S. Ciani

The effects of the macrotetralide actin antibiotics on the electrical properties of phospholipid bilayer membranes.

SummaryThis paper, the last in a series of three, characterizes the electrical properties of phospholipid bilayer membranes exposed to aqueous solutions containing nonactin, monactin, dinactin, and trinactin and Li+, Na+, K+, Rb+, Cs+, and NH4+ ions. Not only are both the membrane resistance at zero current and the membrane potential at zero current found to depend on the aqueous concentrations of antibiotic and ions in the manner expected from the theory of the first paper, but also these measurements are demonstrated to be related to each other in the manner required by this theory for “neutral carriers”. To verify that these antibiotics indeed are free to move as carriers of cations, cholesterol was added to the lipid to increase the “viscosity” of the interior of the membrane. Cholesterol decreased by several orders of magnitude the ability of the macrotetralide antibiotics to lower the membrane resistance; nevertheless, the permeability ratios and conductance ratios remained exactly the same as in cholesterolfree membranes. These findings are expected for the “carrier” mechanism postulated in the first paper and serve to verify it. Lastly, the observed effects of nonactin, monactin, dinactin, and trinactin on bilayers are compared with those predicted in the preceding paper from the salt-extraction equilibrium constants measured there; and a close agreement is found. These results show that the theory of the first paper satisfactorily predicts the effects of the macrotetralide actin antibiotics on the electrical properties of phospholipid bilayer membranes, using only the thermodynamic constants measured in the second paper. It therefore seems reasonable to conclude that these antibiotics produce their characteristic effects on membranes by solubilizing cations therein as mobile positively charged complexes.

The Effects of the Macrotetralide Actin Antibiotics on the Equilibrium Extraction of Alkali Metal Salts into Organic Solvents

SummaryIn order to clarify the mechanism by which neutral molecules such as the macrotetralide actin antibiotics make phospholipid bilayer membranes selectively permeable to cations, we have studied, both theoretically and experimentally, the extraction by these antibiotics of cations from aqueous solutions into organic solvents. The experiments involve merely shaking an organic solvent phase containing the antibiotic with aqueous solutions containing various cationic salts of a lipid-soluble colored anion. The intensity of color of the organic phase is then measured spectrophotometrically to indicate how much salt has been extracted. From such measurements of the equilibrium extraction of picrate and dinitrophenolate salts of Li, Na, K, Rb, Cs, and NH4 into n-hexane, dichloromethane, and hexane-dichloromethane mixtures, we have verified that the chemical reactions are as simple as previously postulated, at least for nonactin, monactin, dinactin, and trinactin. The equilibrium constant for the extraction of each cation by a given macrotetralide actin antibiotic was also found to be measurable with sufficient precision for meaningful differences among the members of this series of antibiotics to be detected. It is noteworthy that the ratios of selectivities among the various cations were discovered to be characteristic of a given antibiotic and to be completely independent of the solvent used. This finding and others reported here indicate that the size and shape of the complex formed between the macrotetralide and a given cation is the same, regardless of the species of cation bound. For such “isosteric” complexes, notable simplifications of the theory become possible which enable us to predict not only the electrical properties of a membrane made of the same solvent and having the thinness of the phospholipid bilayer but also, and more importantly, the electrical properties of the phospholipid bilayer membrane itself. These predictions will be compared with experimental data for phospholipid bilayer membranes in the accompanying paper.

A model for anomalous rectification: Electrochemical-potential-dependent gating of membrane channels

SummaryA model is presented for “anomalous rectification” based upon electrical measurements on the egg cell membrane of the starfish. The objective is to postulate a plausible molecular mechanism which yields an expression for the conductance similar to that deduced empirically by Hagiwara and Takahashi (1974), i.e.,

Permeation of divalent cations through α-latrotoxin channels in lipid bilayers: steady-state current-voltage relationships

G_K = \frac{{Bc_K^{1/2} }}{{1 + \exp \left( {\frac{{\Delta V - \Delta V_h }}{v}} \right)}},

Ion permeation and rectification in ATP-sensitive channels from insulin-secreting cells (RINm5F): effects of K+, Na+ and Mg2+.

