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The role of Exocyst complex in the fusion process of KCa3.1 at the basolateral membrane of epithelial cells
Author(s) -
Farquhar Rachel E.,
McDonald Fiona J.,
Hamilton Kirk L.
Publication year - 2018
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2018.32.1_supplement.750.26
Subject(s) - exocyst , microbiology and biotechnology , vesicle , exocytosis , secretory vesicle , golgi apparatus , syntaxin , secretory pathway , biology , vesicular transport proteins , membrane protein , chemistry , lipid bilayer fusion , biophysics , vacuole , cytoplasm , membrane , biochemistry , endoplasmic reticulum , vacuolar protein sorting
Trafficking of vesicles containing plasma membrane‐directed proteins is accomplished by coordinated processes where specific protein complexes interact with transport vesicle proteins during the tethering, docking and incorporation of the vesicle cargo into the plasma membrane. KCa3.1, a calcium‐activated, intermediate‐conductance potassium channel, is targeted to the basolateral membrane (BLM) in epithelial cells. We have shown that this process is Rab1‐ and Rab8‐dependent (Bertuccio et al. PlosONE 9:e92013, 2014), that Myosin‐Vc is required (Farquhar et al. Front. Physiol. 7:639, 2017), and SNARE proteins (VAMP3, SNAP‐23 and STX‐4) are essential in the trafficking of KCa3.1 to the BLM (Farquhar et al. FASEB J. 31:1007.25, 2017). Rab8 and SNARE proteins interact with the Exocyst complex (eight proteins including Sec‐6 and Sec‐8) that aids in tethering of post‐Golgi secretory vesicles to the plasma membrane. The vesicles are tethered at the BLM by the Exocyst complex prior to SNARE proteins coupling to and promoting fusion of vesicles with the BLM. Therefore, we examined the role of Sec‐6 and Sec‐8 in the trafficking of KCa3.1 to the BLM. We used our Fischer rat thyroid (FRT) epithelial cell line stably expressing KCa3.1‐BLAP‐Bir‐A‐KDEL (cells are grown on filters; channels are biotinylated in the Golgi), immunoblot (IB), siRNA and Ussing chamber (UC) experiments. First, preliminary co‐immunoprecipitation experiments (Co‐IP) revealed that Sec‐6 Co‐IPed with VAMP3, SNAP‐23 and KCa3.1, while Sec‐8 Co‐IPed with VAMP3, SNAP‐23 and STX‐4, but not KCa3.1. Our next approach was to streptavidin label KCa3.1 channels that arrive at the BLM to determine the effects of siRNA knockdown of the Sec proteins on the targeting to the BLM (by IB) and the function (K + current by UC) of KCa3.1 at the BLM. We demonstrated that siRNA reduced cellular expression of Sec‐6 by 49±8% (P < 0.01, n=7), and Sec‐6 knockdown reduced the BLM expression of KCa3.1 by 75±6% (P < 0.001, n=7) compared with the appropriate control cells. With UC experiments, we used 1‐EBIO (100 μM, an agonist of KCa3.1) to stimulate current via KCa3.1. Knockdown of Sec‐6 reduced the K + current of KCa3.1 by 91±4% (P < 0.05, n=5) compared with control cells. Similarly, knockdown of Sec‐8 lowered cellular expression of Sec‐8 by 47±9% (P < 0.05, n=4) and reduced the BLM expression of KCa3.1 by 84±4% (P < 0.05, n=4) compared with the appropriate control cells. In the presence of reduced Sec‐8, the K + current of KCa3.1 was lowered by 86±9% (P < 0.05, n=5) compared with control cells. These data suggest that Sec‐6 and Sec‐8 are critical, along with SNARE proteins, for the docking and fusion of KCa3.1‐containing vesicles at the BLM of polarized epithelial cells. Support or Funding Information This work was supported by a Dean's Fund grant from the School of Biomedical Sciences and an AIM grant from the Department of Physiology, both from the University of Otago. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .