The amiloride sensitive epithelial sodium channel (ENaC) and the CFTR (Cystic Fibrosis Transmembrane conductance Regulator) chloride channel are expressed in the apical membranes of epithelial cells in salivary gland ducts, sweat duct, kidney, distal colon, and airways. Abnormalities in channel functions can be life threatening in diseases such as Liddle's syndrome, pseudohypoaldosteronism (PHA), cystic fibrosis (CF) and renal and cardiovascular pathology. Little is known about the mechanisms regulating ENaC and its interaction with CFTR in a native human tissue due to relative scarcity and inaccessibility of human epithelial tissues expressing these channels in spite of its vital role in health and disease. Recently we showed that basolaterally permeabilized human sweat ducts contain regulated ENaC and CFTR in the apical membrane. Besides, this human tissue is most easily accessible. We found evidence that ENaC activation requires CFTR channel function. This project is designed to explore the molecular interactions between CFTR and ENaC in health and disease by testing the following hypotheses that: 1.) the activities of CFTR and ENaC are coupled, 2.) the stimulation of CFTR C1- channel function is required for ENaC activation, and 3.) the ion composition alters the interaction between CFTR and ENaC. We will investigate the hypotheses by employing electrophysiological, immunocytochemical and fluorescent techniques on basolaterally a-toxin permeabilized sweat ducts derived from normal human subjects and patients with CF, PHA and Liddle's syndrome. We will test the hypotheses by determining whether: 1.) ENaC activation is dependent on CFTR activation by protein kinases A, G, C or G-proteins, 2.) Mutations in CFTR (e.g. DF508, R117H, G551D) alter ENaC function and vice versa (ENaC mutations in PHA and Liddle's syndrome alter CFTR function), 3.) CFTR activation of ENaC requires CFTR C1- channel function induced by phosphorylation and ATP hydrolysis, and 4.) changing ion-composition (Na+, C1-, K+, Ca++ and pH) alter the activities of CFTR and ENaC in parallel. We believe that elucidating the molecular interactions between these channels will contribute to understanding several disease processes evolving from disturbance in NaC1 retention and elimination.
