Premium
The role of SNARE proteins in targeting KCa3.1 to the basolateral membrane of epithelial cells
Author(s) -
Farquhar Rachel,
Hamilton Kirk Lee
Publication year - 2017
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.31.1_supplement.1007.25
Subject(s) - microbiology and biotechnology , epithelial polarity , chemistry , membrane protein , gene knockdown , biology , cell , cell culture , membrane , biochemistry , genetics
Targeting epithelial membrane proteins to a specific membrane is crucial for proper epithelial cell function. KCa3.1, a calcium‐activated, intermediate‐conductance potassium channel, is targeted to the basolateral membrane (BLM) in epithelial cells. A consequence of mutations of KCa3.1 is impaired ion and fluid transport of erythrocytes resulting in hereditary xerocytosis. Surprisingly, the mechanism of membrane targeting of KCa3.1 is not well understood. Recently, we reported that KCa3.1 is targeted to the BLM of epithelial cells by a Myosin‐Vc‐dependent pathway (Farquhar et al. FASEB J. 30:1224.41, 2016) and by a Rab1‐ and Rab8‐dependent process (Bertuccio et al. PlosONE 9:e92013, 2014). Here, we extend our work to examine the role of the SNARE proteins VAMP3, syntaxin 4 (STX‐4) and SNAP‐23 in the targeting of KCa3.1 to the BLM of Fischer rat thyroid (FRT) epithelial cells. We used our FRT epithelial cell line stably expressing KCa3.1‐BLAP/Bir‐A‐KDEL (cells grown on filters), immunoblot (IB), siRNA and Ussing chamber experiments in this study. Our IB approach was to streptavidin label channels that arrive at the BLM to determine the effects of siRNA knockdown of specific SNARE proteins on the targeting to and function (KCa3.1 current) of KCa3.1 at the BLM of the epithelial cells. Using siRNA, we showed that knockdown of VAMP3 reduced the BLM expression of KCa3.1 by 49 ± 8% (P ≤ 0.05, n = 3) compared with control cells. With Ussing chamber experiments, we used 1‐EBIO (100 μM, an agonist of KCa3.1) to stimulate current via KCa3.1. Knockdown of VAMP3 reduced the KCa3.1 current by 86 ± 9% (P ≤ 0.05, n = 3) compared with control cells. Similarly, knockdown of STX‐4 lowered the BLM expression of KCa3.1 by 84 ± 5% (P ≤ 0.05, n = 3) and reduced the KCa3.1 current by 82 ± 8% (P ≤ 0.05, n = 2). Finally, siRNA knockdown of SNAP‐23 reduced the expression of KCa3.1 at the BLM by 52 ± 11% (P ≤ 0.05, n = 6) and reduced the KCa3.1 current by 1.2 ± 0.3 μA (P ≤ 0.05, n = 6) compared with control cells. These data suggest that VAMP3, STX‐4 and SNAP‐23 are critical for the targeting KCa3.1 to BLM of polarized epithelial cells. Support or Funding Information This work was supported by UORG and Dean's Fund grants from the University of Otago and an Aims grant from the Department of Physiology (KLH).