Spontaneous opening at zero membrane potential of sodium channels from eel electroplax reconstituted into lipid vesicles

Summary The voltage-dependent sodium channel from the eel electroplax was purified and reconstituted into vesicles of varying lipid composition. Isotopic sodium uptake experiments were conducted with vesicles at zero membrane potential, using veratridine to activate channels and tetrodotoxin to block them. Under these conditions, channel-dependent uptake of isotopic sodium by the vesicles was observed, demonstrating that a certain fraction of the reconstituted protein was capable of mediating ion fluxes. In addition, vesicles untreated with veratridine showed significant background uptake of sodium; a considerable proportion of this flux was blocked by tetrodotoxin. Thus these measurements showed that a significant subpopulation of channels was present that could mediate ionic fluxes in the absence of activating toxins. The proportion of channels exhibiting this behavior was dependent on the lipid composition of the vesicles and the temperature at which the uptake was measured; furthermore, the effect of temperature was reversible. However, the phenomenon was not affected by the degree of purification of the protein used for reconstitution, and channels in resealed electroplax membrane fragments or reconstituted, solely into native eel lipids did not show this behavior. The kinetics of vesicular uptake through these spontaneously-opening channels was slow, and we attribute this behavior to a modification of sodium channel inactivation.     

Transport-Specific isolation of large channels reconstituted into lipid vesicles

Summary To develop a technique for purifying and identifying pore-forming membrane proteins, we used a transport-specific increase in buoyant density to select for lipid vesicles containing voltage-dependent anion channels (VDAC). Monodisperse, single-walled vesicles were formed by gel filtration from a detergent-solubilized mixture of lipid and protein in a urea buffer. The vesicles were layered on a linear iso-osmolar density gradient formed of urea and sucrose buffers. Since VDAC is open at zerotrans-membrane voltage and is permeable to urea and sucrose, vesicles containing functional VDAC should become more dense as sucrose enters and urea leaves, while those lacking open channels should maintain their original density. Vesicles formed in the absence of VDAC migrated to a characteristic density, while vesicles formed in the presence of VDAC fractionated into two populations in the gradients, one migrating to the same density as the vesicles formed without VDAC, and one at a significantly greater density. In contrast to the lower density vesicles, the higher density vesicles showed a high permeability to calcein, and contained functional VDAC channels (shown by electrophysiological recordings following fusion with a planar bilayer). Thus, vesicles containing open channels were separable from those that did not by a transport-specific shift in density. This technique may be useful for the enrichment of channels of known permeability properties from impure, material.     

Toad bladder amiloride-sensitive channels reconstituted into planar lipid bilayers

In the present study we used established methods to obtain apical membrane vesicles from the toad urinary bladder and incorporated these membrane fragments to solvent-free planar lipid bilayer membranes. This resulted in the appearance of a macroscopic conductance highly sensitive to the diuretic amiloride added to the cis side. The blockage is voltage dependent and well described by a model which assumes that the drug binds to sites in the channel lumen. This binding site is localized at about 15% of the electric field across the membrane. The apparent inhibition constant (K(0)) is equal to 0.98 microM. Ca2+, in the micromolar range on the cis side, is a potent blocker of this conductance. The effect of the divalent has a complex voltage dependence and is modulated by pH. At the unitary level we have found two distinct amiloride-blockable channels with conductances of 160 pS (more frequent) and 120 pS. In the absence of the drug the mean open time is around 0.5 sec for both channels and is not dependent on voltage. The channels are cation selective (PNa/PCl = 15) and poorly discriminate between Na+ and K+ (PNa/PK = 2). Amiloride decreases the lifetime in the open state of both channels and also the conductance of the 160-pS channel.     

