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Regulation of KCNQ5 potassium channels in A7r5 vascular smooth muscle cells involves protein kinase C‐mediated channel phosphorylation
Author(s) -
Olsen Jessica L,
Brueggemann Lioubov I,
Cribbs Leanne L,
Byron Kenneth L
Publication year - 2008
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.22.1_supplement.721.4
Subject(s) - protein kinase c , calphostin c , phosphorylation , activator (genetics) , staurosporine , kinase , potassium channel , protein kinase a , biology , microbiology and biotechnology , calmodulin , chemistry , biochemistry , endocrinology , receptor , enzyme
Protein kinase C (PKC) ‐dependent suppression of KCNQ5 voltage‐dependent K + currents was recently implicated in signal transduction of physiological concentrations of arginine vasopressin (AVP). It has yet to be determined if KCNQ5 channel proteins are directly phosphorylated in response to PKC activation. A FLAG‐ and GFP‐tagged KCNQ5 (FLAG‐KCNQ5) construct was designed and transfected into A7r5 cells. Whole‐cell voltage clamp techniques were used to confirm the expression of functional FLAG‐KCNQ5 channels. FLAG‐KCNQ5 currents were 100‐fold larger than endogenous currents and the currents were abolished by expression of KCNQ5 shRNA. AVP (500 pM) and PKC activator 4β‐phorbol 12‐myristate 13‐acetate (PMA, 1 nM) reduced FLAG‐KCNQ5 currents by about 80%. FLAG‐KCNQ5 channel protein was detected as a band at 105 KDa in anti‐FLAG immunoprecipitates by Western blotting. The same band was recognized by anti‐ phospho‐serine/threonine/tyrosine antibodies following treatment with PMA (1 nM, 15 min). Phosphorylation was abolished by preincubation with the PKC inhibitor calphostin C (250 nM, 2hrs). A‐kinase anchoring protein (AKAP150), which has been implicated in recruitment of protein kinases and channel phosphorylation in other cell systems, was found to co‐immunoprecipitate with FLAG‐KCNQ5 channels. We conclude that KCNQ5 channel proteins are directly phosphorylated in response to PKC activation in A7r5 cells. Supported by NIH R01 HL070670 (KLB) and a 2007 Student Scholarship in Cardiovascular Disease and Stroke (JLO).