IDENTIFICATION OF A PY MOTIF IN THE EPITHELIAL NA CHANNEL SUBUNITS ASA TARGET SEQUENCE FOR MUTATIONS CAUSING CHANNEL ACTIVATION FOUND IN LIDDLE SYNDROME

Liddle syndrome is an autosomal dominant form of hypertension, resulti ng from mutations in the cytoplasmic C-terminus of either the beta or gamma subunits of the amiloride-sensitive epithelial Na channel (ENaC) which lead to constitutively increased channel activity. Most mutatio ns reported to date result in the elimination of 45-75 normal amino ac ids from these segments, leaving open the question of the identity of the precise amino acids in which mutation can lead to an enhanced chan nel activity To address this question, we have performed a systematic mutagenesis study of the C-termini of the alpha, beta and gamma ENaC s ubunits of the rat channel and have analyzed their function by express ion in Xenopus oocytes, The results demonstrate that a short proline-r ich segment present in the cytoplasmic C-terminus of each subunit is r equired for the normal regulation of channel activity Missense mutatio ns altering a consensus PPPXY sequence of the alpha, beta or gamma sub units reproduced the increase in channel activity found in mutants in which the entire cytoplasmic C-termini are deleted. This proline-rich sequence, referred to as the PY motif, is known to be a site of bindin g by proteins bearing a WW domain. These findings show that the three PY motifs in the C-termini of ENaC are involved in the regulation of c hannel activity, probably via protein-protein interactions. This new r egulatory mechanism of channel function is critical for the maintenanc e of normal Na reabsorption in the kidney and of Na+ balance and blood pressure.