Potassium channels in synaptosomal membrane examined using patch-clamp techniques and reconstituted giant proteoliposomes

Synaptosomes isolated from the rat cerebral cortex were mixed with sonicated phospholipid vesicles and subjected to freezing-thawing to acquire giant proteoliposomes. Membranes of these giant proteoliposome could thus be studied using patch-clamp techniques. Single-channel currents were measured with the inside-out patch of the membrane, in KCl solutions. Three different potassium channels were detected and unit conductances were 15.1, 28.6 and 91.0 pS, respectively, in a symmetrical 150 mM KCl solution. All these channels are more permeable to potassium than to sodium ions, the permeability ratio being about 2:1. Tetraethylammonium ions blocked these channels. The gating of these potassium channels is independent of the membrane potential, Presumably, these channels play a role in the resting membrane potential of presynaptic nerve terminals.     

Patch-clamp study of liver nuclear ionic channels reconstituted into giant proteoliposomes

Nuclear ionic channels (NICs) represent ubiquitous structures of living cells, although little is known about their functional properties and encoding genes. To characterize NICs, liver nuclear membrane vesicles were reconstituted into either planar lipid bilayers or proteoliposomes. Reconstitution of nuclear envelope (NE) vesicles into planar lipid bilayer proceeded with low efficiency. NE vesicle reconstitution into proteoliposomes led to NIC observations by the patch-clamp technique. Large conductance, voltage-gated, K(+)-permeant and Cl(-)-permeant NICs were characterized. An 80-105-pS K(+)-permeant NIC with conducting sub-state was also recorded. Our data establish that NICs can be characterized upon reconstitution into giant proteoliposomes and retain biophysical properties consistent with those described for native NICs.     

Potassium transport coupled to ATP hydrolysis in reconstituted proteoliposomes of yeast plasma membrane ATPase

Potassium transport coupled to ATP hydrolysis has been reconstituted in proteoliposomes using a highly purified plasma membrane Mg2+-dependent ATPase of the yeast Schizosaccharomyces pombe. The ATPase activity in the incorporated enzyme was strongly stimulated (2.2-fold) by the H+-conducting agent carbonyl cyanide m-chlorophenylhydrazone (CCCP). The H+/K+ exchanger nigericin (in the presence of K+) stimulated 1.6-fold the ATPase activity. When both ionophores were added together, the stimulation was increased up to 2.7-fold. When a potassium concentration gradient (high K+ in) was applied to the proteoliposome membrane, a significant drop in the CCCP-stimulated ATPase activity was observed. Inversion of the K+ concentration gradient (high K+ out) did not decrease the stimulation by CCCP. High Na+ in also decreased the stimulation induced by CCCP in the absence but not in the presence of external K+. However, high Li+ in had no effect. Direct potassium efflux from the proteolyposomes was detected upon addition of MgATP using a selective K+ electrode. The ATP-dependent potassium efflux was abolished in CCCP and/or nigericin-pretreated proteoliposomes. However, during steady state ATP hydrolysis, a transient and small K+ efflux was observed upon addition of a CCCP pulse. I propose that the plasma membrane Mg2+-dependent ATPase in yeast cells not only carries out electrogenic H+ ejection but also drives the uptake of potassium via a voltage-sensitive gate which is closed in the absence and open in the presence of the membrane potential.     

Purified human MDR 1 modulates membrane potential in reconstituted proteoliposomes

Human multidrug resistance (hu MDR 1) cDNA was fused to a P. shermanii transcarboxylase biotin acceptor domain (TCBD), and the fusion protein was heterologously overexpressed at high yield in K(+)-uptake deficient Saccharomyces cerevisiae yeast strain 9.3, purified by avidin-biotin chromatography, and reconstituted into proteoliposomes (PLs) formed with Escherichia coli lipid. As measured by pH- dependent ATPase activity, purified, reconstituted, biotinylated MDR-TCBD protein is fully functional. Dodecyl maltoside proved to be the most effective detergent for the membrane solubilization of MDR-TCBD, and various salts were found to significantly affect reconstitution into PLs. After extensive analysis, we find that purified reconstituted MDR-TCBD protein does not catalyze measurable H(+) pumping in the presence of ATP. In the presence of physiologic [ATP], K(+)/Na(+) diffusion potentials monitored by either anionic oxonol or cationic carbocyanine are easily established upon addition of valinomycin to either control or MDR-TCBD PLs. However, in the absence of ATP, although control PLs still maintain easily measurable K(+)/Na(+) diffusion potentials upon addition of valinomycin, MDR-TCBD PLs do not. Dissipation of potential by MDR-TCBD is clearly [ATP] dependent and also appears to be Cl(-) dependent, since replacing Cl(-) with equimolar glutamate restores the ability of MDR-TCBD PLs to form a membrane potential in the absence of physiologic [ATP]. The data are difficult to reconcile with models that might propose ATP-catalyzed "pumping" of the fluorescent probes we use and are more consistent with electrically passive anion transport via MDR-TCBD protein, but only at low [ATP]. These observations may help to resolve the confusing array of data related to putative ion transport by hu MDR 1 protein.     

