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Specific phosphorylation sites underlie the stimulation of a large conductance, Ca 2+ ‐activated K + channel by cGMP‐dependent protein kinase
Author(s) -
Kyle Barry D.,
Hurst Soleil,
Swayze Richard D.,
Sheng Jianzhong,
Braun Andrew P.
Publication year - 2013
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/fj.12-223669
Subject(s) - phosphorylation , chemistry , protein kinase a , cgmp dependent protein kinase , nitric oxide , stimulation , patch clamp , microbiology and biotechnology , protein subunit , biophysics , biochemistry , biology , endocrinology , mitogen activated protein kinase kinase , receptor , organic chemistry , gene
Smooth muscle contractility and neuronal excitability are regulated by large conductance, Ca 2+ ‐activated K + (BK Ca ) channels, the activity of which can be increased after modulation by type I cGMP‐dependent protein kinase (cGKI) via nitric oxide (NO)/cGMP signaling. Our study focused on identifying key phosphorylation sites within the BK Ca channel underlying functional enhancement of channel activity by cGKI. BK Ca channel phosphorylation by cGKIα was characterized biochemically using radiolabeled ATP, and regulation of channel activity by NO/cGMP signaling was quantified in rat aortic A7r5 smooth muscle cells by cell‐attached patch‐clamp recording. Serine to alanine substitutions at 3 of 6 putative cGKI phosphorylation sites (Ser691, Ser873, and Ser1112) in the BK Ca α subunit individually reduced direct channel phosphorylation by 25–60% and blocked BK Ca activation by either an NO donor or a membrane‐permeable cGMP by 80–100%. Acute inhibition of cGKI prevented stimulus‐evoked enhancement of BK Ca channel activity. Our data further suggest that augmentation of BK Ca activity by NO/cGMP/cGKI signaling requires phosphorylation at all 3 sites and is independent of elevations in [Ca 2+ ] i . Phosphorylation of 3 specific Ser residues within the murine BK Ca α subunit by cGKIα accounts for the enhanced BK Ca channel activity induced by elevated [cGMP] i in situ. —Kyle, B.D., Hurst, S., Swayze, R. D., Sheng, J., Braun, A.P. Specific phosphorylation sites underlie the stimulation of a large conductance, Ca 2+ ‐activated K + channel by cGMP‐dependent protein kinase. FASEB J. 27, 2027–2038 (2013). www.fasebj.org