indicate that Na⁺–H⁺ exchange activity of proteoliposomes reconstituted with proteins from renal brush border membranes is inhibited by phosphorylation of selected proteins by cAMP-dependent protein kinase. These findings also indicate that the regulatory components of the Na⁺–H⁺ exchanger remain active during the process of solubilization and reconstitution of renal apical membrane proteins.     

Cation Effects on Chloride Fluxes and Accumulation Levels in Barley Roots

Accumulation of Cl^- by excised barley roots, as of K⁺, approaches a maximum level at which the ion influx and efflux rates become equal. The rate of Cl^- influx at this equilibrium is close to the initial rate while the efflux rate increases with time from zero to equality with influx. The Cl^- fluxes are independent of simultaneous exchange flux of the cations, but depend on the nature and concentration of the salt solutions from which they originate. The Cl^- content at equilibrium, however, is largely independent of the external concentrations. The approach to equilibrium reflects the presence of the cation. Cl^- flux equilibrium is attained more rapidly in KCl than in CsCl or CaCl₂. This is presumably an effect of much slower distribution of Cs⁺ and Ca⁺⁺ than of K⁺ within the roots. Accumulated Cs⁺ appears to form a barrier to ion movement primarily within the outermost cells, thereby reducing influx and ultimately efflux rates of both Cl^- and cations. Slow internal mixing and considerable self-exchange of the incoming ions suggest internal transport over a series of steps which can become rate-limiting to the accumulation of ions in roots.     

Partial purification of the Na+-dependentd-glucose transport system from renal brush border membranes

Summary A membrane extract enriched with the Na⁺-dependentd-glucose transport system was obtained by differential cholate solubilization of rat renal brush border membranes in the presence of 120mm Na⁺ ions. Sodium ions were essential in stabilizing the transport system during cholate treatment. This membrane extract was further purified with respect to its Na⁺-coupledd-glucose transport activity and protein content by the use of asolectin-equilibrated hydroxylapatite. The reconstituted proteoliposomes prepared from this purified fraction showed a transient accumulation ofd-glucose in response to a Na⁺ gradient. The observed rate of Na⁺-coupledd-glucose uptake by the proteoliposomes represented about a sevenfold increase as compared to that of the reconstituted system derived from an initial 1.2% cholate extract of the membranes. Other Na⁺-coupled transport systems such asl-alanine, -ketoglutarate and phosphate were not detected in these reconstituted proteoliposomes.     

CLC channels and transporters: Proteins with borderline personalities

Controlled chloride movement across membranes is essential for a variety of physiological processes ranging from salt homeostasis in the kidneys to acidification of cellular compartments. The CLC family is formed by two, not so distinct, sub-classes of membrane transport proteins: Cl^- channels and H⁺/Cl^- exchangers. All CLC's are homodimers with each monomer forming an individual Cl- permeation pathway which appears to be largely unaltered in the two CLC sub-classes. Key residues for ion binding and selectivity are also highly conserved. Most CLC's have large cytosolic carboxy-terminal domains containing two cystathionine β-synthetase (CBS) domains. The C-termini are critical regulators of protein trafficking and directly modulate Cl- by binding intracellular ATP, H+ or oxidizing compounds. This review focuses on the recent mechanistic insights on the how the structural similarities between CLC channels and transporters translate in unexpected mechanistic analogies between these two sub-classes.     

