Cation conductance and efflux induced by polyene antibiotics in the membrane of skeletal muscle fiber.

Cation conductance and efflux induced by polyene antibiotics amphotericin B (AMB), amphotericin B methyl ester (AME), nystatin, mycoheptin, and levorin on frog isolated skeletal muscle fibers and whole sartorius muscles were investigated. Conductance was measured under current-clamp conditions using a double sucrose-gap technique. Cation efflux was studied using flame emission photometry. Some new data were obtained concerning the effects of levorin and mycoheptin on biological membranes. The power dependence of polyene-induced cation transport on antibiotic concentration in muscle membrane was lower than that in bilayers. The decline in the equilibrium conductance caused by polyene removal (except for levorin) was very fast. There was reverse temperature dependence of AMB- and nystatin-induced conductances. Both induced conductance and efflux values demonstrated a correlation with the order of antifungal activities: levorin > AMB, mycoheptin > AME > nystatin, except for AME, which was more potent on yeastlike cells. These effects were interpreted in terms of possible differences in the kinetics of channel formation in biological and model membranes and in light of the role of nonconducting antibiotic forms in biological membranes.     

Effect of Membrane Structure on the Action of Polyenes: I. Nystatin Action in Cholesterol- and Ergosterol-Containing Membranes

A detailed and thorough characterization of nystatin-induced permeability on lipid bilayers of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)-containing ergosterol or cholesterol is presented. The results show that the same collection of transmembrane pores appears in membranes containing either sterol. The concentration range for the appearance of these pores is sterol-dependent. Another mechanism of action, membrane disruption, is also observed in ergosterol-POPC membranes. The greater potency of nystatin present in ergosterol-containing membranes cannot be explained simply by the longer opening times of its pores, as has been suggested; it is also due to an increased number of events in these membranes. The present results and those of a companion paper lead us to propose that membrane structure is the determining factor for drug selectivity in membranes with different sterols.     

Nystatin-induced nitric oxide production in mouse macrophage-like cell line RAW264.7

Nystatin is known to deplete lipid rafts from mammalian cell membranes. Lipid rafts have been reported to be necessary for lipopolysaccharide signaling. In this study, it was unexpectedly found that lipopolysaccharide-induced nitric oxide production was not inhibited, but rather increased in the presence of a non-cytotoxic concentration of nystatin. Surprisingly, treatment with nystatin induced only NO production and iNOS expression in RAW264.7 cells. At the concentration used, no changes in the expression of GM1 ganglioside, a lipid raft marker on RAW264.7 cells, was seen. From studies using several kinds of inhibitors for signaling molecules, nystatin-induced NO production seems to occur via the iκB/NF-κB and the PI3 K/Akt pathway. Furthermore, because nystatin is known to activate the Na-K pump, we examined whether the Na-K pump inhibitor amiloride suppresses nystatin-induced NO production. It was found that amiloride significantly inhibited nystatin-induced NO production. The results suggest that a moderate concentration of nystatin induces NO production by Na-pump activation through the PI3 kinase/Akt/NF-κB pathway without affecting the condition of lipid rafts.     

Activation of K+ channels in renal medullary vesicles by cAMP-dependent protein kinase

Summary ADH, acting through cAMP, increases the potassium conductance of apical membranes of mouse medullary thick ascending limbs of Henle. The present studies tested whether exposure of renal medullary apical membranes in vitro to the catalytic subunit of cAMP-dependent protein kinase resulted in an increase in potassium conductance. Apical membrane vesicles prepared from rabbit outer renal medulla demonstrated bumetanide-and chloride-sensitive²²Na⁺ uptake and barium-sensitive, voltage-dependent⁸⁶Rb⁺-influx. When vesicles were loaded with purified catalytic subunit of cAMP-dependent protein kinase (150 mU/ml), 1mm ATP, and 50mm KCl, the barium-sensitive⁸⁶Rb⁺ influx increased from 361±138 to 528±120pm/mg prot · 30 sec (P<0.01). This increase was inhibited completely when heat-stable protein kinase inhibitor (1 g/ml) was also present in the vesicle solutions. The stimulation of⁸⁶Rb⁺ uptake by protein kinase required ATP rather than ADP. It also required opening of the vesicles by hypotonic shock, presumably to allow the kinase free access to the cytoplasmic face of the membranes. We conclude that cAMP-dependent protein kinase-mediated phosphorylation of apical membranes from the renal medulla increases the potassium conductance of these membranes. This mechanism may account for the ADH-mediated increase in potassium conductance in the mouse mTALH.     