Membrane topology of insulin receptors reconstituted into lipid vesicles

Insulin receptors were incorporated into liposomes by two different procedures, one using dialysis and one using detergent removal by Bio-Beads. Receptor incorporation was analyzed by gradient centrifugation and electron microscopy. Reconstituted receptors projected up to 12 nm above the membrane and exhibited a T-shaped structure compatible with that previously described for the solubilized receptor. Insulin binding and autophosphorylation experiments indicated that approx. 50% of the receptors were incorporated right-side out. Such random orientation was confirmed by immunogold labeling of the - and the -subunit of the receptor. Immunogold labeling of the C-terminus of the -subunit indicates that it resides about 6 nm off the membrane, while two -subunit epitopes were labeled at about twice this distance, confirming that the -subunit is harbored in the cross-bar of the T-structure.We thank Ms. Birthe Nystrøm, Lisette Hansen and Ulla Blankensteiner for excellent technical assistance and Ms. Birgit Risto for skillful work with the photographic prints.     

Polypeptide neurotoxins modify gating and apparent single-channel conductance of veratridine-activated sodium channels in planar lipid bilayers

Summary The effects of scorpion and sea anemone polypeptide toxins on partially purified veratridine (VER)-activated Na channels from rat brain were studied at the single-channel level in planar lipid bilayers. The probability of the VER-activated channel being open (P _o ) increased with depolarization;P _o was 0.5 at –40 to –50 mV. Saxitoxin (STX) blocked VER-activated channels with an apparent dissociation constant of about 1nm at –45 mV. The apparent single-channel conductance was approximately 9 pS, similar to that seen in VER-activated Na channels from skeletal muscle transverse tubules. Addition of sea anemone or scorpion polypeptide toxins to VER-activated Na channels resulted in a 19% increase in apparent single-channel conductance and a hyperpolarizing shift in theP _o vs. V _m relation such that the channels were more likely to be open at potentials <40 mV. These effects of the polypeptide toxins on the single-channel properties of VER-activated Na channels may account for the previously described potentiation of VER action by polypeptide toxins.     

Optical study of active ion transport in lipid vesicles containing reconstituted Na,K-ATPase

Summary A fluorescence method is described for the measurement of ATP-driven ion fluxes in lipid vesicles containing purified Na,K-ATPase. The membrane voltage of enzyme containing vesicles was measured by using a voltage-sensitive indocyanine dye. By addition of valinomycin the vesicle membrane is made selectively permeable to K⁺ so that the membrane voltage approaches the Nernst potential for K⁺. With constant external K⁺ concentration, the time course of internal K⁺ concentration can be continuously measured as change of the fluorescence signal after activation of the pump. The optical method has a higher time resolution than tracer-flux experiments and allows an accurate determination of initial flux rates. From the temperature dependence of active K⁺ transport its activation energy was determined to be 115 kJ/mol. ATP-stimulated electrogenic pumping can be measured as a fast fluorescence change when the membrane conductance is low (i.e., at low or zero valinomycin concentration). In accordance with expectation, the amplitude of the fast signal change increases with decreasing passive ion permeability of the vesicle membrane. The resolution of the charge movement is so high that a few pump turnovers can be easily detected.     

Interaction of charged lipid vesicles with planar bilayer lipid membranes: Detection by antibiotic membrane probes

A technique has been developed for monitoring the interaction of charged phospholipid vesicles with planar bilayer lipid membranes (BLM) by use of the antibiotics Valinomycin, Nonactin, and Monazomycin as surface-charge probes. Anionic phosphatidylserine vesicles, when added to one aqueous compartment of a BLM, are shown to impart negative surface charge to zwitterionic phosphatidylocholine and phosphatidylethanolamine bilayers. The surface charge is distributed asymmertically, mainly on the vesicular side of the BLM, and is not removed by exchange of the vesicular aqueous solution. Possible mechanisms for the vesicle-BLM interactions are discussed.     

