Department of Physiology and Pharmacology and the Libin Cardiovascular Research Institute, University of Calgary, Calgary, Alberta, Canada.
FASEB J. 2013 May;27(5):2027-38. doi: 10.1096/fj.12-223669. Epub 2013 Feb 13.
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.
平滑肌收缩性和神经元兴奋性受大电导、钙激活钾 (BKCa) 通道调节，其活性可通过一氧化氮 (NO)/环鸟苷酸 (cGMP) 信号转导被 I 型 cGMP 依赖性蛋白激酶 (cGKI) 调制后增加。我们的研究重点是确定 BKCa 通道中 cGKI 介导的通道活性增强的关键磷酸化位点。使用放射性标记的 ATP 对 cGKIα 对 BKCa 通道的磷酸化进行了生化特性分析，并通过细胞贴附式膜片钳记录在大鼠主动脉 A7r5 平滑肌细胞中定量测定了 NO/cGMP 信号转导对通道活性的调节。BKCa α 亚基中 6 个假定的 cGKI 磷酸化位点（Ser691、Ser873 和 Ser1112）中的 3 个丝氨酸突变为丙氨酸，分别使直接通道磷酸化减少 25-60%，并使 NO 供体或膜通透性 cGMP 对 BKCa 的激活减少 80-100%。cGKI 的急性抑制作用阻止了刺激引起的 BKCa 通道活性增强。我们的数据进一步表明，NO/cGMP/cGKI 信号转导增强 BKCa 活性需要 3 个位点的磷酸化，并且不依赖于 [Ca(2+)]i 的升高。cGKIα 对小鼠 BKCa α 亚基中 3 个特定 Ser 残基的磷酸化解释了 [cGMP]i 升高时原位诱导的 BKCa 通道活性增强。