Membrane damage by a toxin from the sea anemone Stoichactis helianthus. I. Formation of transmembrane channels in lipid bilayers

The addition of nanomolar amounts of a toxin preparation derived from the sea anemone Stoichactis helianthus to black lipid membranes increases their electrical conductance by one million-fold. In addition, the membranes become permeable predominantly to monovalent cations. The elevated bilayer conductance is voltage-dependent, and the current-voltage curves of these bilayers display rectification as well as a region of negative resistance. The membrane activity of the toxin is proportional to the third power of its concentration, and at very low concentrations the membrane conductance increases in discrete uniform steps. These observations indicate that the mechanism of toxin action involves the formation of transmembrane channels constructed by the aggregation of protein molecules which are inserted in the bilayer. The voltage-dependent membrane conductance arises from two distinct channel characteristics: (1) the unit conductance of individual channels is dependent on the polarity of applied voltage; (2) the number of ion-conducting channels is influenced by the polarity as well as the magnitude of applied potential. It is believed that these effects are due to the influence of an electric field on the insertion of toxin molecules into the bilayer or on their subsequent association with each other to produce channels. Partial chemical characterization of the toxin material has shown that the membrane active factor is a basic protein with a molecular weight of 17 500.     

Membrane damage by a toxin from the sea anemone Stoichactis helianthus. I. Formation of transmembrane channels in lipid bilayers.

The addition of nanomolar amounts of a toxin preparation derived from the sea anemone Stoichactis helianthus to black lipid membranes increases their electrical conductance by one million-fold. In addition, the membranes become permeable predominantly to monovalent cations. The elevated bilayer conductance is voltage-dependent, and the current-voltage curves of these bilayers display rectification as well as a region of negative resistance. The membrane activity of the toxin is proportional to the third power of its concentration, and at very low concentrations the membrane conductance increases in discrete uniform steps. These observations indicate that the mechanism of toxin action involves the formation of transmembrane channels constructed by the aggregation of protein molecules which are inserted in the bilayer. The voltage-dependent membrane conductance arises from two distinct channel characteristics: (1) the unit conductance of individual channels is dependent on the polarity of applied voltage; (2) the number of ion-conducting channels is influenced by the polarity as well as the magnitude of applied potential. It is believed that these effects are due to the influence of an electric field on the insertion of toxin molecules into the bilayer or on their subsequent association with each other to produce channels. Partial chemical characterization of the toxin material has shown that the membrane active factor is a basic protein with a molecular weight of 17,500.     

Effects of proteolytic enzymes on ionic conductances of squid axon membranes.

The effects of proteolytic enzymes on ionic conductances of squid axon membranes have been studied by means of the voltage clamp technique. When perfused internally alpha-chymotrypsin (1 mg/ml) increased and prolonged the depolarizing after-potential. Sodium inactivation was partially inhibited causing a prolonged sodium current, and peak sodium and steady-state potassium currents were suppressed. The time for sodium current to reach its peak was not affected. Leakage conductance increased later. On the other hand, carboxypeptidases A and B, both at 1mg/ml, suppressed the sodium and potassium conductance increases with little or no change in sodium inactivation. The mechanism that controls sodium inactivation appears to be associated with the structure of membrane proteins which is modified by alpha-chymotrypsin but not by carboxypeptidases and is located in a position accessible to alpha-chymotrypsin only from inside the membrane.     