Ouabain-insensitive active sodium transport in rat jejunum: Evidence from ATPase activities,Na uptake by basolateral membrane vesicles and in vitro transintestinal transport

The basolateral membrane of the jejunal enterocyte of the rat was separated by self-orienting Percoll-gradient centrifugation and further purified from brush border contamination. Pellets were analysed for Mg-, Na- and (Na, K)-ATPase activities. The uptake of 0·02 M NaCl was also followed by the rapid micro-filtration technique. Transintestinal transport of fluid and electrolytes, and cell water, Na and K were determined in the in vitro everted and incubated jejunum. There is ouabain-insensitive Na-ATPase in addition to the well-known (Na, K)-ATPase in the basolateral membrane. These are differently inhibited by furosemide and ethacrynate. Na uptake by osmotically active basolateral membrane vesicles is enhanced by ATP and a further enhancement is obtained if there is intravesicular K. The ATP effect is inhibited differently by strophanthidin, furosemide and ethacrynate. In the everted sac preparation, transintestinal transport of Na and fluid still occurs when the Na/K pump is totally inhibited by ouabain. These experimental results suggest that there is also a ouabain-insensitive Na pump, different from the Na/K pump, in the basolateral membrane.     

Dynamic fluorescence polarization studies on lipid mobilities in phospholipid vesicles in the presence of calcium mediators

The influence of Ca²⁺ mediators (nifedipine, verapamil and prostaglandin F_2α) on fluorescence polarization of l-anilino-8-napthalene-sulphonate in dipalmitoyl phosphatidylcholine and dimyristoyl phosphatidylcholine liposomes was studied at various temperatures to understand the dynamic behaviour of membrane lipids. We also studied the effect of change in calcium concentration on the fluorescence polarization of the dye in the liposomes. Our results show increase in polarization (indicative of stiffening of the membrane) in the presence of Ca²⁺ ions. In the case of dimyristoyl phosphatidylcholine liposomes, all 3 drugs caused decrease in fluorescence polarization (increase in fluidity of the membrane) with or without Ca²⁺ ions in the medium. Contrary to this, in the case of dipalmitoyl phosphatidylcholine liposomes, the fluidization effect is observed for all the 3 drugs in the absence of Ca²⁺ ions; in the presence of Ca²⁺ ions stiffening is observed upon addition of nifedipine and verapamil which are antagonists, and fluidization is observed upon addition of prostaglandin F_2α. The role of drug-induced fluidity changes in membranes in therapy planning is discussed in the paper.     

Regulation of the skeletal sarcoplasmic reticulum Ca2+-ATPase by phospholamban and negatively charged phospholipids in reconstituted phospholipid vesicles

The Ca²⁺-ATPase of skeletal sarcoplasmic reticulum was purified and reconstituted in proteoliposomes containing phosphatidylcholine (PC). When reconstitution occurred in the presence of PC and the acidic phospholipids, phosphatidylserine (PS) or phosphatidylinositol phosphate (PIP), the Ca²⁺-uptake and Ca²⁺-ATPase activities were significantly increased (2–3 fold). The highest activation was obtained at a 50:50 molar ratio of PSYC and at a 10:90 molar ratio of PIP:PC. The skeletal SR Ca²⁺-ATPase, reconstituted into either PC or PC:PS proteoliposomes, was also found to be regulated by exogenous phospholamban (PLB), which is a regulatory protein specific for cardiac, slow-twitch skeletal, and smooth muscles. Inclusion of PLB into the proteoliposomes was associated with significant inhibition of the initial rates of Ca²⁺-uptake, while phosphorylation of PLB by the catalytic subunit of cAMP-dependent protein kinase reversed the inhibitory effects. The effects of PLB on the reconstituted Ca²⁺-ATPase were similar in either PC or PC: PS proteoliposomes, indicating that inclusion of negatively charged phospholipid may not affect the interaction of PLB with the skeletal SR Ca²⁺-ATPase. Regulation of the Ca²⁺-ATPase appeared to involve binding with the hydrophilic portion of phospholamban, as evidenced by crosslinking experiments, using a synthetic peptide which corresponded to amino acids 1–25 of phospholamban. These findings suggest that the fast-twitch isoform of the SR Ca²⁺-ATPase may be also regulated by phospholamban although this regulator is not expressed in fast-twitch skeletal muscles.     

