Volume regulation in Spiroplasma floricola: Evidence that Na+ is extruded by a Na+/H+ antiporter

Abstract

Marked cell swelling followed by lysis was observed when Spiroplasma floricola cells were incubated in iso-osmotic solutions of NaCl, KCI, choline chloride or sorbitol in the absence of an energy source. In the presence of an energy source the cells did not swell suggesting that S. floricola relies on an energy-dependent mechanism(s) for cell volume regulation. An ammonium chloride dilution procedure was utilized to generate a pH gradient (inside acid) across the cell membrane of S. floricola cells. The addition of NaCl resulted in an intracellular alkalization suggesting the presence of a Na+/H+ exchange activity. In 22Na+-loaded cells, glucose-dependent 22Na+ extrusion was observed at acidic pH in both the presence and absence of Na+ ions. The extrusion was completely inhibited by carbonyl cyanide m-chlorophenylhydrazone (CCCP, 10 μM) and partially inhibited by dicyclohexylcarbodiimide (DCCD, 100 μM) indicating that in S. floricola, Na+ movement is driven by the electrochemical gradient of H+ via a Na+/H+ antiporter. The specific ATPase activity of S. floricola membranes was at least twofold higher than that described in other mollicutes. Activity was Mg2+-dependent over the pH range (6.5-8.5) tested, but was very little affected by Na+ (up to 100 mM). DCCD (25 μM) markedly inhibited both membrane-bound and solubilized ATPase activity, whereas orthovanadate (50 μM) had only a small inhibitory effect. The properties of the enzyme are consistent with a F0F1-ATPase. It is suggested that the enzyme operates in the direction of hydrolysing ATP formed by glycolysis leading to the generation of a δpH, which is the major driving force for the Na+/H+ antiport activity.