Salt and water transport by rabbit and guinea pig gallbladder: Effect of amphotericin B on NaCl influx

Summary Previous studies have led to the suggestion that salt and water absorption by rabbit and guinea pig gallbladders exposed to Amphotericin B proceeds by a rheogenic Na pump at the basolateral cell membrane. The present studyin vitro was designed to further characterize transport properties of rabbit and guinea pig gallbladders under control conditions and to identify the properties of gallbladder mucosa which are altered by Amphotericin B to allow for the induced serosa-positive electrical potential differences (PD). Potassium is required in the bathing solution at a low concentration to maintain normal tissue O₂ consumption, fluid absorption and the ability of the tissue to develop the maximum Amphotericin B-induced PD; the relative effectiveness of alkali metal cations in substituting for K is KRb>Cs>Li>Na. The carrier mechanism for coupled influx of Na and Cl across the mucosal border of gallbladder appears to be functional in the presence of Amphotericin B; in addition, the diffusional influx of chloride is not significantly altered by the antibiotic. The primary action of Amphotericin B which appears to modify rabbit and guinea pig gallbladders from having transmural PD's of less than ±1 mV to having serosa-positive PD's of 5–30 mV is an increase in the mucosal cell membrane permeability to Na. This permeability change has the effect of partially uncoupling NaCl influx. A rheogenic Na pump mechanism at the basolateral membrane, presumably in operation under control conditions also, may account for the PD.     

Effect of amphotericin B added to one side of a membrane

Amphotericin B which was added to the one side of a membrane in aqueous solutions with low pH 3.0 sharply increases the membrane conductance by the mechanism of channel forming. Long living ionic channels (20 sec) with large conductance dispersion (2-20 Ps) were found at the concentration 2 X 10(-8) M. Amphotericin B concentration which is necessary for obtaining single channels does not depend on cholesterol concentration in the membrane-forming solution in the range from 2 to 10 mg/ml. The effect of amphotericin B on the membrane depends on the phospholipid concentration in the membrane. The one-side selective permeability is the same as one under the symmetric action of the antibiotic. It is assumed that amphotericin B induced the membrane conductance by the mechanism of half-pores formation.     

Ionic permeability of K, Na, and Cl in potassium-depolarized nerve. Dependency on pH, cooperative effects, and action of tetrodotoxin.

The passive ionic membrane conductances (gj) and permeabilities (Pj) of K, Na, and Cl of crayfish (Procambarus clarkii) medial giant axons were determined in the potassium-depolarized axon and compared with that of the resting axon. Passive ionic conductances and permeabilities were found to be potassium dependent with a major conductance transition occurring around an external K concentration of 12-15 mM (Vm = -60 to -65 mV). The results showed that K, Na, and Cl conductances increased by 6.2, 6.9, and 27-fold, respectively, when external K was elevated from 5.4 to 40 mM. Permeability measurements indicated that K changed minimally with K depolarization while Na and Cl underwent an order increase in permeability. In the resting axon (K0 = 5.4 mM, pH = 7.0) PK = 1.33 X 10(-5), PCl = 1.99 X 10(-6), PNa = 1.92 X 10(-8) while in elevated potassium (K0 = 40 mM, pH 7.0), PK = 1.9 X 10(-5), PCl = 1.2 X 10(-5), and PNa = 2.7 X 10(-7) cm/s. When membrane potential is reduced to 40 mV by changes in internal ions, the conductance changes are initially small. This suggests that resting channel conductances depend also on ion environments seen by each membrane surface in addition to membrane potential. In elevated potassium, K, Na, and Cl conductances and permeabilities were measured from pH 3.8 to 11 in 0.2 pH increments. Here a cooperative transition in membrane conductance or permeability occurs when pH is altered through the imidazole pK (approximately pH 6.3) region. This cooperative conductance transition involves changes in Na and Cl but not K permeabilities. A Hill coefficient n of near 4 was found for the cooperative conductance transition of both the Na and Cl ionic channel which could be interpreted as resulting from 4 protein molecules forming each of the Na and Cl ionic channels. Tetrodotoxin reduces the Hill coefficient n to near 2 for the Na channel but does not affect the Cl channel. In the resting or depolarized axon, crosslinking membrane amino groups with DIDS reduces Cl and Na permeability. Following potassium depolarization, buried amino groups appear to be uncovered. The data here suggest that potassium depolarization produces a membrane conformation change in these ionic permeability regulatory components. A model is proposed where membrane protein, which forms the membrane ionic channels, is oriented with an accessible amino terminal group on the axon exterior. In this model the ionizable groups on protein and phospholipid have varied associations with the different ionic channel access sites for K, Na, and Cl, and these groups exert considerable control over ion permeation through their surface potentials.     