Quantification of leakage from large unilamellar lipid vesicles by fluorescence correlation spectroscopy

Fluorescence correlation spectroscopy (FCS) is a powerful experimental technique that in recent years has found numerous applications for studying biological phenomena. In this article, we scrutinize one of these applications, namely, FCS as a technique for studying leakage of fluorescent molecules from large unilamellar lipid vesicles. Specifically, we derive the mathematical framework required for using FCS to quantify leakage of fluorescent molecules from large unilamellar lipid vesicles, and we describe the appropriate methodology for successful completion of FCS experiments. By use of this methodology, we show that FCS can be used to accurately quantify leakage of fluorescent molecules from large unilamellar lipid vesicles, including leakage of fluorescent molecules of different sizes. To demonstrate the applicability of FCS, we have investigated the antimicrobial peptide mastoparan X. We show that mastoparan X forms transient transmembrane pores in POPC/POPG (3:1) vesicles, resulting in size-dependent leakage of molecules from the vesicles. We conclude the paper by discussing some of the advantages and limitations of FCS as compared to other existing methods to measure leakage from large unilamellar lipid vesicles.     

Resistance of Ca2+-ATPase to dilution by excess phospholipid in reconstituted vesicles

Ca²⁺-ATPase and other membrane proteins of the sarcoplasmic reticulum membrane from rabbit skeletal muscle have been reconstituted into lipid vesicles with increasing amounts of phosphatidylcholine. The protein composition and phospholipid concentration of these vesicles were analyzed by determining the density of the reconstituted membrane vesicles on linear H₂O-²H₂O gradients, in a constant concentration of sucrose. In all combinations of the Ca²⁺-ATPase with a weight excess of phosphatidylcholine, the reconstituted vesicles had a phospholipid-to-protein ratio similar to that of the native sarcoplasmic reticulum membrane, even though both solubilization and mixing had occurred. These vesicles of low phospholipid and high protein content exhibited all the original Ca²⁺-ATPase activity and ATP-stimulated calcium transport. The Ca²⁺-ATPase, and the calcium-binding proteins to a lesser extent, may order the lipid in such a manner so as to maintain the initial stoichiometry of lipid to protein observed in the native sarcoplasmic reticulum membrane.     

七叶皂苷钠对肠缺血/再灌注肠过氧化损伤的影响

目的：探讨七叶皂苷钠对肠缺血/再灌注肠过氧化损伤的影响及其机制。方法：复制大鼠肠缺血/再灌注（I/R）损伤模型,观察七叶皂苷钠对血浆和肠组织超氧化物歧化酶（SOD）、丙二醛（MDA）、二胺氧化酶（DAO）、髓过氧化物酶（MPO）的影响,同时观察肠组织水肿和病理损害。结果：七叶皂苷钠可显著改善肠损伤,降低肠组织湿/干比值及含水率,同时升高血浆和肠组织SOD活性,降低血浆和肠组织MPO活性及MDA含量（P〈0.01）。结论：七叶皂苷钠对肠I/R后肠黏膜具有保护作用,其机制可能与抑制中性粒细胞的聚集与活化,对抗脂质过氧化损伤有关。     

Phospholipid dependence of the anion transport system of the human erythrocyte membrane. Studies on reconstituted band 3/lipid vesicles

Band 3 protein extracted from human erythrocyte membranes by Triton X-100 was recombined with the major classes of phospholipid occurring in the erythrocyte membrane. The resulting vesicle systems were characterized with respect to recoveries, phospholipid composition, protein content and vesicle size as well as capacity and activation energy of sulfate transport. Transport was classified into band-3-specific fluxes and unspecific permeability by inhibitors. Transport numbers (sulfate ions per band 3 per minute) served as a measure of functional recovery after reconstitution. The transport properties of band 3 proved to be insensitive to replacement of phosphatidylcholine by phosphatidylethanolamine, while sphingomyelin and phosphatidylserine gradually inactivated band-3-specific anion transport when present at mole fractions exceeding 30 mol%. The activation energy of transport remained unaltered in spite of the decrease in transport numbers. The results, which are discussed in terms of requirements of band 3 protein function with respect to the fluidity and surface charge of its lipid environment, provide a new piece of evidence that the transport function of band 3 protein depends on the properties of its lipid environment just as the catalytic properties of some other membrane enzymes. The well-established species differences in anion transport (Gruber, W. and Deuticke, B. (1973) J. Membrane Biol. 13, 19–36) may to some extent reflect this lipid dependence.     

