Ion and nonelectrolyte permeability properties of channels formed in planar lipid bilayer membranes by the cytolytic toxin from the sea anemone, Stoichactis helianthus.
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Animals↗
Biomedical subjects
A Finkelstein
Publications and source records attributed to A Finkelstein.
Fusion of phospholipid vesicles with planar phospholipid bilayer membranes. I. Discharge of vesicular contents across the planar membrane.
Multilamellar phospholipid vesicles are introduced into the cis compartment on one side of a planar phospholipid bilayer membrane. The vesicles contain a water-soluble fluorescent dye trapped in the aqueous phases between the lamellae. If a vesicle containing n lamellae fuses with a planar membrane, an n-1 lamellar vesicle should be discharged into the opposite trans compartment, where it would appear as a discernible fluorescent particle. Thus, fusion events can be assayed by counting the number of fluorescent particles appearing in the trans compartment. In the absence of divalent cation, fusion does not occur, even after vesicles have been in the cis compartment for 40 min. When CaCl2 is introduced into the cis compartment to a concentration of greater than or equal to 20 mM, fusion occurs within the next 20 min; it generally ceases thereafter because of vesicle aggregation in the cis compartment. With approximately 3 x 10(8) vesicles/cm3 in the cis compartment, about 25-50 fusion events occur following CaCl2 addition. The discharge of vesicular contents across the planar membrane is the most convincing evidence of vesicle-membrane fusion and serves as a model for that ubiquitous biological phenomenon--exocytosis.
Exocytosis↗
Fusion of phospholipid vesicles with planar phospholipid bilayer membranes. II. Incorporation of a vesicular membrane marker into the planar membrane.
Fusion of multilamellar phospholipid vesicles with planar phospholipid bilayer membranes was monitored by the rate of appearance in the planar membrane of an intrinsic membrane protein present in the vesicle membranes. An essential requirement for fusion is an osmotic gradient across the planar membrane, with the cis side (the side containing the vesicles) hyperosmotic to the opposite (trans) side; for substantial fusion rates, divalent cation must also be present on the cis side. Thus, the low fusion rates obtained with 100 mM excess glucose in the cis compartment are enhanced orders of magnitude by the addition of 5-10 mM CaCl2 to the cis compartment. Conversely, the rapid fusion rates induced by 40 mM CaCl2 in the cis compartment are completely suppressed when the osmotic gradient (created by the 40 mM CaCl2) is abolished by addition of an equivalent amount of either CaCl2, NaCl, urea, or glucose to the trans compartment. We propose that fusion occurs by the osmotic swelling of vesicles in contact with the planar membrane, with subsequent rupture of the vesicular and planar membranes in the region of contact. Divalent cations catalyze this process by increasing the frequency and duration of vesicle-planar membrane contact. We argue that essentially this same osmotic mechanism drives biological fusion processes, such as exocytosis. Our fusion procedure provides a general method for incorporating and reconstituting transport proteins into planar phospholipid bilayer membranes.
Calcium Chloride↗
The nonelectrolyte permeability of planar lipid bilayer membranes.
The permeability of lecithin bilayer membranes to nonelectrolytes is in reasonable agreement with Overton's rule. The is, Pd alpha DKhc, where/Pd is the permeability coefficient of a solute through the bilayer, Khc is its hydrocarbon:water partition coefficient, and D is its diffusion coefficient in bulk hydrocarbon. The partition coefficients are by far the major determinants of the relative magnitudes of the permeability coefficients; the diffusion coefficients make only a minor contribution. We note that the recent emphasis on theoretically calculated intramembranous diffusion coefficients (Dm'S) has diverted attention from the experimentally measurable and physiologically relevant permeability coefficients (Pd'S) and has obscured the simplicity and usefulness of Overton's rule.
Butyrates↗
Colicin K acts by forming voltage-dependent channels in phospholipid bilayer membranes.
The bactericidal action of colicins K, E1, Ia, and other functionally related colicins involves disruption of active transport and leakage of ions from the cell. We show that a single colicin K molecule can form a voltage-dependent, relatively nonselective, ion-permeable channel of a few picosiemens conductance in a planar phospholipid bilayer membrane. In a membrane containing many of these channels, the ratio of the number of conducting to nonconducting channels changes e-fold per 3.7 mV. We suggest that the physiological effects of colicin K and functionally related colicins result from their ability to form ion-permeable channels in the bacterial plasma membrane.
Biological Transport, Active↗
Interaction of ions and water in gramicidin A channels: streaming potentials across lipid bilayer membranes.