Membrane channel formation by the lymphocyte pore-forming protein: comparison between susceptible and resistant target cells

The assembly of pores by the pore-forming protein (perforin) of cytolytic T lymphocytes (CTLs) and natural killer cells on the membranes of different cell lines was studied. Using the patch clamp technique in the whole cell configuration, we measured the conductance increase induced by perforin in susceptible cell lines as well as in resistant CTL lines (CTLLs). The results showed that although the amplitudes of the first observed conductance steps produced in both cell types were comparable, CTLLs required at least 10-fold higher doses of perforin to form membrane pores. Outside-out patches excised from CTLL-R8, on the other hand, appeared to be more susceptible to channel formation by perforin than intact cells, as lower doses were able to induce conductance increases. Once channels were induced in CTL membranes, however, their conductances (greater than 1 nS) were indistinguishable from the ones obtained in susceptible cell lines. Fluorescence measurements with quin-2 showed that perforin induced rapid increases in the intracellular Ca2+ concentration in susceptible EL4 cells. In marked contrast, a perforin dose 60-120-fold higher than the minimal dose required to elicit Ca2+ changes in EL4 cells was not able to induce any measurable Ca2+ increase in CTLL-R8. The data suggest that the resistance of CTLs to lysis mediated by their own mediator perforin is at least in part due to their ability to avoid pore formation by this protein. The mechanism underlying this phenomenon is not yet understood, but the observation that outside-out patches excised from CTLL-R8 are more susceptible to channel formation by perforin than intact cells raises the possibility that an intracellular mechanism may be involved.     

Detection of transient capacitance increase associated with channel formation in lipid bilayers.

The insertion of alpha- and beta-latrotoxins and sea anemone (Radianthus macrodactilus) toxin into bilayer lipid membranes (BLMs) was investigated using the method of simultaneous conductance/capacitance measurement. All the toxins investigated induced capacitance changes which preceded toxin-induced conductance increases. The processes that may underlie the observed effect are discussed.     

The effect of hexadecaprenyl diphosphate on phospholipid membranes

In the present study we investigated phospholipid bilayer membranes and phospholipid vesicles made from dioleoylphosphatidylcholine (DOPC) or its mixture with the phosphate ester derivative of long-chain polyprenol (hexadecaprenyl diphosphate, C(80)-PP) by electrophysiological and transmission electron microscopy (TEM) techniques. The membrane conductance-temperature relationships and the membrane breakdown voltage have been measured for different mixtures of C(80)-PP/DOPC. The current-voltage characteristics, the membrane conductance, the activation energy of ion migration across the membrane and the membrane breakdown voltage were determined. Hexadecaprenyl diphosphate decreases the membrane conductance, increases the activation energy and the membrane breakdown voltage for the various values of C(80)-PP/DOPC mole ratio. The analysis of TEM micrographs shows several characteristic structures, which have been described. The data indicate that hexadecaprenyl diphosphate modulates the surface curvature of the membranes by the formation of aggregates in liquid-crystalline phospholipid membranes. The properties of modified membranes can result from the presence of the negative charges in the hydrophilic part of C(80)-PP molecules and can be modulated by the concentration of this compound in membranes. We suggest that the dynamics and conformation of hexadecaprenyl diphosphate in membranes depend on the transmembrane electrical potential.     

Effects of nystatin on membrane conductance and internal ion activities inAplysia neurons

Summary Two methods were used to study effects of the antibiotic, nystatin, on giant neurons ofAplysia. In the first method the effects of various concentrations of nystatin on the current-voltage relationship were evaluated at a fixed time after exposure to the antibiotic using a two-microelectrode voltage clamp. Nystatin increased membrane conductance in a dose-dependent manner. The dose-response relation was very steep, with little or no effect below 15 mg/liter and an effect too large to measure at concentrations greater than 30 mg/liter. Upon return to antibiotic-free solution, membrane conductance returned to pre-treatment levels within 30 minutes. The second type of experiment involved use of ion-specific microelectrodes to measure changes of intracellular univalent ion activities which attended the nystatin-induced permeability. meability was also increased. Nystatin may therefore be used to selectively rearrange the internal ionic milieu to study the effect of such a change on membrane tranpsort or electrical properties.     