Permeation and interaction of monovalent cations with the cGMP-gated channel of cone photoreceptors

We measured the ion selectivity of cGMP-dependent currents in detached membrane patches from the outer segment of cone photoreceptors isolated from the retina of striped bass. In inside-out patches excised from either single or twin cones the amplitude of these currents, under symmetric ionic solutions, changed with the concentration of cGMP with a dependence described by a Hill equation with average values, at +80 mV, of Km = 42.6 microM and n = 2.49. In the absence of divalent cations, and under symmetric ionic solutions, the I-V curves of the currents were linear over the range of -80 to +80 mV. The addition of Ca altered the form of the I-V curve to a new function well described by an empirical equation that also describes the I-V curve of the photocurrent measured in intact photoreceptors. The monovalent cation permeability sequence of the cGMP-gated channels in the absence of divalent ions was PK > PNa = PLi = PRb > PCs (1.11 > 1.0 = 0.99 = 0.96 > 0.82). The conductance selectivity sequence at +80 mV was GNa = GK > GRb > GCs > GLi (1.0 = 0.99 > 0.88 > 0.74 > 0.60). The organic cations tetramethylammonium (TMA) and arginine partially blocked the current, but the larger ion, arginine, was permeant, whereas the smaller ion, TMA, was not. The amplitude of the outward current through the channels increased with the concentration of monovalent cations on the cytoplasmic membrane surface, up to a saturating value. The increase was well described by the adsorption isotherm of a single ion binding site within the channel with average binding constants, at +80 mV, of 104 mM for Na and 37.6 mM for Li. By assuming that the ion channel contains a single ion binding site in an energy trough separated from each membrane surface by an energy barrier, and using Eyring rate theory, we simulated I-V curves that fit the experimental data measured under ionic concentration gradients. From this fit we conclude that the binding site interacts with one ion at a time and that the energy barriers are asymmetrically located within the membrane thickness. Comparison of the quantitative features of ion permeation and interaction between the cGMP-gated channels of rod and cone photoreceptors reveals that the ion binding sites are profoundly different in the two types of channels. This molecular difference may be particularly important in explaining the differences in the transduction signal of each receptor type.     

Monovalent selectivity of the cyclic guanosine monophosphate-activated ion channel

Monovalent cation selectivity has been characterized for the 3',5'-cyclic guanosine monophosphate (cGMP)-activated channel in vertebrate photoreceptor outer segment plasma membranes without divalent cations. Macroscopic currents in excised, inside-out patches were activated with saturating concentrations of cGMP (200 microM). Using a bi-ionic protocol with symmetrical 120 mM ion concentrations across the membrane, alkali metal ions and certain organic cations were substituted for sodium on the cytoplasmic face. The relative permeabilities, determined from shifts in the reversal potential (Erev), were NH4 much greater than Na greater than guanidinium greater than K greater than Li greater than Rb greater than Cs (3.34: 1.0: 0.97: 0.93: 0.92: 0.74: 0.50, respectively). Erev's were also measured as a function of [Na], [NH4], and [Cs], and the slope of the relation was -59.8, -52.1, and -49.1 mV/decade, respectively. The slopes for NH4 and Cs differ significantly from the Nernst-Planck prediction of -58.2 mV/decade expected for a single ion channel. Relative permeabilities were also determined for the alkali metal series of ions with 20 mM ionic concentrations on both sides of the membrane. The permeability sequence at 20 mM was unchanged, but the relative permeability for NH4 and Cs deviated significantly from the measurements at 120 mM with 1.46 and 0.75 ratios, respectively. The dependence of Erev on absolute concentrations and the deviation from Nernst-Planck predictions are best explained by multi-ion occupancy of the cGMP-activated channel. Selectivity was also examined by comparing the conductance ratios as a function of potential.(ABSTRACT TRUNCATED AT 250 WORDS)     

