Ion-transporting properties and ATPase activity of (Na+ + K+)-ATPase large subunit incorporated into bilayer lipid membranes

A purified (Na+ + K+)-ATPase large subunit obtained from microsomes by water-alcohol extraction was incorporated into a bilayer lipid membrane. The protein formed in the membrane conductance channels which were sensitive to ouabain and selective for monovalent cations. ATP activated these channels in the presence of sodium and potassium ions. When sodium ions were eliminated ATP did not change the conductance of the modified membrane whereas p-nitrophenyl phosphate increased it. The (Na+ + K+)-ATPase large subunit incorporated into bilayer lipid membrane possessed an ATPase activity. The presence of a potential on the membrane was a necessary condition for the enzyme incorporated into a bilayer lipid membrane to show high ATPase activity. Increasing the potential above 100 mV resulted in the closing of conductance channels.     

Calcium channel activity in a purified dihydropyridine-receptor preparation of skeletal muscle

A purified dihydropyridine-receptor complex (DHPR) of skeletal muscle consisting of a major polypeptide of Mr 150K under reducing conditions induces divalent cation selective channels when incorporated into planar lipid bilayers. Channels were inserted into preformed planar bilayers by two techniques: (i) direct dilution of detergent-solubilized DHPR into the aqueous chambers adjacent to the bilayer membrane or (ii) reconstitution of DHPR into phospholipid vesicles followed by fusion of the preformed vesicles to the planar bilayer membrane. Unlike native membrane preparations of t-tubules, which only have one major Ca channel type of slope conductance of 12 pS in symmetrical 100 mM Ba, the purified DHPR complex induced at least two channel types with conductances of 12-14 and 22 pS. Some recordings suggest that these two channels are statistically coupled in time, i.e., that they may correspond to substrates of the same DHPR channel. Activity was found to occur spontaneously in the absence of the Ca channel agonist Bay k 8644. The 12-14-pS channel from DHPR exhibits voltage-dependent kinetics, is highly selective for barium ions, and was inhibited by micromolar nitrendipine. The 12-14-pS DHPR channel appears to be identical with functional Ca channels previously described in native t-tubules.     

pH modulation of large conductance potassium channel from adrenal chromaffin granules

We report here that large conductance K(+) selective channel in adrenal chromaffin granules is controlled by pH. We measured electrogenic influx of (86)Rb(+) into chromaffin granules prepared from bovine adrenal gland medulla. The (86)Rb(+) influx was inhibited by acidic pH. Purified chromaffin granule membranes were also fused with planar lipid bilayer. A potassium channel with conductance of 432+/-9 pS in symmetric 450 mM KCl was observed after reconstitution into lipid bilayer. The channel activity was unaffected by charybdotoxin, a blocker of the Ca(2+)-activated K(+) channel of large conductance. It was observed that acidification to pH 6.4 cis side of the membrane lowered the channel open probability and single channel conductance. Whereas only weak influence on the single channel current amplitude and open probability were observed upon lowering of the pH at the trans side. We conclude that a pH-sensitive large conductance potassium channel operates in the chromaffin granule membrane.     

Single channel H+ currents through reconstituted chloroplast ATP synthase CF0-CF1.