Ionic channels induced by surfactin in planar lipid bilayer membranes.

Surfactin is a lipopeptide produced by certain strains of Bacillus subtilis and has potent surface activity. Here, we present the first results showing that ion-conducting pores can be formed by surfactin in artificial lipid membranes. With a low aqueous concentration of surfactin (1 microM) and a restricted membrane area (5.10(-5) cm2) we observed conductance jumps that indicate the formation of individual ionic channels in the presence of K+, Rb+, Cs+, Na+ or Li+ chlorides. Although for every salt concentration (Ci), the distribution in amplitude of the conductance steps (lambda i) may be rather broad, there is always a step amplitude which is more frequent than the others. In addition, the channels corresponding to this most frequent step amplitude are the longest in duration. For Ci = 1 M, the cationic selectivity sequence deduced from these most frequent events is K+ greater than Rb+ greater than Na+ greater than Cs+ = Li+ with respective values for lambda Mi: 130, 110, 80 and 30 pS. In KCl solutions lambda MKCl increases as a function of Ci for low Ci, and shows a plateau for Ci greater than 0.5 M. When measured on larger area membranes (10(-2)cm2) with 1 M solutions of the monovalent salts KCl, NaCl, RbCl and CsCl or the divalent salt CaCl2, the macroscopic low voltage conductance (G0) increases with a slope of 2 on a log-log plot as a function of surfactin concentration. These results demonstrate that surfactin produces selective cationic channels in lipid bilayer membranes and suggest that at higher salt concentration, a dimer is involved in this functional channel-forming process.     

Role of gangliosides in adhesion and conductance changes in large spherical model membranes

The formation of two spherical model membranes at the tips of two syringes has allowed us to study the role of gangliosides in membrane adhesion and look for changes in conductance between two such membranes during the process of adhesion. Membranes were formed in aqueous 100 mM NaCl, 10 mM KCl, 1 mM CaCl2 from 1% (w/v) egg phosphatidylcholine in n-decane, with or without mixed bovine brain gangliosides. After thinning to the 'black' bilayer state, two membranes were moved into contact. With gangliosides, the contact area and conductance increased colinearly with time over a 5 to 20 min period of adhesion. The role of electrostatic bridging by calcium was investigated. In the absence of calcium or in the presence of 2 mM EDTA, adhesion proceeded after a longer lag time at about one-half the normal rate. As the ganglioside concentration was increased from 0 to 15 mol%, the electrical conductance of individual membranes decreased 3-fold from 48 +/- 30 nS/cm2 to 17 +/- 13 nS/cm2. The conductance was pH dependent with a minimum at neutral values. At neutral pH, when two membranes containing 4.1 mol% gangliosides adhered, the region of adhesion had a specific conductance three times that of the nonadhering regions of membranes. Without gangliosides, the specific conductance of the contact region was the same as that of non-adhering regions of the membrane. These data suggest that mixed gangliosides can mediate an adhesion-dependent increase in conductance.     

Dopamine-β-hydroxylase-Induced Changes in the Electrical Conductance of Bimolecular Lipid Membranes

Dopamine-β-hydroxylase (DBH), an enzyme that catalyzes the conversion of dopamine (DA) to norepinephrine (NE) in adrenal medullary chromaffin granules, increases the electrical conductance of bimolecular lipid membranes. The conductance increase requires both DA and Ca²⁺ and occurs in discrete steps. The conductance, which increases as the square of the DBH concentration, is nonselective for cations over anions and requires the native conformation of DBH. NE cannot replace DA.     

