Acute inhibition of brain-specific Na+/H+ exchanger isoform 5 by protein kinases A and C and cell shrinkage

Little is known of the functional properties of the mammalian, brain-specif ic Na+/H+ exchanger isoform 5 (NHE5). Rat NHE5 was stably expressed in NHE- deficient PS120 cells, and its activity was characterized using the fluores cent pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. NH E5 was insensitive to ethylisopropyl amiloride. The transport kinetics disp layed a simple Michaelis-Menten relationship for extracellular Na+ (apparen t K-Na = 27 +/- 5 mM) and a Hill coefficient near 3 for the intracellular p roton concentration with a half-maximal activity at an intracellular pH of 6.93 +/- 0.03. NHE5 activity was inhibited by acute exposure to 8-bromo-cAM P or forskolin (which increases intracellular cAMP by activating adenylate cyclase). The kinase inhibitor H-89 reversed this inhibition, suggesting th at regulation by cAMP involves a protein kinase A (PKA)-dependent process. In contrast, 8-bromo-cGMP did not have a significant effect on activity. Th e protein kinase C (PKC) activator phorbol 12-myristrate 13-acetate inhibit ed NHE5, and the PKC antagonist chelerythrine chloride blunted this effect. Activity was also inhibited by hyperosmotic-induced cell shrinkage but was unaffected by a hyposmotic challenge. These results demonstrate that rat b rain NHE5 is downregulated by activation of PKA and PKC and by cell shrinka ge, important regulators of neuronal cell function.