The purified chloroplast ATP synthase (CF(0)-CF(1)) was reconstituted into azolectin liposomes from which bilayer membranes on the tip of a glass pipette ('dip stick technique')and planar bilayer membranes were form ed. The CF(0)-CF(1) facilitated ion conductance through the bilayer membranes. Our results clearly indicated that the observed single channel currents were carried by H+ through the isolated and reconstituted chloroplast ATPase. We demonstrated that in proteoliposomes it is the whole enzyme complex CF(0)-CF(1) and not the membrane sector CF(0) alone that constitutes a voltagegated, proton-selective channel with a high conductance of 1-5 pS at pH 5.5-8.0. After removal of CF(1) from the liposomes by NaBr treatment the membrane sector CF(0) displayed various kinds of channels also permeable to monovalent cations. The open probability P(0) of the CF(0)-CF(1) channel increased considerable with increasing membrane voltage [from P(0) less than or equal to 1% (V(m) less than or equal to 120 mV) to P(0) less than or equal to 30% (120 mV less than or equal to Vm 200 mV)]. In the presence of ADP (3 microM) and P(i) (5 microM), which specifically bind to CF(1), the open probability decreased and venturicidin (1 microM), a specific inhibitor of H+ flow through CF(0) in thylakoid membranes, blocked the channel almost completely. Our results, which reveal a high channel unit conductance, and at membrane voltages less than 100 mV low open probability with concomitant mean open times in the micros timescale (less than 100 micros) for the energy coupling in the enzyme complex. At physiological membrane voltages for photophosphorylation (about 30 mV) the enzyme complex would then display a time-averaged conductance of about 1 fS.     

Modification of a voltage-gated K+ channel from sarcoplasmic reticulum by a pronase-derived specific endopeptidase

AK+ -selective membrane conductance channel from rabbit sarcoplasmic reticulum (SR) is studied in an artificial planar phospholipid bilayer. Membranes containing many such channels display voltage-dependent conductance, which is well described by a two-state conformational equilibrium with a free energy term linearly dependent on applied voltage. Pronase-derived alkaline proteinase b (APb), when added to the side of the membrane opposite to the SR vesicles (trans side), reduces the voltage dependence of the K+ conductance. Single-channel fluctuation experiments show that after APb treatment, the channel is still able to undergo transitions between its open and closed states, but that the probability of forming the open state is only slightly voltage-dependent. In terms of the conformational model, the enzyme's primary effect is to reduce the effective gating charge of the opening process by over 80%; a second effect of APb is to reduce the internal free energy of opening from +1.2 to +0.4 kcal/mol. The kinetics of APb action are strongly voltage-dependent, so as to indicate that the enzyme can react only with the channel's open state. The results imply that the channel contains a highly charged polypeptide region which moves in the direction perpendicular to the membrane plane when transitions between the open and closed states occur. A lysine or arginine residue in this region becomes exposed to the trans aqueous solution when the channel is in its open conformation.     

叶绿体H^＋—ATP酶在平板脂双层上重组及其质子传导的研究

从菠菜(Spinacia oleracea Mill.)叶中分离获得H~ -ATP酶(CF_0-CF_1)复合体。将CF_0-CF_1重组于平板脂双层上,在电压钳位下,研究CF_0～CF_1的质子传导性能,观察到:(1)当CF_0-CF_1重组于平板脂双层上后,平板膜电阻由10～20GΩ立即下降到1GΩ左右。(2)溶液中蛋白质(CF_0-CF_1)浓度在2mg/L下可记录到单通道电流的涨落,单位电导约在5～10pS。(3)通道电流随膜两侧ΔpH变化而改变,在ΔpH为2～4时,膜电流随ΔpH增加而增大,在ΔpH为4.5时膜电流呈现回落。(4)质子传导抑制剂Dicyclohexyl-carbodiimide(DCCD)显示出迅速地且不可逆地阻断通道电流。(5)无金属离子的溶液中,跨膜(BLM)的ΔpH为3时,在0～ 150mV钳位下,镁离子比钙离子所引起的CF_0-CF_1的通道电流要大得多。以上结果不仅表明CF_0-CF_1已成功地组装于人工膜上,而且也显示出镁离子直接参与了质子传导过程。     