Implication of segment S45 in the permeation pathway of voltage-dependent sodium channels

A 34-mer peptide, encompassing the S4 and S45 segments of domain IV of the electric eel voltage-dependent sodium channel, was synthesized in order to test the potential implication of S45 in the gating or permeation pathway. The secondary structure of peptide S4–S45 assessed by circular dichroism was found mainly helical, both in organic solvents and in lipid vesicles, especially negatively-charged ones. The macroscopic conductance properties of neutral and negatively-charged Montal-Mueller planar lipid bilayers doped with S4–S45 were studied and compared with those of S4. With regard to voltage-dependence, the most efficient system was S4–S45 in neutral bilayers. Voltage thresholds for exponential conductance development were found to correlate with the background or leak conductance. Assuming that the latter reflects interfacial peptide concentration, the mean apparent number of monomers per conducting aggregate could be estimated to be 3–5. In single-channel experiments, the most probable events had amplitudes of 8 pS and 5 pS in neutral and negatively-charged bilayers respectively. Ionic selectivity under salt gradients conditions, both at macroscopic and single-channel levels, was in favour of sodium ions (P_Na/P_K = 3). These properties compare favourably to previous reports dealing with peptide modelling transmembrane segments of voltage-dependent ionic channels. Specifically, when compared to S4 alone, the reduced unit conductance and the increased selectivity for sodium support the implication of the S45 region in the inner lining of the open configuration of sodium channels. Correspondence to: H. Duclohier     

The reconstitution and activity of the small multidrug transporter EmrE is modulated by non-bilayer lipid composition

The ability of multidrug transport proteins within biological membranes to recognise a diverse array of substrates is a fundamental aspect of antibiotic resistance. Detailed information on the mechanisms of recognition and transport can be provided only by in vitro studies in reconstituted bilayer systems. We describe the controlled, efficient reconstitution of the small multidrug transporter EmrE in a simple model membrane and investigate the effect of non-bilayer lipids on this process. Transport activity is impaired, in line with an increase in the lateral pressure within the bilayer. We demonstrate the potential of this lateral pressure modulation method as a general approach to the folding and assembly of membrane proteins in vitro, by recovering functional transporter from a partly denatured state. Our results highlight the importance of optimising reconstitution procedures and bilayer lipid composition in studies of membrane transporters. This is particularly pertinent for multidrug proteins, and we show that the use of a sub-optimal lipid bilayer environment or reconstitution method could lead to incorrect information on protein activity.     

A long isoform of the epithelial sodium channel alpha subunit forms a highly active channel

A long isoform of the human Epithelial Sodium Channel (ENaC) α subunit has been identified, but little data exist regarding the properties or regulation of channels formed by α₇₂₈. The baseline whole cell conductance of oocytes expressing trimeric α₇₂₈βγ channels was 898.1 ± 277.2 and 49.59 ± 13.2 µS in low and high sodium solutions, respectively, and was 11 and 2 fold higher than the conductances of α₆₆₉βγ in same solutions. α₇₂₈βγ channels were also 2 to 5 fold less sensitive to activation by the serine proteases subtilisin and trypsin than α₆₆₉βγ in low and high Na⁺ conditions. The long isoform exhibited lower levels of full length and cleaved protein at the plasma membrane and a rightward shifted sensitivity to inhibition by increases of [Na⁺]_i. Both channels displayed similar single channel conductances of 4 pS, and both were activated to a similar extent by reducing temperature, altogether indicating that activation of baseline conductance of α₇₂₈βγ was likely mediated by enhanced channel activity or open probability. Expression of α₇₂₈ in native kidneys was validated in human urinary exosomes. These data demonstrate that the long isoform of αENaC forms the structural basis of a channel with different activity and regulation, which may not be easily distinguishable in native tissue, but may underlie sodium hyperabsorption and salt sensitive differences in humans.     

