Renal Epithelial Protein (Apx) Is an Actin Cytoskeleton-regulated Na1 Channel

ADRIANA G. PRAT, ELIEZER J. HOLTZMAN, DENNIS BROWN, C. CASEY CUNNINGHAM,IGNACIO L. REISINI, THOMAS R. KLEYMAN, MARGARET MCLAUGHLIN, GEORGE R. JACKSON, JR.,JOHN LYDON AND HORACIO F. CANTIELLO

JOURNAL OF BIOLOGICAL CHEMISTRY

Año: 1996 p. 18045 - 18053

0021-9258

Apx, the amphibian protein associated with renal amiloride-sensitive Na1 channel activity and with properties consistent with the pore-forming 150-kDa subunit of an epithelial Na1 channel complex initially purified by Benos et al. (Benos, D. J., Saccomani, G., and Sariban- Sohraby, S. (1987) J. Biol. Chem. 262, 10613–10618), has previously failed to generate amiloride-sensitive Na1 currents (Staub, O., Verrey, F., Kleyman, T. R., Benos, D. J., Rossier, B. C., and Kraehenbuhl, J.-P. (1992) J. Cell Biol. 119, 1497–1506). Renal epithelial Na1 channel activity is tonically inhibited by endogenous actin filaments (Cantiello, H. F., Stow, J., Prat, A. G., and Ausiello, D. A. (1991) Am. J. Physiol. 261, C882–C888). Thus, Apx was expressed and its function examined in human melanoma cells with a defective actin-based cytoskeleton. Apx-transfection was associated with a 60–900% increase in amiloride-sensitive (Ki 5 3 mM) Na1 currents. Single channel Na1 currents had a similar functional fingerprint to the vasopressin-sensitive, and actin-regulated epithelial Na1 channel of A6 cells, including a 6–7 pS single channel conductance and a perm-selectivity of Na1:K1 of 4:1. Na1 channel activity was either spontaneous, or induced by addition of actin or protein kinase A plus ATP to the bathing solution of excised inside-out patches. Therefore, Apx may be responsible for the ionic conductance involved in the vasopressin-activated Na1 reabsorption in the amphibian kidney.1 channel activity and with properties consistent with the pore-forming 150-kDa subunit of an epithelial Na1 channel complex initially purified by Benos et al. (Benos, D. J., Saccomani, G., and Sariban- Sohraby, S. (1987) J. Biol. Chem. 262, 10613–10618), has previously failed to generate amiloride-sensitive Na1 currents (Staub, O., Verrey, F., Kleyman, T. R., Benos, D. J., Rossier, B. C., and Kraehenbuhl, J.-P. (1992) J. Cell Biol. 119, 1497–1506). Renal epithelial Na1 channel activity is tonically inhibited by endogenous actin filaments (Cantiello, H. F., Stow, J., Prat, A. G., and Ausiello, D. A. (1991) Am. J. Physiol. 261, C882–C888). Thus, Apx was expressed and its function examined in human melanoma cells with a defective actin-based cytoskeleton. Apx-transfection was associated with a 60–900% increase in amiloride-sensitive (Ki 5 3 mM) Na1 currents. Single channel Na1 currents had a similar functional fingerprint to the vasopressin-sensitive, and actin-regulated epithelial Na1 channel of A6 cells, including a 6–7 pS single channel conductance and a perm-selectivity of Na1:K1 of 4:1. Na1 channel activity was either spontaneous, or induced by addition of actin or protein kinase A plus ATP to the bathing solution of excised inside-out patches. Therefore, Apx may be responsible for the ionic conductance involved in the vasopressin-activated Na1 reabsorption in the amphibian kidney.