Probing amphotericin B single channel activity by membrane dipole modifiers

The effects of dipole modifiers and their structural analogs on the single channel activity of amphotericin B in sterol-containing planar phosphocholine membranes are studied. It is shown that the addition of phloretin in solutions bathing membranes containing cholesterol or ergosterol decreases the conductance of single amphotericin B channels. Quercetin decreases the channel conductance in cholesterol-containing bilayers while it does not affect the channel conductance in ergosterol-containing membranes. It is demonstrated that the insertion of styryl dyes, such as RH 421, RH 237 or RH 160, in bilayers with either cholesterol or ergosterol leads to the increase of the current amplitude of amphotericin B pores. Introduction of 5α-androstan-3β-ol into a membrane-forming solution increases the amphotericin B channel conductance in a concentration-dependent manner. All the effects are likely to be attributed to the influence of the membrane dipole potential on the conductance of single amphotericin B channels. However, specific interactions of some dipole modifiers with polyene-sterol complexes might also contribute to the activity of single amphotericin B pores. It has been shown that the channel dwell time increases with increasing sterol concentration, and it is higher for cholesterol-containing membranes than for bilayers including ergosterol, 6-ketocholestanol, 7-ketocholestanol or 5α-androstan-3β-ol. These findings suggest that the processes of association/dissociation of channel forming molecules depend on the membrane fluidity.     

Effect of phloretin on the permeability of thin lipid membranes

Phloretin dramatically increases cation conductances and decreases anion conductances of membranes treated with ion carriers (nonactin, valinomycin, carbonyl-cyanide-m-chlorophenylhydrazone [CCCP], and Hg(C6F5)2) or lipophilic ions (tetraphenylarsonium [tphAs+] and tetraphenylborate [TPhB-]). For example, on phosphatidylethanolamine membranes, 10(-4) M phloretin increases K+ -nonactin and TPhAs+ conductances and decreases CCCP- and TPhB- conductances 10(3)-fold; on lecithin: cholesterol membranes, it increases K+-nonactin conductance 10(5)-fold and decreases CCCP- conductance 10(3)-fold. Similar effects are obtained with p- and m-nitrophenol at 10(-2) M. These effects are produced by the un-ionized form of phloretin and the nitrophenols. We believe that phloretin, which possesses a large dipole moment, adsorbs and orients at the membrane surface to introduce a dipole potential of opposite polarity to the preexisting positive one, thus increasing the partition coefficient of cations into the membrane interior and decreasing the partition coefficient of anions. (Phloretin may also increase the fluidity of cholesterol-containing membranes; this is manifested by its two- to three-fold increase in nonelectrolyte permeability and its asymmetrical effect on cation and anion conductances in cholesterol-containing membranes.) It is possible that pholoretin's inhibition of chloride, urea, and glucose transport in biological membranes results from the effects of these intense intrafacial dipole fields on the translocator(s) of these molecules.     

The electrical potential profile of gallbladder epithelium.

In this study the relative ionic permeabilities of the cell membranes of Necturus gallbladder epithelium have been determined by means of simultaneous measurement of transmural and transmucosal membrane potential differences (PD) and by ionic substitution experiments with sodium, potassium and chloride ions. It is shown that the mucosal membrane is permeable to sodium and to potassium ions. The baso-lateral membrane PD is only sensitive to potassium ions. In both membranes chloride conductance is negligible or absent. The ratio of the resistances of the mucosal and baso-lateral membranes, RM/RS, increases upon reducing the sodium concentration in the mucosal solution. The same ratio decreases when sodium is replaced by potassium which implies a greater potassium than sodium conductance in the mucosal membrane. The relative permeability of the shunt for potassium, sodium and chloride ions is: PK/PNa/PCl=1.81:1.00:0.32. From the results obtained in this study a value for the PK/PNa ratio of the mucosal membrane could be evaluated. This ratio is 2.7. From the same data the magnitude of the electromotive forces generated across the cell membranes could be calculated. The EMF's are -15mV across the mucosal membrane and -81mV across the baso-lateral one. Due to the presence of the low resistance shunt the transmucosal membrane PD is -53.2mV (cell inside negative) and the transmural PD is +2.6mV (serosal side positive). The change in potential profile brought about by the low resistance shunt favors passive entry of Na ions into the cell across the mucosal membrane. Calculations show that this passive Na influx is maximally 64% of the net Na flux estimated from fluid transport measurements. The C-1 conductive of the baso-lateral membrane is too small to allow electrogenic coupling of C1 with Na transport across this membrane. Experiments with rabbit gallbladder epithelium indicate that the membrane properties in this tissue are qualitatively similar to those of Necturus gallbladder epithelium.     

