The Role of the Cytoskeleton and Myosin-Vc in the Targeting of KCa3.1 to the Basolateral Membrane of Polarized Epithelial Cells

Abstract

Understanding the targeting of KCa3.1 to the basolateral membrane (BLM) of polarized epithelial cells is still emerging. Here, we examined the role of the cytoskeleton (microtubules and microfilaments) and Myosin-Vc (Myo-Vc) in the targeting of KCa3.1 in Fischer rat thyroid epithelial cells. We used a pharmacological approach with immunoblot (for the BLM expression of KCa3.1), Ussing chamber (functional BLM expression of KCa3.1) and siRNA experiments. The actin cytoskeleton inhibitors cytochalasin D (10 μM, 5 h) and latrunculin A (10 μM, 5 h) reduced the targeting of KCa3.1 to the BLM by 88 ± 4 and 70 ± 5%, respectively. Colchicine (10 μM, 5 h) a microtubule inhibitor reduced targeting of KCa3.1 to the BLM by 63 ± 7% and decreased 1-EBIO-stimulated KCa3.1 K+ current by 46 ± 18%, compared with control cells. ML9 (10 μM, 5 h), an inhibitor of myosin light chain kinase, decreased targeting of the channel by 83 ± 2% and reduced K+ current by 54 ± 8% compared to control cells. Inhibiting Myo-V with 2,3-butanedione monoxime (10 mM, 5 h) reduced targeting of the channel to the BLM by 58 ± 5% and decreased the stimulated current of KCa3.1 by 48 ± 12% compared with control cells. Finally, using siRNA for Myo-Vc, we demonstrated that knockdown of Myo-Vc reduced the BLM expression of KCa3.1 by 44 ± 7% and KCa3.1 K+ current by 1.04 ± 0.14 μA compared with control cells. These data suggest that the microtubule and microfilament cytoskeleton and Myo-Vc are critical for the targeting of KCa3.1.

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

“…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 ; Farquhar 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, USA) behind the EF-1α and CMV promoters, respectively (Balut et al, 2010a , b , 2011 ; Gao et al, 2010 ).…”

“…Therefore, after assembled, 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 we have previously described (Balut et al, 2010a ; Gao et al, 2010 ; Bertuccio et al, 2014 ; Farquhar 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.…”

“…Since streptavidin is cell impermeable, the cells were labeled by applying streptavidin (10 μg/ml of streptavidin in PBS with 1% BSA) on the desired side (apical or basolateral, or both) for 30 min at 4°C. After labeling, cells were washed three times with PBS with 1% BSA and three times with PBS to eliminate residue streptavidin and incubated for various periods of time at 37°C as indicated in the text (Balut et al, 2010a ; Bertuccio et al, 2014 ; Farquhar et al, 2017 ). Generally, for a given experiment, stably transfected FRT cells were grown on filters for 72 h, channels were labeled basolaterally with streptavidin followed by immediate lysis of cells for t = 0, or filters were returned to the incubator for varying incubation times (1, 3, 5, 8, or 12 h in 37°C) in the presence of a pharmacological inhibitor followed by IB.…”

“…Bertuccio et al ( 2014 ) reported that anterograde trafficking and maintenance of KCa3.1 at the BLM of polarized epithelial cells were dependent upon Rab1 and Rab8. Recently, we demonstrated that the targeting of KCa3.1 to the BLM of epithelial cells was a cytoskeletal- and myosin-Vc-dependent process (Farquhar et al, 2017 ). Further, Bertuccio et al ( 2014 ) have confirmed in polarized epithelia that the KCa3.1 was ubiquitylated prior to and after endocytosis, inhibition of ubiquitylation reduced degradation, and that the trafficking of KCa3.1 was independent of recycling endosomes, and did not require the AP-1 adaptor protein, μ1B for proper trafficking (Bertuccio et al, 2014 ).…”

A self‐organized actomyosin drives multiple intercellular junction disruption and directly promotes neutrophil recruitment in lipopolysaccharide‐induced acute lung injury

“…4. LPS + S + BLEB or LatA or Y27632 or ML-7 or Tb4 groups: media were replaced with neutrophil supernatants 5 h after LPS treatment, cells were treated with LPS, and BLEB (10 mM) (26), LatA (1 mM) (27), Y27632 (10 mM) (26), ML-7 (10 mM) (28), or Tb4 protein (1 mg/ml) (29) was added, respectively. 5.…”