Solubilization and reconsitution of renal brush border Na+−H+ exchanger

Summary In order to permit future characterization and possible isolation of the Na⁺–H⁺ exchanger from the apical membrane of proximal tubular cells, studies were performed to solubilize and reconstitute this transporter. Rabbit brush border membranes were prepared by a magnesium aggregation method, solubilized with the detergent octyl glucoside, and reconstituted into artificial phospholipid vesicles. In the presence of a pH gradient (pH_in 6.0, pH_out 8.0), the uptake of 1mm ²²Na⁺ into the proteoliposomes was five- to sevenfold higher than into liposomes. Amiloride (2mm) inhibited proton gradient-stimulated uptake of sodium by 50%. As compared to proton gradient conditions, the uptake of sodium was lower in the absence of a pH gradient but was significantly higher when the outside and inside pH was 6.0 than 8.0. TheK _a for sodium in reconstituted proteoliposomes studied under pH gradient conditions was 4mm. The uptake of sodium in proteoliposomes prepared from heat-denatured membrane proteins was significantly decreased. These studies demonstrate that proteoliposomes prepared from octyl glucoside-solubilized brush border membrane proteins and asolectin exhibit proton gradient-stimulated, amiloride-inhibitable, electroneutral uptake of sodium. The ability to solubilize and reconstitute the Na⁺–H⁺ exchanger from the apical membrane of the proximal tubule will be of value in isolating and characterizing this transporter.     

Regulation of reconstituted renal Na+/H+ exchanger by calcium-dependent protein kinases

Summary Studies were performed to determine the effect of protein phosphorylation mediated by calcium-calmodulin-dependent multifunctional protein kinase II and calcium-phospholipid-dependent protein kinase on Na⁺/H⁺ exchange activity. Proteins from the apical membrane of the proximal tubule of the rabbit kidney were solubilized in octyl glucoside and incubated in phosphorylating solutions containing the protein kinase.²²Na⁺ uptake was determined subsequently after reconstitution of the proteins into proteoliposomes. Calcium-calmodulin-dependent multifunction protein kinase II inhibited the amiloride-sensitive component of proton gradient-stimulated Na⁺ uptake in a dose-dependent manner. The inhibitory effect of this kinase had an absolute requirement for calmodulin, Ca²⁺, and ATP. Calcium-phospholipid-dependent protein kinase stimulated the amiloride-sensitive component of proton gradient-stimulated Na⁺ uptake in a dose-dependent manner. The stimulating effect of this kinase had an absolute requirement for ATP, Ca²⁺, and an active phorbol ester. These experiments indicate that Na⁺/H⁺ exchange activity of proteoliposomes reconstituted with proteins from renal brush-border membranes are inhibited by protein phosphorylation mediated by calcium-calmodulin-dependent multifunctional protein kinase II and stimulated by that mediated by calcium-calmodulin-dependent protein kinase.     

Muscle: A Three Phase System : The partition of monovalent ions across the cell membrane

The partition of Li⁺, Br^-, and I^- across the membrane of the sartorius muscle of the toad Bufo marinus has been investigated both at the steady state and with kinetic methods. Li⁺ was found to have access to an amount of muscle water similar to that of Na⁺. Br^- and I^- could be regarded as being interchangeable with cellular Cl^-. None of the foreign ions caused significant losses of cellular K⁺. Li⁺ efflux from the cell was slower in muscles which were equilibrated for long periods in Li⁺ than in short equilibrated muscles. Na⁺ efflux from Li⁺-treated muscles was similar in rate to normal controls, but the amount of Na⁺ in the slow fraction was increased by Li⁺. I^- efflux was extremely rapid, and it was not possible to differentiate kinetically between intra- and extracellular material. These results have been found to be consistent with the hypothesis of a three phase system for muscle.     

Ca<Superscript><Emphasis Type="Italic">2+</Emphasis></Superscript>-Induced Cl<Superscript>−</Superscript> Efflux at Rat Distal Colonic Epithelium