Non-transferrin-bound iron uptake by rat liver. Role of membrane potential difference

Non-transferrin-bound iron is efficiently cleared from serum by the liver and may be primarily responsible for the hepatic damage seen in iron-overload states. We tested the hypothesis that transport of ionic iron is driven by the negative electrical potential difference across the liver cell membrane. Extraction of 55Fe-labeled ferrous iron (1 microM) from Krebs bicarbonate buffer by the perfused rat liver was continuously monitored as the transmembrane potential difference (measured using conventional microelectrodes) was altered over the physiologic range by isosmotic ion substitution. Resting membrane potential in Krebs bicarbonate buffer was -28 +/- 1 mV. Perfusion with 1 microM ferrous iron caused a reversible 3 +/- 1 mV depolarization, and higher concentrations of iron caused even greater depolarization. Conversely, depolarization of the liver cells consistently reduced iron extraction. Replacement of sodium with potassium (70 mM) or choline (131 mM) depolarized the hepatocytes to -15 and -20 mV and decreased iron extraction by 28 and 31%, respectively. Perfusion with bicarbonate-free solutions containing tricine buffer (10 mM) reduced the membrane potential to -23 mV and reduced iron extraction by 18%. In contrast, the high basal extraction of iron (91.1 +/- 1.4%) was not further increased by substitution of nitrate for chloride (-46 mV) or infusion of glucagon (-34 mV). All effects were reversible, suggesting that perfusion with 1 microM iron produced little toxicity. These findings are consistent with an electrogenic transport mechanism for uptake of non-transferrin-bound iron that is driven by the transmembrane potential difference.     

Electrophysiological properties of the membrane and acetylcholine receptor in developing rat and chick myotubes

Membrane properties of rat and chick myotubes in various stages of development were studied. Resting membrane potentials (Em) increased from -8 to -55 mV in both rat and chick as the myotubes developed from myoblasts to large multinucleated fibers. In the rat myotubes, this increase was not accompanied by significant changes in specific membrane resistivity or changes in Na+ and K+ ion distribution. Nor have we observed a significant electrogenic component to the resting Em of mature rat myotubes under normal circumstances. A progressive increase in the passive permeability of the membrane to K+ relative to Na+ ions has been observed which can account for the changes in Em with development. In contrast to the changes in the ionic selectivity of the membrane, we have found that the ionic selectivity of the ACh receptor of rat and chick myotubes remains constant during the same period of myotube development.     

Electrolyte transport by gallbladders of rabbit and guinea pig: Effect of amphotericin B and evidence of rheogenic Na transport

Summary Ion transport and electrical properties of rabbit and guinea pig gallbladders were investigated to gain further information about the active transport mechanism that mediates fluid absorption. The intracellular and transepithelial electrical potentials were measured simultaneously using the microelectrode technique. Exposure of the mucosal surface to Amphotericin B resulted in the prompt development of a serosa-positive electrical potential difference (PD) which could not be attributed to an alteration in ion diffusion potentials across either the cell membrane or across the tight junction. Because the Amphotericin B-inducedPD was immediately dependent on warm temperatures and O₂, and was independent of NA and K concentration gradients across the cell membrane, it is suggested that active ion transport is directly responsible for thePD. Since thePD was abolished in the absence of Na in the bathing solutions, a rheogenic Na pump is postulated; this pump also appears to be operative in tissue not exposed to Amphotericin B. The specific tissue properties altered by Amphotericin B to produce a serosa-positivePD remain incompletely defined. The results of the present study indicate that ion transport by rabbit gallbladderin vitro is a consequence of a rheogenic active Na transport mechanism at the basolateral membranes which, in conjunction with a coupled NaCl influx process at the mucosal border, ultimately results in absorption of NaCl and water.     

