Control of the movement of ions and water across epithelia is essential for homeostasis. Changing the number or activity of ion channels at the plasma membrane is a significant regulator of epithelial transport. In polarized epithelia, the intermediate-conductance calcium-activated potassium channel, KCa3.1 is delivered to the basolateral membrane where it generates and maintains the electrochemical gradients required for epithelial transport. The mechanisms that control the delivery of KCa3.1 to the basolateral membrane are still emerging. Herein we investigated the role of the highly conserved tethering complex exocyst. In epithelia, exocyst is involved in the tethering of post-Golgi secretory vesicles with the basolateral membrane, which is required before membrane fusion. In our Fisher rat thyroid cell line that stably expresses KCa3.1, siRNA knockdown of either of the exocyst subunits Sec3, Sec6, or Sec8 significantly decreased KCa3.1-specific current. Additionally, knockdown of exocyst complex subunits significantly reduced the basolateral membrane protein level of KCa3.1. Finally, co-immunoprecipitation experiments, suggest associations between Sec6 and KCa3.1, but not between Sec8 and KCa3.1. Collectively, based on these data and our previous studies, we suggest that components of exocyst complex are crucially important in the tethering of KCa3.1 to the basolateral membrane. After which, SNARE proteins aid in the insertion of KCa3.1-containing vesicles into the basolateral membrane of polarized epithelia.

Trafficking of cargo‐containing vesicles to a specific destination membrane is an intricate process that requires coordinated interactions between vesicle, tethering and docking proteins for the proper delivery of the cargo (e.g., ion channel) to a target membrane. KCa3.1 is a calcium‐activated, intermediate‐conductance K+ channel which is targeted to the basolateral membrane (BLM) of epithelial cells, and it plays various roles in epithelial cell physiology. Previously, we reported that trafficking of KCa3.1 to the BLM is a Rab1‐ and Rab8‐dependent process (Bertuccio et al. PlosONE 9:e92013, 2014); which requires Myosin‐Vc for membrane delivery of KCa3.1 (Farquhar et al. Front. Physiol. 7:639, 2017). At recent EB meetings, we reported that SNARE (Soluble NSF Attachment Protein REceptor) proteins including the target‐SNAREs, SNAP‐23 and syntaxin‐4 (STX4), and the vesicle‐SNARE, VAMP3 (vesicle‐associated membrane protein 3), are all crucial in the delivery of KCa3.1 to the BLM (Farquhar et al. FASEB J. 31:1007.25, 2017). Also, we reported that Sec 6 and Sec 8, which are tethering proteins of the Exocyst complex, are critical for the trafficking of KCa3.1 to the BLM (Farquhar et al. FASEB J 32: Issue 1, #750.27, 2018). Currently, there is a gap in our knowledge as to the protein‐protein interactions that occur between the SNAREs, Exocyst complex proteins and KCa3.1 resulting in the incorporation of KCa3.1 in the BLM. In this study, we hypothesized that there would be protein‐protein interactions between the SNAREs and Exocyst proteins and KCa3.1. In order to test this hypothesis, we used our well‐established Fischer rat thyroid (FRT) epithelial cell line stably expressing KCa3.1‐BLAP‐Bir‐A‐KDEL. Cells were grown on TranswellTM filters, and channels were biotinylated in the endoplasmic reticulum and trafficked to the BLM where channels were labeled with streptavidin. Co‐immunoprecipitation (co‐IP) and Western immunoblot (IB) experiments were performed to identify protein‐protein interactions between the various proteins. A typical experiment was to select one protein (e.g., KCa3.1) as the ‘bait’ with a specific antibody (e.g., streptavidin antibody), then immunoblot for a ‘target’ second protein (e.g., VAMP3). After which, the IB membrane was then stripped and blotted for another ‘target’ protein (e.g., STX4). We determined that KCa3.1 co‐IPed with VAMP3 (n=6) and STX4 (n=6), but not SNAP23 (n=5). Additionally, using SNAP‐23 as ‘bait’, SNAP‐23 co‐IPed with STX4 (n=3) and VAMP3 (n=2), but did not co‐IP with KCa3.1 (n=3). When Sec 8 was used as ‘bait’, Sec 8 co‐IPed with SNAP23 (n=2), STX‐4 (n=2), VAMP3 (n=1), but did not interact with KCa3.1. Finally, when Sec 6 was used as ‘bait’, Sec 6 co‐IPed with VAMP3 (n=2), SNAP‐23 (n=3), KCa3.1 (n=1) and no interaction was found with STX4. Currently, these results suggest that there are multiple interactions between these proteins for the incorporation of KCa3.1 into the BLM.Support or Funding InformationThis work was supported by a Dean's Fund grant from the School of Biomedical Sciences and an AIM grant from the Department of Physiology of the University of Otago.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.