The nature of the voltage-dependent conductance of the hemocyanin channel.

The electrical responses of individual hemocyanain channels in oxidized cholesterol membranes demonstrate that the voltage-dependent conductance of many-chanel membranes arises from two different mechanisms. These are the voltage-dependent redistribution of channels among several discrete single-channel conductance states themselves. The relaxation time for the discrete conductance changes is of the order of seconds nd the relaxation time of the continuous conductance changes is of the order 10(-4) seconds. As salt concentration in the bathing medium is increased, the single-channel conductance first increases lineary and then saturates. The characteristics of the saturation curves suggest that the continuous conductance changes occur at the edges of the channel and that the mean time an ion spends in the channel is 4 nanoseconds...     

Channels in the mitochondrial outer membrane: Evidence from patch clamp studies

Patch clamp techniques were applied to outer mitochondrial membranes of giant mitochondria from mice kept on a cuprizone diet or to vesicles produced by fusing membranes derived from the outer membrane ofNeurospora mitochondria. In the negative range of potentials the conductances decreased with increases in the magnitude of voltage, suggesting the closing of channels. Experiments in which mitochondria were treated with the polyanion polymethacrylate maleate styrene (1:2:3) or succinic anhydride suggest that the channels correspond to VDAC. Although sometimes conductance also decreased with increasing potential over a narrow range of positive potentials, more commonly the conductances increased. Although this phenomenon may represent a detachment of the patch, the changes in conductance are reversible, suggesting that they correspond to the formation or the opening of channels.     

Ionic selectivity, saturation, and block in a K+-selective channel from sarcoplasmic reticulum

The open-channel conductance properties of a voltage-gated channel from sarcoplasmic reticulum were studied in planar phospholipid membranes. The channel is ideally selective for K+ over Cl- and for K+ over Ca++. In symmetrical 1 M solutions, the single-channel conductance (in pmho) falls in the order: K+ (214) > NH4+ (157) > Rb+ (125) > Na+ (72) > La+ (8.1) > Cs+ (< 3). In neutral bilayers, the channel conductance saturates with ion activity according to a rectangular hyperbolic relation, with half-saturation activities of 54 mM for K+ and 34 mM for Na+. Under symmetrical salt conditions, the K+:Na+ channel conductance ratio increases with salt activity, but the permeability ratio, measured by single-channel bi-ionic potentials, is constant between 20 mM and 2.5 M salt; the permeability ratio is equal to the conductance ratio in the limit of low-salt concentration. The channel conductance varies < 5% in the voltage range -100 to +70 mV. The maximum conductance varies K+ and Na+ is only weakly temperature dependent (delta H++ = 4.6 and 5.3 kcal/mol, respectively), but that of Li+ varies strongly with temperature (delta H++ = 13 kcal/mol). The channel's K+ conductance is blocked asymmetrically by Cs+, and this block is competitive with K+. The results are consistent with an Eyring-type barriers as it permeates the channel. The data conform to Lüger's (1973. Biochem. Biophys. Acta. 311:423-441) predictions for a "pure" single-ion channel.