With the aid of the halide-sensitive dye 6-methoxy-N-ethylquinolinium iodide (MEQ), changes in intracellular Cl^- concentration were measured to characterize the role of Ca²⁺-dependent Cl^- channels at the rat distal colon. In order to avoid indirect effects of secretagogues mediated by changes in the driving force for Cl^- exit (i.e., mediated by opening of Ca²⁺-dependent K⁺ channels), all experiments were performed under depolarized conditions, i.e., in the presence of high extracellular K⁺ concentrations. The Ca²⁺-dependent secretagogue carbachol induced a stilbene-sensitive Cl^- efflux, which was mimicked by the Ca²⁺ ionophore ionomycin. Surprisingly, the activation of Ca²⁺-dependent Cl^- efflux was resistant against blockers of classical Ca²⁺ signaling pathways such as phospholipase C, protein kinase C and calmodulin. Hence, alternative pathways must be involved in the signaling cascade. One possible signaling molecule seems to be nitric oxide (NO) as the NO donor sodium nitroprusside could induce Cl^- efflux. Vice versa, the NO synthase inhibitor N-ω-monomethyl-arginine (l-NMMA) reduced the carbachol-induced Cl^- efflux. This indicates that NO may be involved in part of the signaling cascade. In order to test the ability of the epithelium to produce NO, the expression of different isoforms of NO synthase was verified by immunohistochemistry. In addition, the cytoskeleton seems to play a role in the activation of Ca²⁺-dependent Cl^- channels. Inhibitors of microtubule association such as nocodazole and colchicine as well as jasplakinolide, a drug that enhances actin polymerization, inhibited the carbachol-induced Cl^- efflux. Consequently, the activation of apical Cl^- channels by muscarinic receptor stimulation differs in signal transduction from the classical phospholipase C/protein kinase C way.     

Tributylbenzylammonium (TBBA+)-dependent fusicoccin (FC)-induced H+ extrusion in maize roots: relationship between the stimulating effects of TBBA+ on H+ extrusion and on Cl- efflux

The mechanism of the stimulating effect of lipophilic cations on H⁺ extrusion in maize root segments (Zea mays L.) has been investigated. The measurement of the uptake of [³H]tributylbenzylammonium ([³H]TBBA⁺), the most active lipophilic cation on H⁺ extrusion, indicated that although a relevant fraction of TBBA⁺ taken up by the tissue is adsorbed to cell surfaces, a fraction of the cation enters the cells. However no correlation was observed between the rate of TBBA⁺ uptake and that of H⁺ extrusion. On the other hand, the lipophilic cations active on H⁺ extrusion (TBBA⁺ and dibenzyldimethylammonium (DDA⁺)), in the presence of fusicoccin (FC), induced under the same conditions an efflux of Cl^-, while tetramethylammonium (TMA⁺), inactive on H⁺ extrusion, did not. The stimulation of Cl^- efflux by TBBA⁺ was independent of the anion present in the medium and was inhibited by Na-orthovanadate, an inhibitor of plasma membrane ATPase and of TBBA⁺-induced H⁺ extrusion. These results suggest that the stimulation of H⁺ extrusion by TBBA⁺ depends on its effect on Cl^- efflux rather than on its penetration into the cells.Abbreviations DDA⁺ dibenzyldimethylammonium - FC fusicoccin - 3-O-MG 3-O-methyl glucose - PD transmembrane electric potential difference - TBBA⁺ tributylbenzylammonium - TCF tissue concentration factor - TMA⁺ tetramethylammonium - TPB^- tetraphenylboron     

Selective Reconstitution of the Tonoplast H+-ATPase of the Crassulacean-Acid Metabolism Plant Kalanchoë daigremontiana

IA detergent removal technique was used to reconstitute solubilized tonoplast proteins of mesophyll cells of the CAM plant Kalanchoë daigremontiana into phosphatidylcholine liposomes. The proteoliposomes were able to hydrolyse ATP and to pump protons across the vesicle membrane. Both activities were inhibited by nitrate, an inhibitor of V-type ATPases. Freeze-fracture micrographs confirmed the incorporation of membrane proteins into liposomes. Increase of specific ATP-hydrolysis activity compared to solubilized tonoplast proteins and SDS-PAGE analysis of reconstituted proteins in comparison with the polypeptide pattern of the purified tonoplast H⁺-ATPase from the same plant source indicated a highly selective reconstitution of the tonoplast H⁺-ATPase.     

