Structural kinetic and mechanistic aspects of the carboxylic ionophore mediated transmembrane cation transport

Abstract

Maintenance of ionic gradients across biological membranes has been established to be achieved mainly by an interplay of two mechanisms, namely, the carrier mechanism and the channel mechanism. In the carrier mechanism, the carrier ionophore complexes with the cation at one membrane interface to form a lipid?soluble complex, which then diffuses through the hydrophobic core of the membrane interior, releases the cation at the other interface and travels back. In the channel mechanism, the ionophore spans across the membrane like a tunnel, through which the cations go from one side of the membrane to the other. Valinomycin and gramicidin?A are prototypes of carriers and channels, respectively. However, since the chemical structures of the carrier ionophores are wide and varied, the cation binding selectivities, and the rates of complexation/decomplexation and diffusion, vary from one carrier to another. Such details have not yet been fully worked out for the carboxylic ionophores. This thesis is mainly concerned with the structural, kinetic, and mechanistic aspects of the carboxylic ionophore?mediated transmembrane cation transport, in particular lasalocid A and A23187. However, other carrier ionophores — namely valinomycin and nonactin — have also been used in studying the interactions of carrier ionophores with model membrane systems.

A brief description of the importance of membrane transport, various possible mechanisms of cation transport, the physico?chemical studies carried out on the carrier ionophores under consideration, and their transport properties is presented in Chapter?1, followed by a statement of the problem and strategy. This chapter also contains a description of the model membranes and the various techniques employed in studying them.

In Chapter?2, various materials, experimental procedures, techniques, and handling of the experimental data used in these investigations are described.

The results of CD, ^1H and ^13C NMR studies on the interaction of three alkali metal (Li^+, Na^+, and K^+) chlorides and perchlorates with lasalocid A in methanol and acetonitrile are presented in Chapter?3. It has been found that while LiClO? forms complexes of both 1:1 and 2:1 (ionophore:cation) stoichiometries with lasalocid A in acetonitrile of appreciable stability, the other two cations (Na^+ and K^+) form only equimolar 1:1 complexes. The coordination to the cation is mainly provided by the oxygens of the carbonyl, tetrahydrofuran, tetrahydropyran, and the two hydroxyl groups of the ionophore, whereas the salicylic acid part of the molecule does not seem to participate in coordination. The conformations of lasalocid A–LiClO? complexes in acetonitrile are significantly different from those in methanol, whereas there appear to be no significant differences between the conformations of sodium and potassium complexes of lasalocid A in methanol and acetonitrile.

Chapter?4 contains the results of CD, ^1H, and ^13C NMR studies on the interaction of alkaline earth cations (Mg^2+, Ca^2+, Sr^2+, and Ba^2+) with lasalocid A in methanol and acetonitrile. In acetonitrile, while the perchlorates of magnesium and calcium form complexes of both 1:1 and 2:1 stoichiometries, the perchlorates of strontium and barium form predominantly equimolar complexes. Once again, the conformations of the lasalocid A–M^2+ complexes are found to be dependent on cation size, counter?ion identity, and solvent polarity.

The interactions of the trivalent lanthanide ions with lasalocid A in methanol and acetonitrile have been studied using CD and fluorescence techniques, and the results are presented in Chapter?5. The lanthanide ions form both 1:1 and 2:1 complexes in both solvents, with greater stability in acetonitrile than in methanol. Smaller lanthanide ions tend to favor the formation of non?equimolar 2:1 complexes, while larger ions prefer equimolar complexes — a pattern reflected in their stability constants.

In Chapter?6, the conformations of the alkali, alkaline earth, and lanthanide complexes of lasalocid A are compared and discussed in terms of the size, charge, and coordination number of the cations, counter?ions, and solvent polarity.

The interactions of the four carrier ionophores (valinomycin, nonactin, A23187, and lasalocid A) with dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC) liposomes have been investigated by differential scanning calorimetry (DSC), ^1H and ^31P NMR techniques, and these results constitute Chapter?7. The DSC studies indicate interactions of the ionophores with the polar head groups of the lipids.

The transport kinetics of three lanthanide ions (Pr^3+, Nd^3+, and Eu^3+) across DMPC and DPPC unilamellar vesicles (ULVs) in the liquid crystalline phase, mediated by A23187 and the sodium salt of lasalocid A, are studied in Chapter?8. The kinetic data are analyzed to obtain rate constants and the stoichiometry of the transporting species. In the case of lasalocid A, the non?equimolar 2:1 complex is responsible for the transport of lanthanide ions across the vesicles; similarly, for A23187, the 2:1 complex is the active transporting species.

Finally, Chapter?9 presents a summary of the conclusions from these investigations:

Non?equimolar stoichiometry for carrier ionophore–cation complexes appears to be the rule rather than the exception.

The conformations of carboxylic ionophore–cation complexes depend on multiple factors such as cation size, charge, coordination number, counter?ion, solvent polarity, and ionizable group state.

The stoichiometry of the transporting species for carboxylic ionophore?mediated transmembrane cation transport is established to be 2:1 (ionophore:cation).

From the solution studies on lasalocid–cation complexes and ionophore–liposome interactions, a plausible mechanism is proposed for how carboxylic ionophores facilitate transmembrane cation transport.