Fast and slow kinetics of porin channels from Escherichia coli reconstituted into giant liposomes and studied by patch-clamp.

E. coli porins (OmpF and OmpC) were purified and reconstituted into liposomes which were enlarged to giant proteoliposomes by dehydration-rehydration and studied by patch-clamp. The porins could be closed by voltage pulses under -100 mV. The kinetics of closure was slow, with closure events of about 200 pS in 0.1 M KCl. Rapid fluctuations (in the millisecond range) of about one third (60-70 pS) of the large closure steps were also observed. The data are interpreted as follows: an increase in membrane potential favours the cooperation transition of multimers towards an inactivated state, while monomers which have not been inactivated can flicker rapidly between an open and a short-lived closed state.     

Pharmacological studies of Cav3.1 T-type calcium channels using automated patch-clamp techniques

T-type calcium channels are involved in a variety of physiological and pathophysiological processes, and thus could be therapeutic targets. However, there is no T-type channel selective blocker for use in clinical practice, demanding a need for the development of novel drugs where a higher-throughput screening system is required. Here we present pharmacological studies on Ca(v)3.1 T-type channels using automated patch-clamp. The IC(50) values obtained from automated patch-clamp and conventional one showed a good correlation (correlation coefficient of 0.82), suggesting that the automated patch-clamp is an efficient and reliable method for ranking the drug potencies for T-type channels.     

Cyanine and safranine dyes as membrane potential probes in cytochrome c oxidase reconstituted proteoliposomes

Safranine and the cyanine dye, 3',3'-dipropylthiadicarbocyanine (diSC3-5), were examined as membrane potential probes in cytochrome c oxidase vesicles. The spectra of the vesicle-associated dyes resemble those of the same dyes in organic solvents, indicating that safranine and diSC3-5 probably dissolve in a hydrophobic region of the proteoliposomal membrane. This binding of safranine to proteoliposomes occurs with a dye-membrane dissociation constant in the micromolar range. The binding of safranine and of diSC3-5 to liposomes or proteoliposomes is accompanied by fluorescence enhancement. This enhanced fluorescence is quenched by respiration or by the establishment of a K+ diffusion potential by valinomycin (negative interior). An optimal dye/lipid ratio was required to secure maximum fluorescence quenching of the dyes, whether that quenching was active or passive. Calibrations of both the safranine and the diSC3-5 responses with K+ diffusion potentials were also affected by the dye/lipid ratio. At lower dye/lipid ratios, the calibration curve was linear at higher potentials but deviated from linearity at lower potentials. The converse was true at higher dye/lipid ratios. The non-linearity of the calibration curve at higher potential was attributed to a 'saturation' effect; it may also involve increased permeability of proteoliposomal membrane to protons. Destacking of dye at the lower dye/lipid ratio was probably responsible for the non-linearity of the calibration curves at lower potentials. When all these effects are taken into account, the steady-state value of delta psi generated during maximal proteoliposomal respiration was calculated to be between 140 and 160 mV (interior negative) when measured with either safranine or diSC3-5. We conclude that quantitative estimates of delta psi values can be made using these probes in cytochrome c oxidase reconstituted proteoliposomes provided that appropriate precautions are taken.     

NorA functions as a multidrug efflux protein in both cytoplasmic membrane vesicles and reconstituted proteoliposomes