Amphotericin B channels in the bacterial membrane: role of sterol and temperature

Amphotericin B is an antibiotic that forms ion channels in the membrane of a host cell. The change in permeability produced by these channels is greatly improved by sterols; nevertheless, the single channel conductivity remains invariant. Hence, it is proposed that sterols do not act directly, but rather through the modulation of the membrane phase. We look at the formation of these channels in the bacterial membrane to determine the mechanism of its known antibiotic resistance. We found that channels can indeed be formed in this membrane, but a substantial amount of amphotericin B is required. We also study the effects of the antibiotic concentration needed for channel expression as well as the dynamics of channels affected by both sterol and temperature in phosphatidylcholine membranes. The results support the idea that membrane structure is a determining factor in the action of the antibiotic.     

Asymmetry currents and admittance in squid axons.

The complex admittance of squid (Loligo pealei) axon was measured rapidly (within 1 s) with pseudo-random small signals and discrete Fourier transform techniques under guarded, "space-clamp" conditions and during suppression of ion conduction. Asymmetry currents were measured by paired step clam pulses of +/-70 mV from a holding potential of -97 mV and gave an apparent capacitance of 0.36 muF/cm2. However, the admittance data showed no change in capacitance at holding potentials from -97 to -67 mV and gave a decrease of 0.07 of 0.15 muF/cm2 at -37 mV. The failure to observe a capacitance increase at low membrane potentials suggests the following possibilities: (a) the asymmetry current is a displacement current that inactivates completely with time, and (b) the asymmetry current is not a displacement current and arises from large signal effects (i.e., delayed nonlinearity in ionic current) on the membrane.     

Selective modulation of L-type calcium current by magnesium lithospermate B in guinea-pig ventricular myocytes

Magnesium lithospermate B (MLB) is the main water-soluble principle of Salviae Miltiorrhizae Radix (also called as 'Danshen' in the traditional Chinese medicine) for the treatment of cardiovascular diseases. MLB was found to possess a variety of pharmacological actions. However, it is unclear whether and how MLB affects the cardiac ion channels. In the present study, the effects of MLB on the voltage-activated ionic currents were investigated in single ventricular myocytes of adult guinea pigs. MLB reversibly inhibited L-type Ca(2+) current (I(Ca,L)). The inhibition was use-dependent and voltage-dependent (the IC(50) value of MLB was 30 microM and 393 microM, respectively, at the holding potential of -50 mV and -100 mV). In the presence of 100 microM MLB, both the activation and steady-state inactivation curves of I(Ca,L) were markedly shifted to hyperpolarizing membrane potentials, whereas the time course of recovery of I(Ca,L) from inactivation was not altered. MLB up to 300 microM had no significant effect on the fast-inactivating Na(+) current (I(Na)), delayed rectifier K(+) current (I(K)) and inward rectifier K(+) current (I(K1)). The results suggest that the voltage-dependent Ca(2+) antagonistic effect of MLB work in concert with its antioxidant action for attenuating heart ischemic injury.     

How does Amphotericin B Work?: Studies on Model Membrane Systems

Abstract

The drug Amphotericin B is a very important antifungal agent as well as one of the first model systems for transmembrane pore structures. The most widely accepted model for the anticellular activity of this drug involves the formation of 1:1 Amphotericin/ sterol aggregates which subsequently associate into a transmembrane barrel with a large -OH lined aqueous pore down the middle. The stronger association of Amphotericin with ergosterol versus cholesterol explains the higher toxicity toward fungi. However, conflicting membrane permeability data concerning Amphotericin channel ion selectivity, sterol requirements, and mode of delivery has accumulated over the past fifteen years and suggests there exists a multiplicity of AmB channel structures and modes of action. Some of these mechanisms of action may be even more relevant clinically than the Amphotericin/sterol pore structure. Some of the anticellular membrane damage caused by Amphotericin may be due to formation of membrane defects and non-bilayer phases, channels without sterol or even induction of oxidative damage. In this article we present a survey of recent observations on AmB's activity on model membrane systems. As such, we are mostly concerned with liposome and planar bilayer studies. Some of the newer models explaining AmB s differential effects on cholesterol versus ergosterol containing membranes are presented along with a brief overview of membrane disruption models based on current research on membrane-active amphiphilic peptides. A synthesis and reconciliation of many of these diverse observations is attempted in a model which can accommodate most aspects of the classical sterol/Amphotericin barrel model and more recent observations as well.     