whereB, ΔVh andv are constant,cK is the external K+ concentration, and ΔV(=V−V0) is the displacement of the membrane potential from its resting value. It is shown that a similar dependence of the conductance on ΔV is expected for a particular class of models in which the K+ ions are also implicated in “gating”. To give a specific example, we consider the case in which the formation of ion-permeable pores requires a voltage-induced orientation of membrane-bound, electrically-charged groups and subsequent complexation of these groups with the external cations. Furthermore, the proportionality betweenGK andc12/K, when the internal K+ concentration is constant, is accounted for by conventional descriptions of the ionic fluxes using Eyrings rate reaction theory. In terms of the present model,B and ΔVh are explicit functions of the internal K+ concentrations and are thus constant only as long as this is unvaried. The particular value ofv required to fit the data (v≃8.4 mV) is rationalized by the assumption that each of the orientable groups carries three negative elementary charges. In addition, the predictions of the present model are compared with those deduced from an alternative viewpoint, which is related to Armstrongs “blocking particle hypothesis”, in that the probability for opening and closing of the pore is assumed to depend on whether the pore is occupied or empty. Differences and similarities between the two models, as well as ways to discriminate between them, are discussed.

Comparative study of K channel behavior inβ cell lines with different secretory responses to glucose

SummaryA generalized form of the electrodiffusion equation, allowing for any shape of symmetrical energy barrier and any spatial dependence of the diffusion coefficient, is used to deduce theoretically the carrier-mediated conductance for thin (e.g., bilayer) membranes in the limit of low applied current. Both the Nernst-Planck and the Eyring single-barrier treatments are special cases of this more general approach, which allows for the effect of non-uniform properties of the lipid and non-uniform profiles of the forces acting within the membrane interior. Two independent mechanisms for ions to cross the membrane-solution interfaces are considered; namely, (1) the reaction at the interface between ions from solution and carriers from the membrane, and (2) the partition across the interfaces of complexes already formed in the solution. The rates of these reactions are taken into account using the rate equations of chemical kinetics; and the Poisson-Boltzmann equation is integrated in the aqueous solutions to evaluate the effect of charged polar head groups of the lipid. The analysis leads to an expression for the conductance, which, in the approximation of constant field, is an explicit function of such experimentally variable parameters as the concentrations and types of permeant ions and carriers in the aqueous phases, the total ionic strength and the nature of the polar head groups of the lipid. The functional relationship observable in an unknown membrane can, in principle, enable one to deduce such information as the mechanism of ion permeation across the interfaces, the magnitude of the surface charge, and the degree of ion-carrier complexation in the aqueous solutions.

Influence of molecular variations of ionophore and lipid on the selective ion permeability of membranes: II. A theoretical model

Summaryα-Latrotoxin, a polypeptide neurotoxin known to cause massive release of transmitter from vertebrate nerve terminals, is thought to act by forming cation-selective channels in plasma membranes. This paper describes the steady-state current carried by Ca2+, Sr2+ and Ba2+ through pores of α-LaTx molecules incorporated in artificial bilayer membranes made of neutral lipids. Even when the solutions separated by the membrane are identical, theI-V relations rectify strongly, the current being higher when the side to which the toxin is added is positive. The polarity of the rectification is consistent with the hypothesis that the mechanism of action of the toxin is, at least in part, that of promoting inwardly directed flow of cations, and thus, accumulation of Ca2+ and other ions in the intracellular spaces. The dependence of theI-V characteristics on voltage and Ca2+ concentration is well described by a one-site, one-ion model for a channel. Three parameters of the model are deduced: the binding constant of the site for Ca2+,K=1.5m−1 (orK=7m−1 when activities are used instead of concentrations); the “electrical” distance of the site from the toxin-containing solution, α=0.3; the free energy difference between the two barrier peaks, δF =0.26 kT. The values of the parameters deduced by studying the channel in the presence of Ca2+ give theoretical curves that also fit the data with Sr2+ and Ba2+, indicating a low level of discrimination among these three cations.

Characterization of the G protein coupling of a glucagon receptor to the KATP channel in insulin-secreting cells.

Substantial energies in living cells are stored in ionic gradients across membranes. One of the central problems of biological energy transduction is that of understanding how these gradients arise and conversely, how the energy stored in such gradients is utilized to drive chemical reactions. These are two aspects of the reversible coupling of the energy of transmembrane ionic concentration gradients with the energy of chemical reactions—an electrochemical problem which may be thought of as involving, on the one hand, a mechanism for selective ion permeation and, on the other, a means of coupling that mechanism to the appropriate chemical reaction.