CELL-SURFACE EXPRESSION OF THE EPITHELIAL NA CHANNEL AND A MUTANT CAUSING LIDDLE SYNDROME - A QUANTITATIVE APPROACH

The epithelial amiloride-sensitive sodium channel (ENaC) controls tran sepithelial Na+ movement in Na+-transporting epithelia and is associat ed with Liddle syndrome, an autosomal dominant form of salt-sensitive hypertension. Detailed analysis of ENaC channel properties and the fun ctional consequences of mutations causing Liddle syndrome has been, so far, limited by lack of a method allowing specific and quantitative d etection of cell-surface-expressed ENaC. We have developed a quantitat ive assay based an the binding of I-125-labeled M(2) anti-FLAG monoclo nal antibody (M(2)Ab) directed against a FLAG reporter epitope introd uced in the extracellular loop of each of the alpha, beta, and gamma E NaC subunits. Insertion of the FLAG epitope into ENaC sequences did no t change its functional and pharmacological properties. The binding sp ecificity and affinity (K-d = 3 nM) allowed as to correlate in individ ual Xenopus oocytes the macroscopic amiloride-sensitive sodium current (I-Na) with the number of ENaC wild-type and mutant subunits expresse d at the cell surface. These experiments demonstrate that: (i) only he teromultimeric channels made of alpha, beta and gamma ENaC subunits ar e maximally and efficient-hv expressed at the cell surface; (ii) the o verall ENaC open probability is one order of magnitude lower than prev iously observed in single-channel recordings; (iii) the mutation causi ng Liddle syndrome (beta R564stop) enhances channel activity by two me chanisms, i.e., by increasing ENaC cell surface expression and by chan ging channel open probability. This quantitative approach provides new insights on the molecular mechanisms underlying one form of salt-sens itive hypertension.