Functional Expression of p64, an Intracellular Chloride Channel Protein

p64 is a protein identified as a chloride channel by biochemical purification from kidney microsomes. We expressed p64 in HeLa cells using a recombinant vaccinia virus/T7 RNA polymerase driven system. Total cell membranes were prepared from infected/transfected cells and fused to a planar lipid bilayer. A novel chloride channel activity was found in cells expressing p64 and not in control cells. The p64-associated activity shows strong anion over cation selectivity. Single channels show prominent outward rectification with single channel conductance at positive potentials of 42 pS. The chloride channel activity is activated by treatment of the membranes with alkaline phosphatase and inhibited by DNDS and by TS-TM calix(4)arene. Whole membrane anion permeability was determined by a chloride efflux assay, revealing that membranes from cells expressing p64 showed a small but highly significant increase in chloride permeability, consistent with expression of a novel chloride channel activity. Received: 17 November 1997/Revised: 9 February 1998     

Proton-Conductivity of CF0-CF1 Reconstructed into Planar Lipid Bilayer

The proton conductable ATP synthase (CF0-CF1) is the key enzyme of energy conversion in the membrane of bacteria, mitochondria and chloroplast. In spite of a large body of studies, the structure and molecular mechanism of ATP synthases are still elusive. In order to learn the mechanism of ATP synthases, the authors used voltage-olamp technique to study the effect of different conditions on the proton conductance of F0-F1 into planar lipid bilayer membrane. The results obtained were as follows: (1) When CF0-CF1 was reconstructed into planar lipid bilayer membrane, the resistance decreased by 10 times. (2) Channel-like current was recorded at the low concentration of CF0-CFl(protein 2 mg/L) in the solution. (3) In metal ion-free solution, the channel currents changed with the trans-membrane proton gradient (ApH). Under holding potential from 0 to + 150 mV, the stimulation of △pH on channel current increased with a rise in the ApH from 2 to 4, the stimulation of 4.5 △pH on channel current was weaker than that of △pH 4.0. (4) The proton conduetance inhibitor, dicyclohexylcarbodiimide (DCCD), showed a rapid and irreversible inhibition effect on the channel current. (5) In metal ion-free solution (10 mmol/L Tris-HC1), when the ApH across the black lipid membrane (BLM) maintained at 3.0, the addition of Mg2 + caused a alger channel current of CF0-CF1 than the addition of Ca2+ , with holding potential from 0 to + 150 mV. The results indicated that reconstruction of CF0-CF1 was successful and Mg2 + was directly involved in the proton conductance pathways.     

Purification and reconstitution in lipid bilayer membranes of an outer membrane, pore-forming protein of Aeromonas salmonicida.

We have purified a major outer membrane protein from Aeromonas salmonicida. This 42-kilodalton protein shared several physical characteristics with enterobacterial porins in that it was noncovalently associated with the peptidoglycan, it was released from the peptidoglycan in the presence of 0.1 M NaCl and sodium dodecyl sulfate, and its mobility on sodium dodecyl sulfate-polyacrylamide gels was dependent on the solubilization temperature before electrophoresis. When added to the aqueous solution bathing a planar bilayer membrane it caused the conductance of the membrane to increase by several orders of magnitude. At lower protein concentrations, single channels with an average conductance of 1.6 nS in 1 M KCl were incorporated into the membrane in a stepwise fashion. Evidence that the protein formed a large, relatively nonselective, water-filled channel was obtained by performing single-channel experiments at different NaCl concentrations and in a variety of different salts. Current through the channel was a linear function of the applied voltage, and no evidence of voltage gating was observed. In addition, we obtained evidence for a 43-kilodalton channel-forming protein in the outer membrane of A. hydrophila with a similar single-channel conductance as the 42-kilodalton protein in 1 M NaCl.     

Transmembrane segment M2 of glycine receptor as a model system for the pore-forming structure of ion channels

The glycine receptor belongs to the ligand-gated ion channel superfamily. It is a chloride conducting channel composed of four transmembrane domains. It was previously shown that the second transmembrane domain (M2) of the glycine receptor forms an ion conduction pathway throughout lipid bilayers. The amino-acid sequence of the transmembrane segment M2 of the glycine receptor has a high homology to all receptors of the ligand-gated ion channel superfamily. In our report, we have used a synthetic M2 peptide. It was incorporated into a planar membrane of known lipid composition and currents induced by M2 were measured by the Black Lipid Membrane technique. When the planar lipid bilayer was composed of 75% phosphatidylethanolamine and 25% phosphatidylserine, the reversal potential measured in a 150/600 mM KCl (cis/trans) gradient was -19 mV suggesting that the examined >pore was preferential to anions, P(K)/P(Cl) = 0.25. In contrast, when 75% phosphatidylserine and 25% phosphatidylethanolamine was used, the reversal potential was +20 mV and the >pore was preferential to cations, P(K)/P(Cl) = 4.36. Single-channel currents were recorded with two predominant amplitudes corresponding to the main-conductance and sub-conductance states. Both conductance states (about 12 pS and 30 pS) were measured in a symmetric solution of 50 mM KCl. The observed single-channel properties suggest that the selectivity and conductance of the pore formed by the M2 peptide of the glycine receptor depend on the lipid composition of the planar bilayer.     

The role of specific surface loop regions in determining the function of the imipenem-specific pore protein OprD of Pseudomonas aeruginosa.

Pseudomonas aeruginosa OprD is a specific porin which facilitates the uptake of basic amino acids and imipenem across the outer membrane. In this study, we examined the effects of deletions in six of the proposed eight surface loops of OprD on the in vivo and in vitro functions of this protein. Native OprD formed very small channels in planar lipid bilayers, with an average single-channel conductance in 1.0 M KCl of 20 pS. When large numbers of OprD channels were incorporated into lipid bilayer membranes, addition of increasing concentrations of imipenem to the bathing solutions resulted in a progressive blocking of the membrane conductance of KCl, indicating the presence of a specific binding site(s) for imipenem in the OprD channel. From these experiments, the concentration of imipenem value of resulting in 50% inhibition of the initial conductance was calculated as approximately 0.6 microM. In contrast, no decrease in channel conductance was observed for the OprDdeltaL2 channel upon addition of up to 2.4 microM imipenem, confirming that external loop 2 was involved in imipenem binding. Deletion of four to eight amino acids from loops 1 and 6 had no effect on antibiotic susceptibility, whereas deletion of eight amino acids from loops 5, 7, and 8 resulted in supersusceptibility to beta-lactams, quinolones, chloramphenicol, and tetracycline. Planar lipid bilayer analysis indicated that the OprDdeltaL5 channel had a 33-fold increase in single-channel conductance in 1 M KCl but had retained its imipenem binding site. The disposition of these loop regions in the interior of the OprD channel is discussed.     

Incorporation of ion channels from bovine rod outer segments into planar lipid bilayers.

Membranes vesicles, prepared from bovine rod outer segments were fused with planar lipid bilayers. Two different ion channels were identified by recording currents from single channels. Both types of channels were selective for sodium rather than potassium and were impermeable to chloride ions. Unit conductances were 20 and 120 pS, respectively, in 150 mM sodium chloride. The channel with the larger unit conductance was sensitive to the transmembrane potential. This channel rapidly activated within less than 10 ms after a voltage jump to a more negative membrane potential and then inactivated after several seconds. The duration of the active period and the properties of the channel depended on the amplitude of the voltage jump. The channel of smaller unit conductance did not show any voltage-dependent activation or inactivation. Both types of channels were insensitive to light in the planar bilayer system. Channels incorporated into planar bilayers on a Teflon sandwich septum or on the tip of a glass micropipette gave similar results.     

