Functional characterization of delta subunit of the epithelial sodium channel

Abstract

The central aim of this work is to functionally characterize the δ ENaC activity modulation using various intracellular and extracellular regulators which confers novel biophysical properties to ENaC. Test for amiloride sensitivity showed that the deletion of carboxy terminus assists channel homomerization of δ 561 , and also that the heteromeric δ 561 βγ channel is highly active in comparison to wildtype δ 638 βγ channel. The newly sequenced δ802- ENaC forms a very low activity heteromeric channel. Gradient in extracellular Na + /K + was used to understand the whole cell behavior of X.laevis oocytes expressing δ ENaC to approximately reflect the extracellular Na + affinity of δ ENaC isoforms. Extracellular proteolytic activation using trypsin revealed that δ 638 βγ and δ 802 βγ ENaC are activated to a significantly higher degree in comparison to the δ561 and δ 561 βγ ENaC which were activated to lower but comparable levels. Extracellular acidification increased the sodium transport in all three heteromeric isoforms but the degree of activation was significantly lower in the homomeric δ 561 channels. Activity of δ ENaC isoforms at the membrane was found to be elevated upon stimulation of intracellular cAMP dependent PKA levels thus suggesting that δ ENaC may mediate some of the PKA response in vivo. On the other hand, δ ENaC isoforms exhibited deviation from classical PKC mediated ENaC endocytosis, where specific activation of δ ENaC was observed amidst βγ- dependent endocytosis, unlike α ENaC.