Binding of thallium and other cations to the gramicidin A channel

Gramicidin A is a linear peptide antibiotic which forms dimer transmembrane channels selective for small monovalent cations, including thallium ions (Tl+) which are strongly bound. While there is great interest in the number of ion-binding sites per channel and the affinities of the sites for the various cations, measurements of the kinetics of ion permeation yield these equilibrium parameters only as indirect estimates dependent on the model assumed for the channel. Sonicated lipid vesicles. containing 1 mole of gramicidin per 30 moles of dimyristoylphosphatidylcholine. can be prepared with 5 mm-gramicidin. Evidence from our previous spectroscopic studies strongly supports the belief that this gramicidin is in the form of symmetrical dimer channels. Lipid vesicles containing gramicidin were dialyzed against control vesicles without gramicidin in the presence of a constant amount of radioactive 201Tl+ and increasing amounts of non-radioactive Tl+. The ratio of 201Tl+ free in solution to 201Tl+ bound to the channel was measured after equilibrium (≥ 48 h) at 23 °C, and this ratio was plotted as a function of the free Tl+ concentration. The inverse of the slope yielded 0.8 to 1.1 for the maximum number of simultaneously occupied highest affinity sites per channel, and the inverse of the intercept yielded a highest affinity constant of 500 to 1000 m−1 for each site. It appears that direct electrostatic repulsion prevents ions from binding simultaneously to the identical channel ends for thallium ion concentrations up to 20 mm. Estimates of the highest affinity constants for Rb+ and Na+ were also obtained.