A long isoform of the epithelial sodium channel alpha subunit forms a highly active channel

A long isoform of the human Epithelial Sodium Channel (ENaC) α subunit has been identified, but little data exist regarding the properties or regulation of channels formed by α₇₂₈. The baseline whole cell conductance of oocytes expressing trimeric α₇₂₈βγ channels was 898.1 ± 277.2 and 49.59 ± 13.2 µS in low and high sodium solutions, respectively, and was 11 and 2 fold higher than the conductances of α₆₆₉βγ in same solutions. α₇₂₈βγ channels were also 2 to 5 fold less sensitive to activation by the serine proteases subtilisin and trypsin than α₆₆₉βγ in low and high Na⁺ conditions. The long isoform exhibited lower levels of full length and cleaved protein at the plasma membrane and a rightward shifted sensitivity to inhibition by increases of [Na⁺]_i. Both channels displayed similar single channel conductances of 4 pS, and both were activated to a similar extent by reducing temperature, altogether indicating that activation of baseline conductance of α₇₂₈βγ was likely mediated by enhanced channel activity or open probability. Expression of α₇₂₈ in native kidneys was validated in human urinary exosomes. These data demonstrate that the long isoform of αENaC forms the structural basis of a channel with different activity and regulation, which may not be easily distinguishable in native tissue, but may underlie sodium hyperabsorption and salt sensitive differences in humans.     

Electrical currents generated by a partially purified Na/Ca exchanger from lobster muscle reconstituted into liposomes and adsorbed on black lipid membranes: Activation by photolysis of caged Ca2+

The Na/Ca exchanger from lobster muscle crossreacts specifically with antibodies raised against the dog heart Na/Ca exchanger. Immunoblots of the lobster muscle and mammalian heart exchangers, following SDS-PAGE, indicate that the invertebrate and mammalian exchangers have similar molecular weights: about 120 kDa. The exchanger from lobster muscle was partially purified and functionally reconstituted into asolectin vesicles which were loaded with 160 mm NaCl. ⁴⁵Ca uptake by these proteoliposomes was promoted by replacing 160 mm NaCl in the external medium with 160 mm KCl to produce an outwardly-directed Na⁺ concentration gradient. When the proteoliposomes were adsorbed onto black lipid membranes (BLM), and DMNitrophen-Ca²⁺ (caged Ca²⁺) was added to the KCl medium, photolytically-evoked Ca²⁺ concentration jumps elicited transient electric currents. These currents corresponded to positive charge exiting from the proteoliposomes, and were consistent with the Na/Ca exchanger-mediated exit of 3 Na⁺ in exchange for 1 entering Ca²⁺. The current was dependent upon the Ca²⁺ concentration jump, the protein integrity, and the outwardly directed Na⁺ gradient. KCl-loaded proteoliposomes did not produce any current. Low external Na⁺ concentrations augmented the current, whereas Na⁺ concentrations >25 mM reduced the current. The dependence of the current on free Ca²⁺ was Michaelis-Menten-like, with halfmaximal activation (K_M(Ca)) at <10 m Ca²⁺. Caged Sr²⁺ and Ba²⁺, but not Mg²⁺, also supported photolysisevoked outward current, as did Ni²⁺, but not Mn²⁺. However, Mg²⁺ and Mn²⁺ augmented the Cadependent current, perhaps by facilitating the adsorption of proteoliposomes to the BLM. The Ca-dependent current was irreversibly blocked by La³⁺ (added as 200 m DMN-La³⁺). The results indicate that the properties of the Na/Ca exchanger can be studied with these electrophysiological methods.The technical assistance of Verena Heiselpetz in some experiments is gratefully acknowledged. This work was partly supported by the Deutsche Forschungsgemeinschaft (SFB 169) and by National Institutes of Health grants HL30315 and GM39500 to JHK and HL45215 and NS16106 to MPB. MPB was the recipient of a Senior Scientist Award from the Alexander von Humboldt Stiftung.