Actin filament organization is required for proper cAMP-dependent activation of CFTR

ADRIANA G. PRAT, C. CASEY CUNNINGHAM, G. ROBERT JACKSON, JR., STEVEN C. BORKAN, YIHAN WANG,DENNIS A. AUSIELLO AND HORACIO F. CANTIELLO

AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY

Año: 1999 p. 1160 - 1169

0363-6143

Actin filament organization is required for proper cAMP-dependent activation of CFTR. Am. J. Physiol. 277 (Cell Physiol. 46): C1160–C1169, 1999.—Previous studies have indicated a role of the actin cytoskeleton in the regulation of the cystic fibrosis transmembrane conductance regulator (CFTR) ion channel. However, the exact molecular nature of this regulation is still largely unknown. In this report human epithelial CFTR was expressed in human melanoma cells genetically devoid of the filamin homologue actin-cross-linking protein ABP-280 [ABP(2)]. cAMP stimulation of ABP(2) cells or cells genetically rescued with ABP-280 cDNA [ABP(1)] was without effect on whole cell Cl2 currents. In ABP(2) cells expressing CFTR, cAMP was also without effect on Cl2 conductance. In contrast, cAMP induced a 10-fold increase in the diphenylamine- 2-carboxylate (DPC)-sensitive whole cell Cl2 currents of ABP(1)/CFTR(1) cells. Further, in cells expressing both CFTR and a truncated form of ABP-280 unable to cross-link actin filaments, cAMP was also without effect on CFTR activation. Dialysis of ABP-280 or filamin through the patch pipette, however, resulted in a DPC-inhibitable increase in the whole cell currents of ABP(2)/CFTR(1) cells. At the single-channel level, protein kinase A plus ATP activated single Cl2 channels only in excised patches from ABP(1)/ CFTR(1) cells. Furthermore, filamin alone also induced Cl2 channel activity in excised patches of ABP(2)/CFTR(1) cells. The present data indicate that an organized actin cytoskeleton is required for cAMP-dependent activation of CFTR.Am. J. Physiol. 277 (Cell Physiol. 46): C1160–C1169, 1999.—Previous studies have indicated a role of the actin cytoskeleton in the regulation of the cystic fibrosis transmembrane conductance regulator (CFTR) ion channel. However, the exact molecular nature of this regulation is still largely unknown. In this report human epithelial CFTR was expressed in human melanoma cells genetically devoid of the filamin homologue actin-cross-linking protein ABP-280 [ABP(2)]. cAMP stimulation of ABP(2) cells or cells genetically rescued with ABP-280 cDNA [ABP(1)] was without effect on whole cell Cl2 currents. In ABP(2) cells expressing CFTR, cAMP was also without effect on Cl2 conductance. In contrast, cAMP induced a 10-fold increase in the diphenylamine- 2-carboxylate (DPC)-sensitive whole cell Cl2 currents of ABP(1)/CFTR(1) cells. Further, in cells expressing both CFTR and a truncated form of ABP-280 unable to cross-link actin filaments, cAMP was also without effect on CFTR activation. Dialysis of ABP-280 or filamin through the patch pipette, however, resulted in a DPC-inhibitable increase in the whole cell currents of ABP(2)/CFTR(1) cells. At the single-channel level, protein kinase A plus ATP activated single Cl2 channels only in excised patches from ABP(1)/ CFTR(1) cells. Furthermore, filamin alone also induced Cl2 channel activity in excised patches of ABP(2)/CFTR(1) cells. The present data indicate that an organized actin cytoskeleton is required for cAMP-dependent activation of CFTR.