Gating of the bacterial sodium channel, NaChBac: voltage-dependent charge movement and gating currents

The bacterial sodium channel, NaChBac, from Bacillus halodurans provides an excellent model to study structure-function relationships of voltage-gated ion channels. It can be expressed in mammalian cells for functional studies as well as in bacterial cultures as starting material for protein purification for fine biochemical and biophysical studies. Macroscopic functional properties of NaChBac have been described previously (Ren, D., B. Navarro, H. Xu, L. Yue, Q. Shi, and D.E. Clapham. 2001. Science. 294:2372-2375). In this study, we report gating current properties of NaChBac expressed in COS-1 cells. Upon depolarization of the membrane, gating currents appeared as upward inflections preceding the ionic currents. Gating currents were detectable at -90 mV while holding at -150 mV. Charge-voltage (Q-V) curves showed sigmoidal dependence on voltage with gating charge saturating at -10 mV. Charge movement was shifted by -22 mV relative to the conductance-voltage curve, indicating the presence of more than one closed state. Consistent with this was the Cole-Moore shift of 533 micros observed for a change in preconditioning voltage from -160 to -80 mV. The total gating charge was estimated to be 16 elementary charges per channel. Charge immobilization caused by prolonged depolarization was also observed; Q-V curves were shifted by approximately -60 mV to hyperpolarized potentials when cells were held at 0 mV. The kinetic properties of NaChBac were simulated by simultaneous fit of sodium currents at various voltages to a sequential kinetic model. Gating current kinetics predicted from ionic current experiments resembled the experimental data, indicating that gating currents are coupled to activation of NaChBac and confirming the assertion that this channel undergoes several transitions between closed states before channel opening. The results indicate that NaChBac has several closed states with voltage-dependent transitions between them realized by translocation of gating charge that causes activation of the channel.     

alpha-Bungarotoxin-sensitive hippocampal nicotinic receptor channel has a high calcium permeability.

The hippocampal nicotinic acetylcholine receptor (nAChR) is a newly identified ligand-gated ion channel that is blocked by the snake toxin alpha-bungarotoxin (alpha-BGT) and that probably contains the alpha 7 nAChR subunit in its structure. Here its ion selectivity was characterized and compared with that of the N-methyl-D-aspartate (NMDA) receptor channel. The reversal potentials (VR) of acetylcholine- and NMDA-activated whole-cell currents were determined under various ionic conditions. Using ion activities and a Goldman-Hodgkin-Katz equation for VR shifts in the presence of Ca2+, permeability ratios were calculated. For the alpha-BGT-sensitive nAChR, PNa/PCs was close to 1 and Cl- did not contribute to the currents. Changing the [Ca2+]0 from 1 to 10 mM, the VRs of the nAChR and NMDA currents were shifted by +5.6 +/- 0.4 and +8.3 +/- 0.4 mV, respectively, and the nAChR current decay was accelerated. These shifts yielded PCa/PCss of 6.1 +/- 0.5 for the nAChR channel and 10.3 +/- 0.7 for the NMDA channel. Thus, the neuronal alpha-BGT-sensitive nAChR is a cation channel considerably selective to Ca2+ and may mediate a fast rise in intracellular Ca2+ that would increase in magnitude with membrane hyperpolarization.     

Effect of changing temperature on the ionic permeation through the cyclic GMP-gated channel from vertebrate photoreceptors.

