Interactions Between SNARE and Exocyst Complex Proteins with KCa3.1 in the Tethering and Fusion of KCa3.1‐containing Vesicles to the Basolateral Membrane of Epithelial Cells

Abstract

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.