Calcium-permeable channel in sarcoplasmic reticulum of rabbit skeletal muscle

Membrane vesicles from sarcoplasmic reticulum of rabbit skeletal muscle were incorporated into a bilayer lipid membrane. With this system, single current fluctuation was observed in the presence of 50 mM Ba-gluconate. This channel activity was observed only in vesicles from terminal cisternae. The single channel conductance was 14.1 pS, and the channel state was almost wholly open. The open-close transition of the channel obeyed simple two-state kinetics and was voltage-independent. The ionic selectivity was also studied, and the channel showed no selectivity among Ba, Ca, Mn, and Mg. On the other hand, it was less permeable to Cs than to Ba. Based on these results, the relation of the Ca channel to excitation-contraction coupling is discussed.     

pH modulation of large conductance potassium channel from adrenal chromaffin granules

We report here that large conductance K⁺ selective channel in adrenal chromaffin granules is controlled by pH. We measured electrogenic influx of ⁸⁶Rb⁺ into chromaffin granules prepared from bovine adrenal gland medulla. The ⁸⁶Rb⁺ influx was inhibited by acidic pH. Purified chromaffin granule membranes were also fused with planar lipid bilayer. A potassium channel with conductance of 432±9 pS in symmetric 450 mM KCl was observed after reconstitution into lipid bilayer. The channel activity was unaffected by charybdotoxin, a blocker of the Ca²⁺-activated K⁺ channel of large conductance. It was observed that acidification to pH 6.4 cis side of the membrane lowered the channel open probability and single channel conductance. Whereas only weak influence on the single channel current amplitude and open probability were observed upon lowering of the pH at the trans side. We conclude that a pH-sensitive large conductance potassium channel operates in the chromaffin granule membrane.     

Properties of the mitochondrial peptide-sensitive cationic channel studied in planar bilayers and patches of giant liposomes.

A voltage-dependent cationic channel of large conductance is observed in phospholipid bilayers formed by the tip-dip method from proteoliposomes derived from mitochondrial membranes. It is blocked by peptide M, a 13 residue peptide having the properties of a mitochondrial signal sequence. To verify the reliability of the experimental approach, mitochondrial membranes from bovine adrenal cortex or porin-deficient mutant yeast were either fused to planar bilayers or incorporated in giant liposomes which were studied by patch clamp. Cationic channels were found with both techniques. They had the same conductance levels and voltage-dependence as those which have been described using the tip-dip method. Moreover, they were similarly blocked by peptide M. The voltage-dependence of block duration was analyzed in planar bilayer and tip-dip records. Results strengthen the idea that peptide M might cross the channel. Other mitochondrial channels were observed in planar bilayers and patch clamp of giant liposomes. Because they were never detected in tip-dip records, they are likely to be inactivated at the surface monolayer used to form the bilayer in this type of experiment.     

Reconstitution in planar lipid bilayers of a voltage-dependent anion-selective channel obtained from paramecium mitochondria

Summary We have incorporated into planar lipid bilayer membranes a voltage-dependent, anion-selective channel (VDAC) obtained fromParamecium aurelia. VDAC-containing membranes have the following properties: (1) The steady-state conductance of a many-channel membrane is maximal when the transmembrane potential is zero and decreases as a steep function of both positive and negative voltage. (2) The fraction of time that an individual channel stays open is strongly voltage dependent in a manner that parallels the voltage dependence of a many-channel membrane. (3) The conductance of the open channel is about 500 pmho in 0.1 to 1.0m salt solutions and is ohmic. (4) The channel is about 7 times more permeable to Cl^– than to K⁺ and is impermeable to Ca⁺⁺. The procedure for obtaining VDAC and the properties of the channel are highly reproducible.VDAC activity was found, upon fractionation of the paramecium membranes, to come from the mitochondria. We note that the published data on mitochondrial Cl^– permeability suggest that there may indeed be a voltage-dependent Cl^– permeability in mitochondria.The method of incorporating VDAC into planar lipid bilayers may be generally useful for reconstituting biological transport systems in these membranes.     

