Characterization of Na+-permeable Cation Channels in LLC-PK1Renal Epithelial Cells

MALAY K. RAYCHOWDHURY, CRISTINA IBARRA, ALICIA DAMIANO, GEORGE R. JACKSON, JR.,PETER R. SMITH, MARGARET MCLAUGHLIN, ADRIANA G. PRAT, DENNIS A. AUSIELLO,ALAN S. LADER AND HORACIO F. CANTIELLO

JOURNAL OF BIOLOGICAL CHEMISTRY

Año: 2004 p. 20137 - 20146

0021-9258

In this study, the presence of Na-permeable cation channels was determined and characterized in LLC-PK1 cells, a renal tubular epithelial cell line with proximal tubule characteristics derived from pig kidney. Patchclamp analysis under cell-attached conditions indicated the presence of spontaneously active Na-permeable cation channels. The channels displayed nonrectifying single channel conductance of 11 pS, substates, and an 3:1 Na/K permeability-selectivity ratio. The Na-permeable cation channels were inhibited by pertussis toxin and reactivated by G protein agonists. Cation channel activity was observed in quiescent cell-attached patches after vasopressin stimulation. The addition of protein kinase A and ATP to excised patches also induced Na channel activity. Spontaneous and vasopressin- induced Na channel activity were inhibited by extracellular amiloride. To begin assessing potential molecular candidates for this cation channel, both reverse transcription-PCR and immunocytochemical analyses were conducted in LLC-PK1 cells. Expression of porcine orthologs of the ENaC and ApxL genes were found in LLC-PK1 cells. The expression of both gene products was confirmed by immunocytochemical analysis. Although ENaC labeling was mostly intracellular, ApxL labeled to both the apical membrane and cytoplasmic compartments of subconfluent LLC-PK1 cells. Vasopressin stimulation had no effect on ENaC immunolabeling but modified the cellular distribution of ApxL, consistent with an increased membrane-associated ApxL. The data indicate that proximal tubular LLC-PK1 renal epithelial cells express amiloride-sensitive, Na-permeable cation channels, which are regulated by the cAMP pathway, and G proteins. This channel activity may implicate previously reported epithelial channel proteins, although this will require further experimentation. The evidence provides new clues as to potentially relevant Na transport mechanisms in the mammalian proximal nephron.-permeable cation channels was determined and characterized in LLC-PK1 cells, a renal tubular epithelial cell line with proximal tubule characteristics derived from pig kidney. Patchclamp analysis under cell-attached conditions indicated the presence of spontaneously active Na-permeable cation channels. The channels displayed nonrectifying single channel conductance of 11 pS, substates, and an 3:1 Na/K permeability-selectivity ratio. The Na-permeable cation channels were inhibited by pertussis toxin and reactivated by G protein agonists. Cation channel activity was observed in quiescent cell-attached patches after vasopressin stimulation. The addition of protein kinase A and ATP to excised patches also induced Na channel activity. Spontaneous and vasopressin- induced Na channel activity were inhibited by extracellular amiloride. To begin assessing potential molecular candidates for this cation channel, both reverse transcription-PCR and immunocytochemical analyses were conducted in LLC-PK1 cells. Expression of porcine orthologs of the ENaC and ApxL genes were found in LLC-PK1 cells. The expression of both gene products was confirmed by immunocytochemical analysis. Although ENaC labeling was mostly intracellular, ApxL labeled to both the apical membrane and cytoplasmic compartments of subconfluent LLC-PK1 cells. Vasopressin stimulation had no effect on ENaC immunolabeling but modified the cellular distribution of ApxL, consistent with an increased membrane-associated ApxL. The data indicate that proximal tubular LLC-PK1 renal epithelial cells express amiloride-sensitive, Na-permeable cation channels, which are regulated by the cAMP pathway, and G proteins. This channel activity may implicate previously reported epithelial channel proteins, although this will require further experimentation. The evidence provides new clues as to potentially relevant Na transport mechanisms in the mammalian proximal nephron.