Analysis of Blocker-labeled Channels Reveals the Dependence of Recycling Rates of ENaC on the Total Amount of Recycled Channels
Trafficking is one of the primary mechanisms of epithelial Na+ channel (ENaC) regulation. Although it is known that ENaCs are recycled between the apical membrane and the intracellular channel pool, it has been difficult to investigate the recycling of ENaCs; especially endogenously expressed ENaCs. The aim of the present study is to investigate if the recycling rates of ENaCs depend on the total amount of recycled ENaCs. To accomplish this point, we established a novel method to estimate the total amount of recycled ENaCs and the ENaC recycling rates by using a specific blocker (benzamil) of ENaC with a high-affinity for functional label of the channels in recycling. Applying this method, we studied if a decrease in the total amount of ENaCs caused by brefeldin A (5 µg/mL, 1 h) affects respectively the rates of insertion and endocytosis of ENaCs to and from the apical membrane in monolayers of renal epithelial A6 cells. Our observations indicate that: 1) both insertion and endocytosis rates of ENaC increase when the total amount of ENaCs decreases, 2) the increase in the insertion rate is larger than that in the endocytosis rate, and 3) this larger increase in the insertion rate than the endocytosis rate caused by the decrease in the total amount of ENaCs plays an important role in preventing Na+ transport from drastically diminishing due to a decrease in the total amount of ENaCs. The newly established analysis of blocker-labeled ENaCs in the present study provides a useful tool to investigate the recycling of endogenously expressed ENaCs.