Visualization by Freeze-Fracture Electron Microscopy of Intramembraneous Particles corresponding to the Tonoplast H+-Pyrophosphatase and H+-ATPase of Kalanchoë daigremontiana Hamet et Perrier de la Bâthie*

The H⁺-PPase and the H⁺-ATPase of the vacuolar membrane were separated during purification of tonoplast proteins of Kalanchoë daigremontiana Hamet et Perrier de la Bǎthie. Three membrane protein fractions prepared contained firstly, the H⁺-PPase protein without any subunits of the H⁺-ATPase, secondly, the H⁺-PPase protein with only minute traces of the intramembraneous 16 kDa c-subunit of the H⁺-ATPase, and thirdly, the H⁺-ATPase subunits without H⁺-PPase peptides as verified by SDS-PAGE. These three preparations were reconstituted into soybean (Glycine max L.)-phospholipid vesicles, and compared with proteoliposomes obtained by reconstitution of total solubilized tonoplast proteins as well as with native tonoplast vesicles. Analysis of freeze-fracture replicas prepared from these five different types of vesicles showed that there are two populations of intramembraneous particles, one with a diameter of 6.7-7.2 nm corresponding to the H⁺-PPase, and one with an average diameter of 9.1 nm belonging to the H⁺-ATPase. Thus, freeze-fracture electron microscopy allows one to visualize H⁺-PPase particles in addition to H⁺-ATPase particles in the tonoplast of Kalanchoë daigremontiana.     

Localization of Hydrogen Ion and Chloride Ion Fluxes in Nitella

Alternating bands of acid and base formation have been detected along the length of the internodal cell of Nitella clavata when it is illuminated, while in the dark this phenomenon is minimal. Chloride influx occurs only or largely in the acid-extruding regions, and this is also a light-dependent ion movement. Chloride efflux is slightly dependent on illumination and is not localized as are H⁺ efflux and Cl^- influx. The results obtained support Kitasato's (1968) proposal that a large passive H⁺ influx is balanced by an active efflux of this ion. Transport mechanisms suggested by the correlations of Cl^- and HCO₃^- influxes with H⁺ extrusion are discussed.     

Effect of limited trypsin digestion on the renal Na+−H+ exchanger and its regulation by cAMP-Dependent protein kinase

Summary The Na⁺–H⁺ exchanger from solubilized rabbit renal brush border membranes is inhibited by cAMP-dependent protein kinase (PKA) mediated protein phosphorylation. To characterize this inhibitory response and its sensitivity to limited proteolysis, the activity of the transporter was assayed after reconstitution of the proteins into artificial lipid vesicles. Limited trypsin digestion increased the basal rate of proton gradient-stimulated, amiloride-inhibitable sodium uptake in reconstituted proteoliposomes and blocked the inhibitory response to PKA-mediated protein phosphorylation. To determine if the inhibitory response to PKA-mediated protein phosphorylation could be restored to the trypsin-treated solubilized proteins, nontrypsinized solubilized brush border membrane proteins were separated by column chromatography. The addition of small molecular weight polypeptides, fractionated on Superose-12 FPLC (V _e=0.7), to trypsinized solubilized brush border membrane proteins restored the inhibitory response to PKA-mediated protein phosphorylation. Similarly, the addition of the 0.1m NaCl fraction from an anion exchange column, Mono Q-FPLC, also restored the inhibitory response to PKA. Both protein fractions contained a common 42–43 kDa protein which was preferentially phosphorylated by PKA.These results indicate that limited trypsin digestion dissociates the activity of the renal Na⁺–H⁺ exchanger from its regulation by PKA. It is suggested that trypsin cleaves an inhibitory component of the transporter and that this component is the site of PKA-mediated regulation. Phosphoprotein analysis of fractions that restored PKA regulation raises the possibility that a polypeptide of 42–43 kDa is involved in the inhibition of the renal Na⁺–H⁺ exchanger by PKA-mediated, protein phosphorylation.     

Renal cortical basolateral Na+/HCO 3 − cotransporter: I. Partial purification and reconstitution

