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Plasma Membrane Insertion of KCa2.3 (SK3) is Dependent upon the SNARE Proteins, Syntaxin‐4 and SNAP‐23
Author(s) -
Condliffe Steven,
Bertuccio Claudia,
Devor Daniel
Publication year - 2015
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.29.1_supplement.845.12
Subject(s) - syntaxin , chemistry , gene knockdown , endosome , microbiology and biotechnology , snare complex , biotinylation , endocytosis , biophysics , vesicle , biochemistry , biology , membrane , cell , apoptosis
We previously demonstrated that endocytosis of KCa2.3 is caveolin‐1‐, dynamin‐ and Rab5‐dependent. KCa2.3 then enters Rab35/EPI64C‐ and RME‐1‐containing recycling endosomes and is returned to the plasma membrane (PM). Here, we report our studies on the mechanism by which KCa2.3 is inserted into the PM during recycling and following exit from the Golgi. We demonstrate that KCa2.3 colocalizes with SNAP‐23 and Syntaxin‐4 in the PM of HEK and endothelial cells by confocal immunofluorescence. We further show that KCa2.3 can be co‐immunoprecipitated with SNAP‐23 and Syntaxin‐4. Overexpression of either Syntaxin‐4 or SNAP‐23 increased PM expression of KCa2.3, whereas siRNA‐mediated knockdown of these SNARE proteins significantly decreased PM KCa2.3 expression. Whole‐cell patch clamp studies confirmed that knockdown of SNAP‐23 significantly decreased the apamin sensitive, KCa2.3 current. Using standard biotinylation/stripping methods, we demonstrate that shRNA mediated knockdown of SNAP‐23 inhibits recycling of KCa2.3, whereas scrambled shRNA had no effect. Finally, using biotin ligase acceptor peptide‐tagged KCa2.3, coupled with ER‐resident biotin ligase, channels could be biotinylated in the ER after which we evaluated their rate of insertion from the Golgi into the PM. We demonstrate that knockdown of SNAP‐23 significantly slows the rate of Golgi to PM delivery of KCa2.3. The inhibition of both recycling and PM delivery of newly synthesized channels likely accounts for the decreased PM expression observed following knockdown of these SNARE proteins. In total, our results suggest that insertion of KCa2.3 into the PM depends upon the SNARE proteins, Syntaxin‐4 and SNAP‐23.