The degradation of KCa3.1 in FRT polarised epithelium
|Institution:||University of Otago|
|Keywords:||Physiology; KCa3.1; Potassium; Ubiquitin; ESCRT|
|Full text PDF:||http://hdl.handle.net/10523/5457|
The intermediate conductance calcium activated potassium channel (KCa3.1) has a prominent role in numerous physiological functions, and has recently emerged as the main therapeutic target for many disorders and diseases ranging from lymphoma to Alzheimer’s disease. Much is known about the physiology and pharmacology of this channel, but how the numbers of the channel are regulated at the cell surface remains elusive today. Previous studies aimed to unravel the degradation pathway of KCa3.1 are predominantly based on non-polarised models, as KCa3.1 is widely expressed in polarised epithelial tissues, it is likely that the degradation pathway can present different patterns. In this study, I investigated the key molecular components that regulate the degradation of KCa3.1 using polarised epithelium generated by Fisher rat thyroid (FRT) cells stably expressing KCa3.1. Using immunoblot and Ussing chamber techniques, I demonstrated that KCa3.1 is degraded via an ubiquitin dependent pathway. Inhibition of E1 ubiquitin activating enzyme via UBEI-41 crippled the ability for the cells to internalise the channel, shown by the increase of surface channel expression and DC-EBIO sensitive K+ current. Furthermore, the involvement of deubiquitinases and degradation by the lysosome were also confirmed by treating the cells with PR-619 and leupeptin/pepstatin, respectively; which significantly decreased the rate of membrane KCa3.1 degradation. These findings are the first to demonstrate the involvement of ubiquitin in the degradation of KCa3.1 within a polarised epithelium.