The renal basolateral Na⁺/HCO ₃ ^– cotransporter is the main system responsible for HCO ₃ ^– transport from proximal tubule cells into the blood. The present study was aimed at purifying and functionally reconstituting the Na⁺/HCO ₃ ^– cotransporter protein from rabbit renal cortex. Highly purified rabbit renal cortical basolateral membrane vesicles (hereafter designated as original basolateral membrane), enriched 12-fold in Na-K-ATPase, were solubilized in 2% octylglucoside, and then reconstituted in l--phosphatidylcholine (proteoliposomes). Na⁺/HCO ₃ ^– cotransporter activity was assessed as the difference in ²²Na uptake in the presence of HCO ₃ ^– and gluconate. The activity of the Na⁺/HCO ₃ ^– cotransporter was enhanced 18-fold in the solubilized protein reconstituted into proteoliposomes compared to the original basolateral membranes. The reconstituted solubilized purified protein exhibited kinetic properties similar to the cotransporter from original basolateral membranes. In addition, it was like the original cotransporter, inhibited by disulfonic stilbene SITS, and was eleetrogenic. The catalytic subunit of protein kinase A significantly inhibited Na⁺/HCO ₃ ^– cotransporter activity in proteoliposomes. The octylglucoside-solubilized protein was further purified by hydroxylapatite column chromatography, and this resulted in an additional enhancement of Na⁺/HCO ₃ ^– cotransporter activity of 80-fold over the original basolateral membranes. The fractions containing the highest activity were further processed by glycerol gradient centrifugation, resulting in a 124- to 300-fold increase in Na⁺/HCO ₃ ^– cotransporter activity compared to the original basolateral membranes. SDS-PAGE analysis showed an enhancement of a protein doublet of 56 kD MW in the glycerol gradient fraction. Our results demonstrate that we have partially purified and reconstituted the renal Na⁺/HCO ₃ ^– cotransporter and suggest that the 56 kD doublet protein may represent the Na⁺/HCO ₃ ^– cotransporter.This work was supported by the Merit Review Program from the Veterans Administration Central Office (J.A.L.A.), and the National Kidney Foundation of Illinois (A.A.B.).     

Abscisic-acid-induced turion formation in Spirodela polyrrhiza L. IV. Comparative ion flux characteristics of the turion and the vegetative frond and the effect of ABA during early turion development

Abstract Using the method of compartmental analysis, the ion fluxes and compartment concentrations of Ca²⁺, K⁺ and Cl^- have been compared in the untreated vegetative frond and the abscisic acid (ABA) induced turion of Spirodela polyrrhiza. The ABA-induced turion is characterized by reduced Ca²⁺ exchange across the tonoplast and low vacuolar Ca²⁺ concentration relative to the vegetative frond. In addition the turion exhibits a higher plasmalemma flux with a correspondingly high Ca²⁺ concentration in the cytoplasm. The concentration of K⁺ and Cl^- is much lower in the cytoplasm of the ABA-induced turion than in the vegetative frond with the influx/efflux ratio at both the plasmalemma and the tonoplast being less than 1, a finding exhibited also in dormant storage tissue. Treatment of vegetative fronds with ABA for 18 h resulted in a reduced K⁺ plasmalemma efflux relative to untreated vegetative fronds and a concomitant increase in the cytoplasmic concentration. There was no rapid effect of ABA on Ca²⁺, K⁺ or Cl^- fluxes through either membrane. These results are consistent with the notion that drastic changes in ion fluxes and concentrations in the turion are a secondary consequence of ABA-induced development, possibly due to prior regulation by ABA of enzymes inherent to processes involved in membrane transport.     

Induction of increased calcium uptake in liposomes having membrane proteins of chicken erythrocytes by S-adenosylmethionine

Liposomes having membrane proteins of chicken erythrocytes were prepared and the effect of S-adenosylmethionine on ⁴⁵Ca²⁺ uptake into the liposomes was investigated. S-Adenosylmethionine, a donor of methyl groups in enzymatic methylation, induced an increase of ⁴⁵Ca²⁺ uptake into the proteoliposomes with membrane proteins but not into the liposomes without membrane proteins. Increased release of ⁴⁵Ca²⁺ from the inside of the proteoliposomes was also induced by S-adenosylmethionine. These increases of uptake and release of ⁴⁵Ca²⁺ were inhibited by S-adenosylhomocystein, an inhibitor of enzymatic methylation. Furthermore, membrane proteins from chicken erythrocytes showed protein and phospholipid methyltransferase activities. The uptake of other materials, 3-0-[methyl-³H]glucose, α-[1-¹⁴C]aminoisobutyric acid, ⁴²K⁺ and ⁵⁴Mn²⁺, into the proteoliposomes was not increased by S-adenosylmethionine. These results suggest that enzymatic methylation of membrane components may have an important role in the regulation of calcium transport in the chicken erythrocyte membrane and this regulation is rather specific for calcium.     