Overexpression of NorA, an endogenous efflux transporter of Staphylococcus aureus, confers resistance to certain fluoroquinolone antimicrobials and diverse other substrates. The norA gene was amplified by PCR and cloned in the expression vector pTrcHis2. Histidine-tagged NorA (NorA-His) was overexpressed in Escherichia coli cells to prepare two experimental systems, everted membrane vesicles enriched with NorA-His and proteoliposomes reconstituted with purified NorA-His. In membrane vesicles, NorA-His actively transported Hoechst 33342, a dye that is strongly fluorescent in the membrane but has low fluorescence in an aqueous environment. Transport was activated by the addition of ATP or lactate and reversed by the addition of nigericin, with the addition of K(+)-valinomycin having little effect. Transport of Hoechst 33342 was inhibited competitively by verapamil, a known inhibitor of NorA, and by other NorA substrates, including tetraphenyl phosphonium and the fluoroquinolones norfloxacin and ciprofloxacin. In contrast, sparfloxacin, a fluoroquinolone whose antimicrobial activity is not affected by NorA expression, exhibited noncompetitive inhibition. NorA induction and overexpression yielded 0.5 to 1 mg of a largely homogeneous 40- to 43-kDa protein per liter of culture. NorA-His incorporated into proteoliposomes retained the ability to transport Hoechst 33342 in response to an artificial proton gradient, and transport was blocked by nigericin and verapamil. These data provide the first experimental evidence of NorA functioning as a self-sufficient multidrug transporter.     

Carnitine transport in submitochondrial particles and reconstituted proteoliposomes.

Influx and efflux measurements of carnitine with submitochondrial particles lead to the conclusion that carnitine can cross the inner mitochondrial membrane by either facilitated diffusion or more rapidly by a carnitine-carnitine exchange. Both, the facilitated diffusion and the exchange are inhibited by N-ethylmaleimide or mersalyl at low concentrations. Reconstituted particles prepared from liposomes and either submitochondrial particles or an octyl β-glucoside-solubilized preparation were active in catalyzing carnitine-carnitine exchange.     

ATP-dependent phosphate transport in sarcoplasmic reticulum and reconstituted proteoliposomes

During uptake of Ca2+ by rabbit sarcoplasmic reticulum, about 1 mumol of 32Pi was taken up per mumol 45Ca2+ transported. The uptake of Pi was dependent on external Ca2+, Mg2+ and ATP. Intravesicular Ca2+ did not substitute for external Ca2+. In contrast to the accumulation of Ca2+ which was abolished by the ionophore A23187, the uptake of Pi continued to take place provided sufficient Ca2+ was present in the medium. Thus, a Ca2+ gradient did not seem to be required. Similar observations were made with proteoliposomes reconstituted with membrane preparations of sarcoplasmic reticulum and soybean phospholipids. However, when purified Ca2+ -ATPase was used for reconstitution, there was ATP-dependent Ca2+ uptake but no ATP-dependent Pi transport was observed. These data show that the mechanism of Pi transport cannot be a passive movement in response to a Ca2+ gradient but appears to be catalyzed by a specific protein, which is inactivated during purification of the Ca2+ -ATPase. A protein that catalyzes Pi transport in reconstituted vesicles has been solubilized by extraction of sarcoplasmic reticulum with sodium cholate.     

Curvature-electric effects in artificial and natural membranes studied using patch-clamp techniques

Methods for applying sound pressure to membrane patches formed at the tips of patch-clamp pipettes have been developed. Artificial membrane patches were formed from diphytanoyl phosphatidylcholine using a pipette dipping technique. Natural membrane patches were excised (inside-out mode) from collagenase-treated locust muscle membrane. Curvature-electric signals were registered under both voltage clamp and current clamp conditions. The phenomenon of flexoelectricity in membranes has previously been attributed to curvature-induced polarization originating from the liquid crystalline properties of membranes. The estimated magnitude (2·10^-18 C) of the flexoelectric coefficient of the artificial lipid bilayers is consistent with previous findings while that of the muscle membrane was in certain cases several times larger. The present study is the first to report on flexoelectricity in a natural membrane and raises the question of the biological significance of this phenomenon.     

The interaction of ethanol with reconstituted synaptosomal plasma membrane Ca2+ -ATPase

The primary effect of ethanol is on the central nervous system. However, the molecular mechanisms responsible for the physiological symptoms of ethanol intoxication are still unknown. Low concentrations of ethanol were observed to stimulate the activity of the calcium pump from reconstituted synaptosomal plasma membrane Ca2+ -ATPase (PMCA), and ethanol inhibited Ca2+ -ATPase activity at concentrations above 5%. The greatest stimulating effect was obtained with 5% (v/v) ethanol and was lipid-dependent, being 74% when the protein had been reconstituted in phosphatidylcholine (PC) and less when the reconstituted protein had previously been activated by calmodulin or after removal of a 9-kDa autoinhibitory site by controlled trypsinization. Stimulation of the pump by ethanol was lower for the native or trypsin-digested protein in the presence of phosphatidylserine than in PC. These results suggest a direct ethanol-protein interaction, because the activating effect depended on the state of Ca2+ -ATPase (native or truncated, or in presence of calmodulin). The activating mechanism of ethanol may involve opening an autoinhibitory domain located close to the calmodulin binding domain.     

