Antibiotic-mediated transport of alkali ions across lipid barriers.

Investigations of the effects of valinomycin on mitochondria" 2 led to the discovery of antibiotic-mediated ion transport.3 Subsequent work revealed that this property is shared by valinomycin, the enniatins, the macrolide actins, and the gramicidins, including an extensive array of natural and synthetic analogues.4-6 Passive ion permeability effects have been reported obtained with these agents on other systems including erythroeytes,7 synthetic lipid vesicles,7 and artificial bimolecular lipid leaflets.8' 9 The macrolide actins can even mediate cation transport, as detected by biionic potential development across a bulk phase of CCl4.l1 All these antibiotics, which have in common low molecular weights (ca. 500-1500), a curious alternation of D and L configurations, lipid solubility, and a lack of ionizable groups, will be referred to collectively as the valinomycin class of antibiotics. A second class of transport-mediating antibiotics, including nigericin dianemycin, and others, has been found to reverse transport induced by the valinomycin class."1-'4 All members of the second class possess low molecular weights (450950), and lipid solubility, and contain an ionizable carboxyl group.'4 This communication will establish that both classes of antibiotics act analogously in many respects, both in mitochondria and in a variety of other lipid barrier systems. Methods and Materials.-Multiparameter measurements of rat liver mitochondria and other systems, during antibiotic-induced ion movements, were carried out with the apparatus described in detail previously.'5 K+ was monitored with the Beckman 39047 electrode, 02 by a Clark-type membrane electrode, pH by the A. H. Thomas 4858 combination electrode, fluorescence by 450-ma light excited by a 366-mn beam, and light-scattering at 650 mis. Mitochondria were prepared and protein was determined as described previously.'6 Samples of nigericin were obtained from H. A. Lardy, R. Harned (Commercial Solvents Corp.), and M. Gorman (Eli Lilly Co.), dianemycin from Lardy and Gorman, oligomycin from F. M. Strong, and p-trifluoromethoxy arboxylcyanide phenylhydrazone (FCCP) from P. Heytler (duPont). Valinomycin was prepared by means of a Streptomyces culture donated by J. C. McDonald according to a modification of his procedure.'7 Results.-The responses of mitochondria to valinomycin addition as shown in Figure 1 have been detailed previously.4 A subsequent addition of nigericin induces a rapid discharge of K+ accompanied by a countermovement of H+ and an increase in light-scattering, indicative, of mitochondrial contraction, as reported by Graven et al.11 Under the conditions of Figure 1A, respiration is stimulated initially signifying an increased energy load, before becoming strongly inhibited as the loss of K+ progresses. In these experiments the concentration of dissolved oxygen does not become rate limiting. The transient stimulation of respiration was presumably not apparent in previous work because of the presence of phosphate in the medium, which increases respiration in the presence of valinomycintype antibiotics3 thereby obscuring any subsequent stimulation by nigericin.