title: Specific phosphorylation sites underlie the stimulation of a large conductance, Ca(2+)-activated K(+) channel by cGMP-dependent protein kinase.
abstract: Smooth muscle contractility and neuronal excitability are regulated by large conductance, Ca(2+)-activated K(+) (BKCa) 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 BKCa channel underlying functional enhancement of channel activity by cGKI. BKCa 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 BKCa α subunit individually reduced direct channel phosphorylation by 25-60% and blocked BKCa activation by either an NO donor or a membrane-permeable cGMP by 80-100%. Acute inhibition of cGKI prevented stimulus-evoked enhancement of BKCa channel activity. Our data further suggest that augmentation of BKCa 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 BKCa α subunit by cGKIα accounts for the enhanced BKCa channel activity induced by elevated [cGMP]i in situ.
authors: Department of Physiology and Pharmacology and the Libin Cardiovascular Research Institute, University of Calgary, Calgary, Alberta, Canada.
authors: Barry D
authors: BD
authors: Kyle
authors: Soleil
authors: S
authors: Hurst
authors: Richard D
authors: RD
authors: Swayze
authors: Jianzhong
authors: J
authors: Sheng
authors: Andrew P
authors: AP
authors: Braun
mesh_terms: D000818:Animals; D006152:Cyclic GMP / Q000378:metabolism; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D051036:Large-Conductance Calcium-Activated Potassium Channels / Q000378:metabolism*; D051379:Mice; D009569:Nitric Oxide / Q000378:metabolism; D020030:Nitric Oxide Donors / Q000494:pharmacology; D010766:Phosphorylation; D051381:Rats; D012694:Serine / Q000378:metabolism; D015398:Signal Transduction
chemical_list: D051036:Large-Conductance Calcium-Activated Potassium Channels; D020030:Nitric Oxide Donors; D009569:Nitric Oxide; D012694:Serine; D017869:Cyclic GMP-Dependent Protein Kinases; D006152:Cyclic GMP