Structure and energy-linked activities in reconstituted bacteriorhodopsin--yeast ATPase proteoliposomes.

Bacteriorhodopsin-F₁·F₀ (mitochondrial oligomycin-sensitive ATPase complex) proteoliposomes have poor proton pumping and photophosphorylation activities when reconstituted by cholate dialysis. A considerable proportion of the bacteriorhodopsin is not incorporated by cholate dialysis, the particles being too large to be combined into liposomes. Much better reconstitution is achieved where the purple membranes are first fragmented by sonication. Optimal incorporation occurs where bacteriorhodopsin and the phospholipids are sonicated together, suggesting that some perturbation of the liposomes is necessary for successful integration. Since F₁·F₀ is denatured by sonication a two-step reconstitution procedure has been developed wherein bacteriorhodopsin is first incorporated by sonication, then F₁·F₀ by cholate dialysis. The vesicles have high phosphorylation rates and also catalyze postillumination [³²P]ATP formation where pyridine is present during first stage illumination.F₁·F₀ can also be incorporated into sonicated bacteriorhodopsin vesicles by “direct incorporation.” This depends on the presence of negatively charged amphiphiles such as cholate or phosphatidylserine in the membranes, and is stimulated by divalent metal cations. Optimum conditions for the various reconstitution procedures are described.     

Flippase activity in proteoliposomes reconstituted with Spinacea oleracea endoplasmic reticulum membrane proteins: evidence of biogenic membrane flippase in plants

Phospholipid translocation (flip-flop) in biogenic (self-synthesizing) membranes such as the endoplasmic reticulum of eukaryotic cells (rat liver) and bacterial cytoplasmic membranes is a fundamental step in membrane biogenesis. It is known that flip-flop in these membranes occurs without a metabolic energy requirement, bidirectionally with no specificity for phospholipid headgroup. In this study, we demonstrate for the first time ATP-independent flippase activity in endoplasmic reticulum membranes of plants using spinach as a model system. For this, we generated proteoliposomes from a Triton X-100 extract of endoplasmic reticulum membranes of spinach and assayed them for flippase activity using fluorescently labeled phospholipids. The half-time for flipping was found to be 0.7-1.0 min. We also show that (a) proteoliposomes can flip fluorescently labeled analogues of phosphatidylcholine and phosphatidylethanolamine, (b) flipping activity is protein-mediated, (c) more than one class of lipid translocator (flippase) is present in spinach membranes, based on the sensitivity to protease and protein-modifying reagents, and (d) translocation of PC and PE is affected differently upon treatment with protease and protein-modifying reagents. Ca (2+)-dependent scrambling activity was not observed in the vesicles reconstituted from plant ER membranes, ruling out the possibility of the involvement of scramblase in translocation of phospholipids. These results suggest the existence of biogenic membrane flippases in plants and that the mechanism of membrane biogenesis is similar to that found in animals.     

Ionic channels in the plasma membrane ofSchizosaccharomyces pombe: Evidence from patch-clamp measurements

Patch-clamp studies of the yeastSchizosaccharomyces pombe reveal that the plasma membrane contains a voltage-gated channel mildly selective for potassium over sodium, lithium, and chloride. The channel exhibits several conductances with a maximum of 153 pS. The channel gates in the region of physiologically relevant voltages, being closed at hyperpolarizing and open at depolarizing voltages. It is not inhibited by tetraethylammonium, quinine, or quinidine applied from the cytoplasmic side of the membrane; similarly, ATP and stretch have no effect. The frequency of its occurrence in patches implies that about 35 channels of this kind are present in the plasma membrane of a single cell.     

Epithelial sodium channels: characterization by using the patch-clamp technique

The patch-clamp technique was used to resolve currents through individual Na-selective ion channels in the apical membrane of the rat cortical collecting tubule. The channels had a single unit conductance of 5 pS under control conditions (cell-attached patches, room temperature, 140 mM NaCl in the pipette). They appeared to be highly selective for Na, as K conduction through them was not measurable in inside-out patches. The channels underwent spontaneous transitions between open and closed states, both states being long-lived. At physiological temperature (37C) the conductance increased to 9 pS and the spontaneous transitions became more rapid. In the presence of amiloride on the luminal side of the membrane, the channel flickered rapidly between open and blocked states. The size of the current transitions did not change. This channel activity was observed only in rats that were fed a low-Na diet to elevate aldosterone secretion. In addition to mineralocorticoid control, the activity of the channels in inside-out patches were modulated by the pH on the cytoplasmic side of the membrane. Alkalinization from pH 6.4 to 7.4 increased the probability of channels' being open by eightfold. Changes in Ca concentration on the cytoplasmic side of the membrane did not directly affect the channels. However, addition of ionomycin, a Ca ionophore, to the bath resulted in a decrease in channel activity measured in cell-attached patches. This suggests that high cytoplasmic Ca may indirectly down-regulate Na channels in this tissue.     

