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Mutation of PKC phosphorylation site 1075T on the BKCa channel α subunit modulates BKCa channel activity
Author(s) -
Zhu Shu,
White Richard E.,
Browning Darren,
Hou Yali,
Meadows Mary L.,
Barman Scott
Publication year - 2006
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.20.5.a1113-c
Subject(s) - hek 293 cells , protein subunit , transfection , phosphorylation , patch clamp , chemistry , microbiology and biotechnology , biology , biochemistry , gene , receptor
Although protein kinase C (PKC) inhibits the activity of the large conductance, calcium‐ and voltage‐activated potassium (BKCa) channel in hypertensive pulmonary arterial smooth muscle, the effect of BKCa channel subunit expression and activation on this phenomenon is unknown. Therefore, to determine the effect of specific BKCa channel subunit expression on PKC regulation of channel activity, phospho‐null (1075T‐A) and phospho‐mimetic (1075T‐E) mutations were made on a PKC phosphorylation site of the BKCa channel α subunit. Human embryonic kidney (HEK)293 cells were co‐transfected with wild type BKCa channel α and β1 subunit (WTαβ), phospho‐null mutated α subunit and β1 subunit (MTαT‐Aβ), or phospho‐mimetic mutated α subunit and β1 subunit (MTαT‐Eβ), respectively. Western blot analysis confirmed mutated BKCa channel subunit expression, and single‐channel patch‐clamp was performed on transfected HEK293 cells. In cell‐attached patches MTαT‐Aβ exhibited less channel activity compared to WTαβ, while baseline BKCa channel activity was increased in MTαT‐Eβ. Addition of 100nM PMA decreased MTαT‐Aβ BKCa channel activity compared to WTαβ. In inside‐out patches the cGMP‐dependent protein kinase (400U) did not activate BKCa channels in MTαT‐Aβ, as observed previously in WTαβ. In addition, BKCa channel activity in MTαT‐Eβ, which was not diminished by lowering calcium at the intracellular face of the membrane, was completely blocked by 1mM TEA, a specific inhibitor of BKCa channels. These results suggest that mutation of specific BKCa channel subunit PKC phosphorylation site may be important in the development of pulmonary hypertension. Supported by HL‐68026 and HL‐73890.