RESUMO
The cystic fibrosis transmembrane conductance regulator (CFTR) is expressed in many segments of the mammalian nephron, where it may interact with and modulate the activity of a variety of apical membrane proteins, including the renal outer medullary potassium (ROMK) K(+) channel. However, the expression of CFTR in apical cell membranes or its function as a Cl(-) channel in native renal epithelia has not been demonstrated. Here, we establish that CFTR forms protein kinase A (PKA)-activated Cl(-) channels in the apical membrane of principal cells from the cortical collecting duct obtained from mice. These Cl(-) channels were observed in cell-attached apical patches of principal cells after stimulation by forskolin/3-isobutyl-1-methylxanthine. Quiescent Cl(-) channels were present in patches excised from untreated tubules because they could be activated after exposure to Mg-ATP and the catalytic subunit of PKA. The single-channel conductance, kinetics, and anion selectivity of these Cl(-) channels were the same as those of recombinant mouse CFTR channels expressed in Xenopus laevis oocytes. The CFTR-specific closed-channel blocker CFTR(inh)-172 abolished apical Cl(-) channel activity in excised patches. Moreover, apical Cl(-) channel activity was completely absent in principal cells from transgenic mice expressing the DeltaF508 CFTR mutation but was present and unaltered in ROMK-null mice. We discuss the physiologic implications of open CFTR Cl(-) channels on salt handling by the collecting duct and on the functional CFTR-ROMK interactions in modulating the metabolic ATP-sensing of ROMK.
Assuntos
Canais de Cloreto/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Túbulos Renais Coletores/metabolismo , Animais , Benzoatos/farmacologia , Canais de Cloreto/antagonistas & inibidores , Regulador de Condutância Transmembrana em Fibrose Cística/antagonistas & inibidores , Regulador de Condutância Transmembrana em Fibrose Cística/deficiência , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Feminino , Técnicas In Vitro , Córtex Renal/metabolismo , Cinética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CFTR , Camundongos Knockout , Camundongos Transgênicos , Mutação , Oócitos/metabolismo , Técnicas de Patch-Clamp , Canais de Potássio Corretores do Fluxo de Internalização/deficiência , Canais de Potássio Corretores do Fluxo de Internalização/genética , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tiazolidinas/farmacologia , Xenopus laevisRESUMO
The cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel plays vital roles in fluid transport in many epithelia. While CFTR is expressed along the entire nephron, its function in renal tubule epithelial cells remains unclear, as no specific renal phenotype has been identified in cystic fibrosis. CFTR has been proposed as a regulator of the 30 pS, ATP-sensitive renal K channel (Kir1.1, also known as renal outer medullar K [ROMK]) that is critical for K secretion by cells of the thick ascending limb (TAL) and distal nephron segments responsive to aldosterone. We report here that both ATP and glibenclamide sensitivities of the 30 pS K channel in TAL cells were absent in mice lacking CFTR and in mice homozygous for the deltaF508 mutation. Curcumin treatment in deltaF508-CFTR mice partially reversed the defect in ATP sensitivity. We demonstrate that the effect of CFTR on ATP sensitivity was abrogated by increasing PKA activity. We propose that CFTR regulates the renal K secretory channel by providing a PKA-regulated functional switch that determines the distribution of open and ATP-inhibited K channels in apical membranes. We discuss the potential physiological role of this functional switch in renal K handling during water diuresis and the relevance to renal K homeostasis in cystic fibrosis.