Vacuolar ion channel of the yeast, Saccharomyces cerevisiae

Ionic flux is most likely to regulate the chemiosmotic potential differences across vacuolysosomal membranes in animal, plant, and fungal cells. We found a membrane potential-dependent cation channel in yeast vacuolar membrane and characterized its several features by an electrophysiological method using artificial planar bilayer membranes incorporated with isolated yeast vacuolar membrane vesicles. This ion channel conducts K+ (single channel conductance, 435 pS in 0.3 M KCl) and several other monovalent cations (Cs+, Na+, and Li+) with broad selectivity, but does not conduct Cl-. The opening of this channel is regulated by the membrane potential and the presence of calcium ion on the cytoplasmic face. These characteristics suggested that the vacuolar cation channel functions as one of essential components for formation and regulation of the chemical and electrical potential differences across the vacuolar membrane.     

Reconstitution of the influenza virus M2 ion channel in lipid bilayers

M₂, an integral membrane protein of influenza A virus, was purified from either influenza A virus-infected CV-1 cells or from Spodoptera frugiperda (Sf9) cells infected with a recombinant-M₂ baculovirus. The purified protein, when incorporated into phospholipid bilayer membranes, produced ion-permeable channels with the following characteristics: (1) The channels appeared in bursts during which unit conductances of diverse magnitudes (25–500 pS) were observed. (2) The most probable open state was usually the lowest unit conductance (25–90 pS). (3) The channels were selective for cations; t _Na = 0.75 when 150 mm NaCl bathed both sides of the membrane. (4) Amantadine reduced the probability of opening of the high conductance state and also the conductance of the most probable state. (5) Reducing pH increased the mean current through the open channel as well as the conductance of the most probable state. (6) The sequence of selectivity for group IA monovalent cations was Rb > K > Cs ～ Na > Li. The pH activation, amantadine block and ion selectivity of the M₂ protein ion channel in bilayers are consistent with those observed on expression of the M₂ protein in oocytes of Xenopus laevis as well as for those predicted for the proposed role of an ion channel in the uncoating process of influenza virus. The finding that the M₂ protein has intrinsic ion channel activity supports the hypothesis that it has ion channel activity in the influenza virus particle.     

Specific interaction between follitropin and GM1 ganglioside incorporated into lipid membranes.

A specific interaction was demonstrated between a monosialo-ganglioside (galactosyl-N-acetylgalactosaminyl-(N-acetylneuraminyl)-galactosylglucosylceramide) incorporated in glycerol monoleate planar bilayer membranes and follitropin. Conductance of glycerol monoleate planar bilayer membranes containing gangliosides were measured before and after addition of follitropin in the aqueous phase. A 5 fold increase of membrane conductance was observed in presence of a specific monosialo-ganglioside. No significant effect was obtained with di- and trisialogangliosides. Membrane conductance changes were suppressed by the presence of an equimolar mixture of the saccharide residues (galactose, N-acetylgalactosamine and N-acetylneuraminic acid) present in the hydrophilic moeity of the ganglioside. The results favour the hypothesis that gangliosides may contribute to the formation of functional glycoproteins receptors.     

Channel-closing activity of porins from Escherichia coli in bilayer lipid membranes

The opening and closing of the ompF porin from Escherichia coli JF 701 was investigated by reconstituting the purified protein into planar bilayer membranes. The electrical conductance changes across the membranes at constant potential were used to analyze the size and aggregate nature of the porin channel complexes and the relative number of opening and closing events. We found that, when measured at pH 5.5, the channel conductance diminished and the number of closing events increased when the voltage was greater than 100 mV. The results suggest that the number of smaller sized conductance channels increases above this potential. There was also an increase in the smaller subunits and in the closing events when the pH was lowered to 3.5, and these changes were further enhanced by increasing the voltage. We propose that both lowering the pH and elevating the potential across the membrane stabilize the porin in a conformation in which the subunits are less tightly associated and the subunits open in a non-cooperative manner. These same conditions also appear to stabilize the closed state of the pore.