Native cGMP-gated channels were studied in rod outer segments of the larval tiger salamander Ambystoma tigrinum. The alpha subunit of the cGMP-gated channel from bovine rods, here referred to as the wild type (w.t.), and mutant channels were heterologously expressed in Xenopus laevis oocytes. These channels were studied in excised membrane patches in the inside-out configuration and were activated by the addition of 100 or 500 microM cGMP. The effect of temperature on the ionic permeation was studied. The macroscopic current flowing through the native channel at +100 mV had an activation energy of 35.8, 30, 31.8, 34.5, 41.3, and 22.4 kJ mol-1 in the presence of Li+, Na+, K+, Rb+, Cs+, and NH4+, respectively. The macroscopic current flowing through the w.t. channel at +100 mV had an activation energy of 45.2, 38.2, 37.5, 47.3, 49.4, and 38.9 kJ mol-1 in the presence of Li+, Na+, K+, Rb+, Cs+, and NH4+, respectively. The activation energy of the macroscopic current flowing through the native and w.t. channels did not vary significantly when the ionic concentration of the permeant ion was changed between 2.5 and 110 mM. The activation energy of the single-channel current of the w.t. channel at +100 mV was 40.4 and 33 kJ mol-1 for Na+ and NH4+, respectively. The reversal potential of biionic solutions changed significantly with temperature. These results can be used to obtain an estimate of the enthalpic and entropic contributions to the barrier of the Gibbs free energy experienced by an ion during its permeation through the open channel. These estimates indicate that the ionic permeation and selectivity of the cGMP-gated channel are controlled both by enthalpic and entropic factors and that the selectivity of the native channel for Li+ over Na+ is primarily caused by entropic effects.     

Effects of applying oxytocin to Helix pomatia neurons. II. Hyperpolarization

The ionic mechanisms of hyperpolarization produced by applying oxytocin (OT) were investigated at the membrane of identifiedHelix pomatia neurons. Two types of neuron were known to exist, in one of which hyperpolarization is produced by a reduction in chloride ions at the membrane and a rise in membrane permeability to potassium ions in the other. In the first of these, response to OT had a reversal potential of –40 mV and decreased when furosemide and tolbutamide were added to the external medium. In the second case, the potential of the reversal of the response to OT was –70 mV. Upon doubling of potassium ion concentration in the external solution it was shifted towards depolarization by 15 mV. It is sugested thatHelix pomatia neurons have different types of OT receptors, some of which, when activated, manifest reduced chloride permeability at the membrane (probably through the cell cyclase system) with a rise in potassium permeability at the membrane in others.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 20, No. 5, pp. 659–666, September–October, 1988.     

Microelectrode studies of amphotericin B on Na+ and K+ conductance in bullfrog cornea

Addition of 10(-5) M amphotericin B to the tear solution of an in vitro preparation of the frog cornea increased the transepithelial conductance, gt, and decreased the apical membrane fractional resistance, f(R0), in the presence or absence of tear Na+ and Cl-. In the presence of tear Na+ and Cl-, amphotericin B increased the short-circuit current, Isc, from 3.9 to 8.8 microA.cm-2 and changed the intracellular potential, V0, from -48.5 to -17.9 mV probably due to a higher increase in the Na+ than in the K+ conductance. In the absence of tear Na+ and Cl-, amphotericin B decreased Isc from 5.5 to about 0 microA.cm-2 due to K+ (and possibly Na+) flux from cell to tear and changed V0 from -35.4 to -63.6 mV due to the increase in conductance of both ions. Increase in the tear K+ from 4 to 79 mM (in exchange for choline), in the presence of amphotericin B and absence of tear Na+ and Cl-, decreased f(R0) from 0.09 to 0.06, increased gt from 0.23 to 0.31 mS, increased Isc from 0.63 to 7.3 microA.cm-2, and changed V0 from -65.5 to -17.3 mV due to the change in EK in the presence of a high conductance in the tear membrane. Similar effects were observed with an increase of tear Na+. Results support the concept that the Na+ conductance opened by amphotericin B in the apical membrane is greater than the K+ conductance. Previously observed transepithelial effects of the ionophore may be explained mostly on the basis of its effect on the apical membrane.     

