On the mechanism of action of an ion channel mimic
Date
1991
Authors
Kaye, Katharine Clare
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Abstract
The transport of alkali metal cations across the lipid bilayers of large unilamellar vesicles is mediated by a synthetic ion channel mimic, (G8TrgP)6. The transport mechanism is determined by comparison to the behaviours of gramicidin D (an ion channel), and valinomycin (an ion carrier), in the same experimental system. The transport properties examined are concentration dependence, temperature dependence, cation selectivity and concentration dependence, and inhibition of the transport activity by octylammonium sulphate.
The concentration dependence of the mimic was examined over the range 0.008 - 0.154 mole percent of lipid. A similar set of experiments examined the behaviour of gramicidin over a concentration range of (0.08 - 3.2) x 10·3 mole percent of lipid, and for valinomycin, in the range of 0.017 - 0.17 mole percent of lipid. The results for the mimic showed a trend of increasing rate for increasing concentrations up to a saturation rate. This behaviour was observed for gramicidin, but not for valinomycin. Increasing concentrations of valinomycin increased the transport rates without reaching a saturation rate. Both the mimic and gramicidin were observed to have increasing extents of transport for increasing concentrations, but valinomycin had a constant extent of transport independent of its concentration.
The temperature dependence of all three transporters was investigated over the range 5-35 °C. The Arrhenius activation energies derived from the temperature dependence study were: (G8TrgP)6 mimic, 16 ± 3 kJ/mol; gramicidin, 16 ± 2 kJ/mol; and valinomycin, 47 ± 7 kJ/mol. The difference between the activation energies of gramicidin and valinomycin is indicative of their different transport mechanisms. The activation energies of the mimic and gramicidin are identical, and on the order of thermal energies for the temperature range used in this study.
The selectivity sequences were determined for the transporters by comparing rates and extents of transport for equal concentrations of the sulphate salts of lithium, sodium, potassium, rubidium, and cesium. The cation selectivity sequences for the transporters were all different: the mimic, Cs+ > Rb+ > K+ > Na+ > Li+; valinomycin, Cs+ = K+ = Rb+ >>> Na+ = Li+ = 0; and gramicidin had no apparent selectivity in this experimental system. The selectivity sequence of the mimic appears related to the size of the bare cation and/or the ease of dehydration of the cation. The sequence is not the same as for complexation to 18-crown-6 crown ether, nor is it the same as the sequence for the extraction of the cations into the lipid bilayer.
The dependence of rate of transport and extent of transport as a function of concentration of cesium and potassium cations was investigated. The Michaelis-Menton parameters, Vmax and Km, for transport of potassium were determined by a Lineweaver-Burk analysis. The parameters, for potassium transport, of Vmax and Km, respectively, were: for the mimic, 0.19 ± 0.03 x 10--2s-1 and 3 ± 0.2 mM potassium; for gramicidin, 0.30 ± 0.09 x 10-2s-1 and 5 ± 0.4 mM potassium; and for valinomycin, 0.38 ± 0.02 x 10-2s-1 and 34 ± 0.7 mM potassium. In general, the cation selectivity sequences and the Michaelis-Menton parameters were not indicative of transporter mechanism. It was observed that the mimic was capable of mediating the transport of magnesium and calcium cations.
The transport activity of the (G8TrgP)6 mimic was inhibited in the presence of octylammonium sulphate. The inhibition of potassium transport was examined over the octylammonium concentration range of 0 - 230 times the concentration of mimic. At a 10:1 mole ratio of octylammonium cation to mimic, potassium transport was virtually blocked. At lower ratios, the rate of transport increased. Gramicidin activity was also inhibited with octylammonium sulphate. The activity of valinomycin was not affected.
In this experimental system, the (G8TrgP)6 mimic had critical dissimilarities to the behaviour of valinomycin, and insignificant differences compared to grarnicidin. There were no similarities to valinomycin that could sustain an argument that the mimic acted with a carrier mechanism. The experiments produced strong evidence in support of the mechanism of the mimic being analogous to that of gramicidin.