P. Läuger

Formation of large, ion-permeable membrane channels by the matrix protein (porin) of Escherichia coli

One of the major proteins of the outer membrane of Escherichia coli, the matrix protein (porin), has been isolated by detergent solubilisation. When the protein is added in concentrations of the order 10 ng/cm3 to the outer phases of a planar lipid bilayer membrane, the membrane conductance increases by many orders of magnitude. At lower protein concentrations the conductance increases in a stepwise fashion, the single conductance increment being about 2 nS (1 nS = 10(-9) siemens = 10(-9) omega -1) in 1 MKCl. The conductance pathway has an ohmic current vs. voltage character and a poor selectivity for chloride and the alkali ions. These findings are consistent with the assumption that the protein forms large aqueous channels in the membrane. From the average value of the single-channel conductance a channel diameter of about 0.9 nm is estimated. This channel size is consistent with the sugar permeability which has been reported for lipid vesicles reconstituted in the presence of the protein.

Ion transport through pores: A rate-theory analysis

This paper gives a treatment of ion transport through pores based on the theory of reaction rates. According to Eyring, the pore is considered as a sequence of energy barriers over which the ion has to jump. The present analysis differs from previous treatments in that it allows for saturation effects in the pore, i.e. it is assumed that for electrostatic reasons the pore may contain no more than one ion. The saturation phenomena are described by introducing the equilibrium constant for the occupancy of the pore. The theory leads to expressions which relate experimentally accessible quantities such as electrical conductivity, tracer permeability and biionic potential to the microscopic properties of the pore. It is shown that certain information on the single rate constants may be obtained from stationary conductance measurements. The specificity of the pore for different ions depends on thermodynamic quantities (equilibrium constants) as well as on kinetic parameters (rate constants). The experimental results with the gramicidin A pore are compared with the theory. The jump rates of K+ and Na+ in the gramicidin A pore are estimated to be of the order of 1·108−1·109 s−1.

Electrical capacity of black lipid films and of lipid bilayers made from monolayers

Planar bilayer membranes were formed from monolayers of a series of mono-unsaturated monoglycerides and lecithins. The hydrocarbon thickness of these membranes, as calculated from the electrical capacity, increases with the length of the fatty acid chain. The specific capacity of monoolein bilayers was found to be 0.745 muF/cm-2 which is nearly twice that of a monoolein black film made in the presence of decane, but is close to that obtained after freezing out the solvent from the black film. The hydrocarbon thickness of the bilayer, as calculated with a dielectric constant of 2.1, is considerably less than twice the length of the extended hydrocarbon chain of the monoglyceride. The specific capacity (Cm) of bilayers made from monoolein monolayers showed a negligible voltage dependence, whereas the Cm increased significantly at a voltage of 150 mV in the case of Mueller-Rudin-type monoolein films with n-decane as a solvent.

Channel formation kinetics of gramicidin A in lipid bilayer membranes

SummaryPrevious studies have given evidence that the active form of gramicidin A in lipid bilayer membranes is a dimer which acts as an ion channel; it has been further shown that the mean lifetime of the channel strongly depends on the membrane thickness. As the thickness slightly decreases when a voltage is applied to the membrane, the equilibrium between conducting dimers and nonconducting monomers may be displaced by a voltage jump. From the relaxation of the electrical current after the voltage jump, information about the kinetics of channel formation is obtained. For a dioleoyllecithin/n-decane membrane the rate constant of association is found to be 2×1014 cm2 mole−1 sec−1, which is by three orders of magnitude below the limiting value of a diffusion-controlled reaction in a two-dimensional system. The dissociation rate constant is equal to 2 sec−1, a value which is consistent with the channel lifetime as obtained from electrical fluctuation measurements.

Ionic selectivity of pores formed by the matrix protein (porin) of Escherichia coli.

Incorporation of the matrix protein (porin) from the outer membrane of Escherichia coli into black lipid films results in the formation of ion-permeable pores with a single-pore conductance of the order of 2 nS (in 1 M KCl). Information on the structure of this pore has been obtained by determining the selectivity for various species differing in charge and size. From the permeability of the pore for large organic ions (Tris+, glucosamine+, Hepes-) a minimum pore diameter of 0.8 nm is estimated. At neutral pH the pore is two to four times more permeable for alkali ions than for chloride. On the basis of the observed pH dependence of permeability, this cationic selectivity is explained by the assumption that the pore contains fixed negative charges.

The Rate Constants of Valinomycin-Mediated Ion Transport through Thin Lipid Membranes

Electrical relaxation experiments have been performed with phosphatidylinositol bilayer membranes in the presence of the ion carrier valinomycin. After a sudden change of the voltage a relaxation of the membrane current with a time constant of about 20 musec is observed. Together with previous stationary conductance data, the relaxation amplitude and the relaxation time are used to evaluate the rate constants of valinomycin-mediated potassium transport across the lipid membrane. It is found that the rate constants of translocation of the free carrier S and the carrier-ion complex MS(+) are nearly equal (2.10(4) sec(-1)) and are of the same order as the dissociation rate constant of MS(+) in the membrane-solution interface (5.10(4) sec(-1)). The equilibrium constant of the heterogeneous association reaction M(+) (solution) + S (membrane) --> MS(+) (membrane) is found to be approximately 1 M(-1), about 10(6) times smaller than the association constant in ethanolic solution.

Kinetic properties of ion carriers and channels

ConclusionA unified description of ion carriers, channels, and pumps seems possible based on the concept of channels with multiple conformational states. The notion of a channel with variable energy profile is suggested by recent work on the dynamics of proteins. With the exception of mobile, translatory carriers of the valinomycin type which represent a separate class of ion translocators, most transport systems in biological membranes seem to be built-in proteins. Transmembrane proteins may differ in their mode of operation by the extent to which conformational changes are involved in the translocation of the permeant. While carriers and channels in the usual sense are limiting cases of a multistate channel, many real transport systems probably function by an intermediate mechanism.

Relaxation studies of ion transport systems in lipid bilayer membranes.

Relaxation techniques have been widely used in kinetic studies of chemical reactions in homogeneous solution (Eigen & DeMayer, 1963). The principle of this method is well known: an external variable such as temperature or pressure is suddenly changed and the time course of a state parameter of the system such as concentration is recorded as it approaches a new steady value. Relaxation techniques can also be used for studying the rate of elementary processes in membranes. This method has proved particularly useful for the investigation of ion transport systems (ion carriers, channels, pumps) in artificial planar bilayer membranes. In this review we describe different relaxation techniques which have been developed for this purpose during the last years, as well as applications to a number of ion transport systems.

Valinomycin-mediated ion transport through neutral lipid membranes: Influence of hydrocarbon chain length and temperature

SummaryStationary electrical conductance experiments together with nonstationary relaxation experiments allow a quantitative determination of rate constants describing carrier-mediated ion transport. Valinomycin-induced ion transport across neutral lipid membranes was studied. The dependence of the transport parameters on the chain length of the lipid molecules, on the kind of alkali ion, and on the temperature was determined. The relaxation time τ the current following a voltage jump shows a marked increase with decreasing temperature or with increasing chain length of the lipid molecules. This variation of τ is interpreted on the basis of a varying membrane fluidity. It is shown that under favorable circumstances the equilibrium constant of complex formation in the aqueous phase may be obtained from membrane experiments. Furthermore, the kinetics of exchange of valinomycin between membrane and water was studied. We found a marked influence of the totus surrounding the black film on the kinetics as well as on the total amount of valinomycin molecules in the membrane. The problem of location of the free carrier molecules inside the membrane is discussed.

Formation of ion channels by a negatively charged analog of Gramicidin A

SummaryO-pyromellitylgramicidin is a derivative of gramicidin in which three carboxyl groups are introduced at the terminal hydroxyl end of the peptide. Experiments with artificial lipid membranes indicate that this negatively charged analog forms ion-permeable channels in a way similar to that of gramicidin. If O-pyromellitylgramicidin is added to only one aqueous solution, the membrane conductance remains small, but increases by several orders of magnitude if the same amount is also added to the other side. In accordance with the dimer model of the channel, the membrane conductance under symmetrical conditions is proportional to the square of the aqueous concentration of O-pyromellitylgramicidin over a wide range. The ratioΜPG/ΜG of the single-channel conductance of O-pyromellitylgramicidin to that of gramicidin is close to unity at high ionic strength, but increases more than fivefold at smaller ionic strength (0.01m). This observation is explained in terms of an electrostatic effect of the fixed negative charges localized near the mouth of the channel. In a mixture of O-pyromellitylgramicidin and gramicidin, unit conductance steps of intermediate size are observed in addition to the conductance steps corresponding to the pure compounds, indicating the formation of hybrid channels. Hybrid channels with preferred orientation may be formed if small amounts of gramicidin and O-pyromellitylgramicidin are added to opposite sides of the membrane. These hybrid channels show a distinct asymmetry in the current-voltage characteristic.