Nystatin-induced increase in photocurrent in the system ‘bacteriorhodopsin proteoliposome/bilayer planar membrane’

Proteoliposomes were reconstituted from bacteriorhodopsin sheets, asolectin and cholesterol with or without nystatin. Bacteriorhodopsin-mediated electrogenesis was monitored using (1) a proteoliposome suspension and phenyldicarbaundecaborane (PCB^?) probe or (2) proteoliposomes associated with planar bilayer membrane and orthodox electrometer techniques. In the light, PCB^? was shown to be taken up by proteoliposomes. The PCB^? uptake was inhibited by addition of nystatin to an incubation mixture with proteoliposomes if they were reconstituted in the presence of nystatin. Extraproteoliposomal nystatin was without influence if nystatin was omitted from the reconstitution mixture. The nystatin-containing proteoliposomes were associated with a planar bilayer asolectin membrane in the presence of Ca²⁺. It was found that in such a system, bacteriorhodopsin generated a photocurrent charging the proteoliposome-containing (cis-side) compartment negatively and the trans-side compartment positively. The photoresponse was shown to be increased several-fold by addition of nystatin to the trans-side solution. Nystatin addition was ineffective if proteoliposomes were reconstituted without nystatin. Taking into account that nystatin forms ion-permeable pores in a membrane only if present on both sides of the membrane and that this membrane is bilayer, one can explain the above data assuming that (1) the intraproteoliposomal solution does not mix with the extraproteoliposomal one when proteoliposomes are attached to a planar black membrane and (2) the attached proteoliposomes are separated from the trans-side bathing solution by a bimolecular membrane. If this is the case, nystatin in the trans-side bathing solution and inside the attached proteoliposome can form pores across that part of the planar membrane which separates the proteoliposome interior from the trans-side solution. Through these pores, H⁺ (pumped by bacteriorhodopsin from the cis-side solution into the proteoliposome interior) or some other intraproteoliposomal ions can be equilibrated with those in the trans-side solution. As a result, the bacteriorhodopsin-generated photocurrent increases.     

Bioenergetics of Neurotransmitter Transport

Neurotransmitter transporters are essential components in the recycling of neurotransmitters released during neuronal activity. These transporters are the targets for important drugs affecting mood and behavior. They fall into at least four gene families, two encoding proteins in the plasma membrane and two in the synaptic vesicle membrane, although the known vesicular transporters have not all been cloned. Each of these transporters works by coupling the downhill movement of small ions such as Na⁺, Cl^–, K⁺, and H⁺ to the uphill transport of neurotransmitter. Plasma membrane transporters move the transmitter into the cytoplasm by cotransport with Na⁺. Many transporters also couple Cl^– cotransport to transmitter influx and these all belong to the NaCl-coupled family, although within the family the coupling stoichiometry can vary. Transporters for glutamate couple influx of this excitatory amino acid to Na⁺ and H⁺ influx and K⁺ efflux. Transporters in synaptic vesicles couple H⁺ efflux to neurotransmitter transport from the cytoplasm to the vesicle lumen.     

Effects of local anesthetics on properties of tetrodotoxin-sensitive protein reconstituted into liposomes

The effects of some local anesthetics on properties of tetrodotoxin (TTX)-sensitive protein reconstituted into liposomes in such a manner that its TTX-sensitive center is located at the internal surface of the liposome membrane were studied. It was shown that tetracaine, lidocaine and its derivative QX-314 decreased the rate of efflux of radioactive sodium from the²²Na-preloaded proteoliposomes with the same efficiency as TTX acted from the inside of liposomes. The results confirm our earlier suggestion that TTX-sensitive protein is a soluble precursor of the protein forming voltage-dependent sodium channels.Neirofiziologiya/Neurophysiology, Vol. 27, No. 4, pp. 299–302, July–August, 1995.