For very narrow channels in which ions and water cannot overtake one another (single-file transport), electrokinetic measurements provide information about the number of water molecules within a channel. Gramicidin A is believed to form such narrow channels in lipid bilayer membranes. In 0.01 and 0.1 M solutions of CsCl, KCL, and NaCl, streaming potentials of 3.0 mV per osmolal osmotic pressure difference (created by urea, glycerol, or glucose) appear across gramicidin A-treated membranes. This implies that there are six to seven water molecules within a gramicidin channel. Electroosmotic experiments, in which the water flux assoicated with current flow across gramicidin-treated membranes is measured, corroborate this result. In 1 M salt solutions, streaming potentials are 2.35 mV per osmolal osmotic pressure difference instead of 3.0 mV. The smaller value may indicate multiple ion occupancy of the gramicidin channel at high salt concentrations. Apparent deviations from ideal cationic selectivity observed while attempting to measure single-salt dilution potentials across gramicidin-treated membranes result from streaming potential effects.
Gramicidin↗
Water permeability of gramicidin A-treated lipid bilayer membranes.
In membranes containing aqueous pores (channels), the osmotic water permeability coefficient, P f, is greater than the diffusive water permeability coefficient, P d. In fact, the magnitude of P f/P d is commonly used to determine pore radius. Although, for membranes studied to date, P f/P d monotonically declines with decreasing pore radius, there is controversy over the value it theoretically assumes when that radius is so small that water molecules cannot overtake one another within the channel (single-file transport). In one view it should equal 1, and in another view it should equal N, the number of water molecules in the pore. Gramicidin A forms, in lipid bilayer membranes, narrow aqueous channels through which single-file transport may occur. For these channels we find that P f/P d approximately 5. In contrast, for the wider nystatin and amphotericin B pores, P f/P d approximately 3. These findings offer experimental support for the view that P f/P d = N for single-file transport, and we therefore conclude that there are approximately five water molecules in a gramicidin A channel. A similar conclusion was reached independently from streaming potential data. Using single-channel conductance data, we calculate the water permeability of an individual gramicidin A channel. In the Appendix we report that there is a wide range of channel sizes and lifetimes in cholesterol-containing membranes.
Electric Conductivity↗
Reconstitution in planar lipid bilayers of a voltage-dependent anion-selective channel obtained from paramecium mitochondria.
We have incorporated into planar lipid bilayer membranes a voltage-dependent, anion-selective channel (VDAC) obtained from Paramecium 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 1.0 M 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.
Animals↗
Inactivation of monazomycin-induced voltage-dependent conductance in thin lipid membranes. I. Inactivation produced by long chain quaternary ammonium ions.
The voltage-dependent conductance induced in thin lipid membranes by monazomycin undergoes inactivation upon the introduction of quaternary ammonium ions (QA) having a long alkyl chain (e.g. dodecyltrimethylammonium [C12]) to the side containing monazomycin. That is, in response to a step of voltage the conductance rises to a peak and then falls to a much lower steady-state value. We demonstrate that the basis of this phenomenon is the ability of QA to pass through the stimulated membrane and bind to the opposite surface. As a consequence, the surface potential on that side becomes more positive, thus reducing the voltage across the membrane proper and turning off the monazomycin-induced conductance. Because the flux of QA through the membrane increases linearly with conductance, we believe that these ions pass through the monazomycin channels. QA permeability increases with alkyl chain length; remarkably, in spite of its much larger size, C12 is about 150 times more permeant than K+. It appears, therefore, that there is a hydrophobic region of the cahnnel that favors the alkyl chain; we propose that this region is formed by the hydrophobic faces of the monazomycin channels in lipid bilayers to QA inactivation of potassium channels in the squid giant azon, and suggest that there may be a common structural feature for the two channels. It is possible that some of the inactivation phenomena in excitable cells may arise from local field changes not measurable by the recording electrodes.
Anti-Bacterial Agents↗
Inactivation of monazomycin-induced voltage-dependent conductance in thin lipid membranes. II. Inactivation produced by monazomycin transport through the membrane.
At sufficiently large conductances, the voltage-dependent conductance induced in thin lipid membranes by monazomycin undergoes inactivation. This is a consequence of depletion of monazomycin from the membrane solution interface, as monazomycin crosses the membrane to the opposite (trans) side from which it was added. The flux of monazomycin is directly proportional to the monazomycin-induced conductance; at a given conductance it is independent of monazomycin concentration. We conclude that when monazomycin channels break up, some or all of the molecules making up a channel are deposited on the trans side. We present a model for the monazomycin channel: approximately five molecules, each spanning the membrane with its NH3+ on the trans side and an uncharged hydrophilic (probably sugar) group anchored to the cis side, form an aqueous channel lined by--OH groups. The voltage dependence arises from the flipping by the electrical field of molecules lying parallel to the cis surface into the "spanned state;" the subsequent aggregation of these molecules into channels is, to a first approximation, voltage independent. The channel breakup that deposits monomers on the trans side involves the collapsing of the channel in such a way that the uncharged hydrophilic groups remain in contact with the water in the channel as they close the channel from behind. We also discuss the possibility that inactivation of sodium channels in nerve involves the movement from one side of the membrane to the other of the molecules (or molecule) forming the channel.