Inhibition of biogenic membrane flippase activity in reconstituted ER proteoliposomes in the presence of low cholesterol levels

Biogenic membranes or self-synthesizing membranes are the site of synthesis of new lipids such as the endoplasmic reticulum (ER) in eukaryotes. Newly synthesized phospholipids (PLs) at the cytosolic leaflet of ER need to be translocated to the lumen side for membrane biogenesis and this is facilitated by a special class of lipid translocators called biogenic membrane flippase. Even though ER is the major site of cholesterol synthesis, it contains very low amounts of cholesterol, since newly synthesized cholesterol in ER is rapidly transported to other organelles and is highly enriched in plasma membrane. Thus, only low levels of cholesterol are present at the biosynthetic compartment (ER), which results in loose packing of ER lipids. We hypothesize that the prevalence of cholesterol in biogenic membranes might affect the rapid flip-flop. To validate our hypothesis, detergent solubilized ER membranes from both bovine liver and spinach leaves were reconstituted into proteoliposomes with varying mol% of cholesterol. Our results show that (i) with increase in the cholesterol/PL ratio, the half-life time of PL translocation increased, suggesting that cholesterol affects the kinetics of flipping, (ii) flipping activity was completely inhibited in proteoliposomes reconstituted with 1 mol% cholesterol, and (iii) FRAP and DSC experiments revealed that 1 mol% cholesterol did not alter the bilayer properties significantly and that flippase activity inhibition is probably mediated by interaction of cholesterol with the protein.     

Electron microscopic observations of reconstituted proteoliposomes with the purified major intrinsic membrane protein of eye lens fibers

The purified major intrinsic protein of the lens fiber plasma membrane (MP26) reconstituted into liposomes favored membrane-to-membrane close contacts as visualized by freeze fracture and immunoelectron microscopy. Reconstituted apposed unilamellar vesicles formed pentalaminar profiles, and multilamellar liposomes showed regions of stacked bilayers. Immunogold labeling, using antibody directed against MP26, demonstrated that this polypeptide is present in regions of membrane-to-membrane close interaction. Fracture faces displayed both randomly distributed clusters of 8-nm polygonal intramembrane particles and membrane domains where a bidimensional lattice of repeating subunits was present. The structural pleomorphism which characterized the MP26-reconstituted proteoliposomes seems quite comparable to that visualized in natural fiber plasma membrane domains.     

Preparation and some properties of giant liposomes and proteoliposomes

Optimal conditions for formation of giant liposomes and proteoliposomes were investigated. A suspension of small unilamellar vesicles made of various phospholipids in a buffer of 0-3 M KCl, 0.1 mM EDTA, and 20 mM MOPS (pH 7.0) was subjected to a freeze-thaw treatment. Giant multilamellar liposomes of diameter ranging from 10 to 60 microns were found to form from phospholipid mixtures containing phosphatidylethanolamine as a major component and phosphatidylserine as a minor component. The concentration of KCl optimal for the giant vesicle formation was 30-500 mM. By applying a patch-pipette to a giant liposome, suitable conditions for obtaining a high-resistance (giga-ohm) seal were sought. It was found that use of a patch-pipette of relatively small tip diameter (less than 1 micron), the presence of divalent metal cations in the suspension medium and inflation of vesicles in a hypotonic solution facilitated giga-seal formation. In a suspension of asolectin (soybean phospholipid) vesicles which had been subjected to the freeze-thaw treatment, giant unilamellar vesicles were found. They could be held on the tip of a suction pipette and impaled with a microelectrode filled with an EGTA solution. Small unilamellar proteoliposomes were prepared by the cholate-dialysis method from asolectin and sarcoplasmic reticulum vesicles, and were subjected to a freeze-thaw cycle. When the ratio of exogenous phospholipid to protein was larger than 10, giant multilamellar vesicles were formed.(ABSTRACT TRUNCATED AT 250 WORDS)