Ionic currents and ion fluxes in Neurospora crassa hyphae

Voltage dependence of ionic currents and ion fluxes in a walled, turgor-regulating cell were measured in Neurospora crassa. The hyphal morphology of the model organism Neurospora simplifies cable analysis of ionic currents to determine current density for quantitative comparisons with ion fluxes. The ion fluxes were measured directly and non-invasively with self-referencing ion-selective microelectrodes. Four ions (H(+), Ca(2+), K(+), and Cl(-)) were examined. H(+) net uptake and Ca(2+) net release were small (10.2 nmol m(-2) s(-1) and 1.1 nmol m(-2) s(-1), respectively) and voltage independent. K(+) and Cl(-) fluxes were larger and voltage dependent. Maximal K(+) net release ( approximately 1440 nmol m(-2) s(-1)) was observed at positive voltages (+15 mV), while maximal Cl(-) net release ( approximately 905 nmol m(-2) s(-1)) was observed at negative voltage (-210 mV). A possible function of the net outward K(+) and Cl(-) fluxes is regulation of the plasma membrane potential. Total ion fluxes were 37-58% of the total ionic current density (about +/-244 mA m(-2), equivalent to +/-2500 nmol m(-2) s(-1), at 0 mV and -200 mV) so other ions must contribute significantly to the ionic currents.     

Properties of amphotericin B channels in a lipid bilayer

Properties of individual ionic channels formed by polyene antibiotic Amphotericin B were studied on brain phospholipid membranes containing cholesterol. The ionic channels have a closed state and an open one (with conductance of about 6.5 pS in 2 M KCl). The conductance value of an open channel is independent of cholesterol concentration in the membrane and of pH in the range from 3.5 to 8.0. The voltage-current characteristics of a single channel are superlinear. Zero current potential value in the case of different KCl concentrations in the two solutions indicates preferential but not ideal anionic selectivity of a single channel. Channel conductivity grows as the electrolyte concentration is increased and tends to a limiting value at high concentrations. A simple model having only one site for an ion was shown to represent satisfactorily an open channel behaviour under different conditions. An individual ionic channel performs a large number of transitions between the open and closed states during its life-time of several minutes. Rate constants of these transitions depend on the kind and concentration of salt in aqueous solutions. The switching system functioning is not influenced by an ion situated inside the pore.     

Cyclic AMP-induced chloride permeability in the apical membrane of necturus gallbladder epithelium

The effects of theophylline, 8-Br-cAMP, and cAMP on necturus gallbladder epithelium were investigated using microelectrode techniques. Each of these substances depolarized the cell membranes by approximately 15 mV and decreased the apparent ratio of apical to basolateral membrane resistances to a value not significantly different from zero. Examination of the ionic selectivity of the apical membrane by ion substitutions in the mucosal bathing medium revealed a large increase in Cl permeability with no apparent changes in K and Na permeabilities. Intracellular Cl activity ((a)CL(i)) was measured using Cl- sensitive liquid ion-exchanger microelectrodes. Under control conditions, (a)Cl(i) was approximately 20 mM, 2.5 times higher than the value expected for equilibrium distribution ((a)Cl(i/eq). After addition of 8-Br-cAMP, (a)Cl(i) decreased within less than 60 s to approximately 13 mM, a value not significantly different from ((a)Cl(i/eq)). Virtually identical results were obtained with theophylline. Under control conditions, luminal Cl removal caused (a)Cl(i) to fall at an initial rate of 1.8 mM/min, whereas in tissues exposed to 8-Br- cAMP or theophylline a rate of 11.6 mM/min was observed. The apical membrane Cl transference number was estimated from the change of (a)Cl(i) upon exposure to 8-Br-cAMP as well as from the changes in apical membrane potential during variation of the luminal Cl concentration. The results, 0.91 and 0.88, respectively, are indicative of a high Cl permeability of the apical membrane during cAMP. This effect may explain, at least in part, the complete inhibition of fluid absorption produced by theophylline in this tissue. Moreover, enhancement of apical membrane Cl permeability may account for a variety of cAMP effects in epithelial tissues.