Modulation of Retrograde Trafficking of KCa3.1 in a Polarized Epithelium

Abstract

In epithelia, the intermediate conductance, Ca2+-activated K+ channel (KCa3.1) is targeted to the basolateral membrane (BLM) where this channel plays numerous roles in absorption and secretion. A growing body of research suggests that the membrane resident population of KCa3.1 may be critical in clinical manifestation of diseases. In this study, we investigated the key molecular components that regulate the degradation of KCa3.1 using a Fisher rat thyroid cell line stably expressing KCa3.1. Using immunoblot, Ussing chamber, and pharmacological approaches, we demonstrated that KCa3.1 is targeted exclusively to the BLM, provided a complete time course of degradation of KCa3.1 and degradation time courses of the channel in the presence of pharmacological inhibitors of ubiquitylation and deubiquitylation to advance our understanding of the retrograde trafficking of KCa3.1. We provide a complete degradation profile of KCa3.1 and that the degradation is via an ubiquitin-dependent pathway. Inhibition of E1 ubiquitin activating enzyme by UBEI-41 crippled the ability of the cells to internalize the channel, shown by the increased BLM surface expression resulting in an increased function of the channel as measured by a DCEBIO sensitive K+ current. Additionally, the involvement of deubiquitylases and degradation by the lysosome were also confirmed by treating the cells with PR-619 or leupeptin/pepstatin, respectively; which significantly decreased the degradation rate of membrane KCa3.1. Additionally, we provided the first evidence that KCa3.1 channels were not deubiquitylated at the BLM. These data further define the retrograde trafficking of KCa3.1, and may provide an avenue for therapeutic approach for treatment of disease.

Role of SNARE Proteins in the Insertion of KCa3.1 in the Plasma Membrane of a Polarized Epithelium

“…As seen in Figure 1, KCa3.1 was highly expressed in the BLM (Lane 3) of the polarized epithelial cells (n = 5). These results confirm previous findings that KCa3.1 is targeted to the BLM in our stably-transfected FRT KCa3.1-BLAP cell line (Bertuccio et al, 2014;Farquhar et al, 2017;Lee et al, 2017).…”

“…The biotin ligase acceptor peptide (BLAP) sequence (GLNDIFEAQKIEWHE) was inserted into the extracellular loop of KCa3.1 between the transmembrane domains S3 and S4 as previously described ( Balut et al, 2010a ; Gao et al, 2010 ; Farqhuar et al, 2017 ; Lee et al, 2017 ). KCa3.1-BLAP and BirA (biotin ligase) with an endoplasmic reticulum (ER) retention sequence, KDEL (kindly provided by Dr. Alice Ting, Massachusetts Institute of Technology, Cambridge, MA, Chen et al, 2005 ; Howarth and Ting, 2008 ), were subcloned into a bicistronic plasmid, pBudCE4.1 (Invitrogen, Carlsbad, CA, United States) behind the EF-1α and CMV promoters, respectively ( Balut et al, 2010a ; 2010b ; Gao et al, 2010 ; Balut et al, 2011 ).…”

“…Therefore, after assembly, KCa3.1-BLAP is biotinylated in the ER prior to being trafficked out to the plasma membrane. Streptavidin labeling of surface KCa3.1-BLAP was performed as previously described ( Balut et al, 2010a ; Gao et al, 2010 ; Bertuccio et al, 2014 ; Farquhar et al, 2017 ; Lee et al, 2017 ). Briefly, upon reaching confluence (72 h after cell seeding), cells were taken out of the incubator for labeling; all procedures and solutions were maintained at 4°C to prevent channel internalization.…”

“…Immunoblot (IB) experiments were performed as described previously ( Jones et al, 2004 , 2007 ; Balut et al, 2010a , 2010b ; Gao et al, 2010 ; Bertuccio et al, 2014 ; Farquhar et al, 2017 . ; Lee et al, 2017 ). Cells were lysed and the protein was harvested and protein concentrations were determined by the bicinchoninic acid protein assay technique ( Walker, 1994 ).…”

“…Ussing chamber experiments were conducted to examine the effect of knockdown of SNARE proteins on the functional expression of KCa3.1 at the BLM, as measured as K + currents (I K ). I K was measured by a VCC MC Ussing chamber system that consisted of an Easymount chamber system and an 8-channel voltage/current clamp unit (Physiologic Instruments, San Diego, CA, United States) as previously described ( Farquhar et al, 2017 ; Lee et al, 2017 ). FRT cells were grown on Snapwell™ filters for 5 days prior to an experiment and transfection of control or siRNA occurred 48 h prior to the experiments (details are stated in the text).…”