gramicidin-a and dodecylamine

Inhibitors of Na(+)/Ca(2+) exchange (NCX) such as KB-R7943 and SEA0400 are thought to act by promoting an inactive state of the exchanger induced by elevated cytosolic Na(+) concentrations (Na(+)-dependent inactivation). This conclusion is based mainly on experiments in excised patches from frog oocytes expressing NCX and has not been fully tested in intact cells. Here we characterize the inhibitory effects of KB-R7943 and other amphiphilic cations on NCX activity in transfected Chinese hamster ovary (CHO) cells expressing the cardiac isoform of the Na(+)/Ca(2+) exchanger (NCX1.1). Cytosolic Na(+) concentrations were varied using the channel-forming ionophore gramicidin. KB-R7943 (10microM) inhibited NCX activity at high (140mM) but not at low (10mM) cytosolic Na(+) concentrations. Cells expressing NCX mutants that were either more (F223E) or less (K229Q) susceptible than the wild-type to Na(+)-dependent inactivation were either hypersensitive or resistant, respectively, to KB-R7943 inhibition. We recently reported that Na-dependent inactivation in transfected CHO cells could be partially but transiently reversed by increasing the external Ca(2+) concentration (Chernysh et al., 2008). The inhibitory effects of KB-R7943 were likewise partially reversed by elevated Ca(2+). Other amphiphilic cations such as dodecylamine (20microM) and sphingosine (10microM) closely mimicked the effects of KB-R7943 on NCX activity. Our results support the hypothesis that KB-R7943 inhibits NCX activity in intact cells by promoting Na(+)-dependent inactivation and suggest that this is a general mode of inhibition by amphiphilic cations.

Topics: Amines; Animals; Anti-Bacterial Agents; Calcium; CHO Cells; Cricetinae; Cricetulus; Gramicidin; Protein Isoforms; Sodium; Sodium-Calcium Exchanger; Sphingosine; Thiourea; Transfection

The effect of the antibiotic gramicidin S and the synthetic cationic amphipath dodecylamine on membranes was studied with large unilamellar vesicles containing phosphatidylcholine and varying concentrations of cardiolipin. Fusion of vesicles composed of equal amounts of the two phospholipids occurred with both drugs at concentrations lower than 10 microM. Fusion was accompanied by leakage of the contents, while higher drug concentrations caused complete loss of vesicle contents. Drug concentrations at least one order of magnitude lower were needed to induce leakage from vesicles containing only phosphatidylcholine. Under these conditions, contents leakage occurred with no measurable aggregation or membrane intermixing. On the other hand, much higher concentrations of both drugs were required to induce leakage from vesicles containing predominantly cardiolipin. Release of contents occurred upon aggregation of the vesicles and collapse of the vesicular organization, as well as formation of paracrystalline structure when dodecylamine was employed or amorphous material when gramicidin A was used. In contradistinction to other model systems, phosphatidylcholine was needed for fusion induced by the cationic amphipaths, and its presence reduced the threshold concentration of the drugs needed to induce leakage of the contents. The similar effects of the two drugs on membranes imply that, at least in these model membranes, the relevant feature of both drugs is only their amphiphatic nature.

Topics: Amines; Cardiolipins; Gramicidin; Liposomes; Membrane Fusion; Microscopy, Electron; Phosphatidylcholines; Structure-Activity Relationship