Anti-Bacterial Agents↗
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.
Anti-Bacterial Agents↗
Water and nonelectrolyte permeability of lipid bilayer membranes.
Both the permeability coefficients (Pd's) through lipid bilayer membranes of varying composition (lecithin [L], lecithin:cholesterol [LC], and spingomyelin:cholesterol [SC]) and the n-hexadecane:water partition coefficients (Knc's) of H2O and seven nonelectrolytes (1,6 hexanediol, 1,4 butanediol, n-butyramide, isobutyramide, acetamide, formamide, and urea) were measured. For a given membrane compositiin, Pd/DKnc (where D is the diffusion constant in water) is the same for most of the molecules tested. There is no extraordinary dependence of Pd on molecular weight; thus, given Pd(acetamide), Pd(1,6 hexanediol) is correctly predicted from the Knc and D values for the two molecules. The major exceptions are H2O, whose value of Pd/DKnc is about 10-fold larger, and urea, whose value is about 5-fold smaller than the general average. In a "tight" membrane such as SC, Pd(n-butyramide)/Pd(isobutyramide)=2.5; thus this bilayer manifests the same sort of discrimination between branched and straight chain molecules as occurs in many plasma membranes. Although the absolute values of the Pd's change by more than a factor of 100 in going from the tightest membrane (SC) to the loosest (L), the relative values remain approximately constant. The general conclusion of this study is that H2O and nonelectrolytes cross lipid bilayer membranes by a solubility-diffusion mechanism, and that the bilayer interior is much more like an oil (a la Overton) than a rubber-like polymer (a la Lieb and Stein).
Acetamides↗
Nature of the water permeability increase induced by antidiuretic hormone (ADH) in toad urinary bladder and related tissues.
In artificial lipid bilayer membranes, the ratio of the water permeability coefficient (Pd(water)) to the permeability coefficient of an arbitrary nonelectrolyte such as n-butyramide (Pd(n-butyramide)) remains relatively constant with changes in lipid composition and temperature, even though the individual Pd's increase more than 100-fold. I propose that this is a general rule that also holds for the lipid bilayers of cells and tissues, and that therefore if Pd(water)/Pd(solute greatly exceeds the value found for artifical lipid bilayers (where "solute" is a molecule, such as 1,6 hexanediol or n-butyramide, that crosses the cell membrane by a solubility-diffusion mechanism without the aid of a special transporting system), then water crosses the cell membrane via aqueous pores. Applying this criterion to the toad urinary bladder, we find that even in the unstimulated bladder, water probably crosses the luminal membrane primarily through small aqueous pores, and that this almost certainly the case after antidiuretic hormone (ADH) stimulation. I suggest that ADH stimulation ultimately leads either to formation (or enlargement) of pores, by the rearrangement of preexisting subunits, or to an unplugging of these pores.
Animals↗
Membrane channels and conductance. Discussion paper.
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Biological Transport↗
Pores formed in lipid bilayer membranes by nystatin, Differences in its one-sided and two-sided action.
Nystatin and amphotericin B induce a cation-selective conductance when added to one side of a lipid bilayer membrane and an anion-selective conductance when added to both sides. The concentrations of antibiotic required for the one-sided action are comparable to those employed on plasma membranes and are considerably larger than those required for the two-sided action. We propose that the two-sided effect results from the formation of aqueous pores formed by the hydrogen bonding in the middle of the bilayer of two "half pores," whereas the one-sided effect results from the half pores alone. We discuss, in terms of the flexibility of bilayer structure and its thickness, how it is possible to have conducting half pores and "complete pores" in the same membrane. The role of sterol (cholesterol and ergosterol) in pore formation is also examined.
Amphotericin B↗
Bilayers: formation, measurements, and incorporation of components.
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Animals↗
The electrostatic basis of Mg++ inhibition of transmitter release.
The inhibition by Mg(++) of stimulus-evoked transmitter release is attributed to a decrease in surface potential, Psi(0), on the outer surface of the presynaptic terminal and hence a lower surface calcium concentration, [Ca(++)](0). Data on the frog neuromuscular junction are quantitatively fit by assuming that there is a negative charge density, sigma, on the outer surface of the presynaptic terminal of 6.5 x 10(13) charges per cm(2) and that simple diffuse double layer theory is applicable. No specific binding of Mg(++) or Ca(++) is required. Without any additional assumptions, the inhibitory effect of univalent cations is also quantitatively predicted.
Acetylcholine↗
Aqueous pores created in thin lipid membranes by the polyene antibiotics nystatin and amphotericin B.
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Amphotericin B↗