Patel Leena S, Mitchell Cheryl K, Dubinsky William P, O'Brien John
Department of Ophthalmology and Visual Science, University of Texas Health Science Center, Houston, 77030, USA.
Cell Commun Adhes. 2006 Jan-Apr;13(1-2):41-54. doi: 10.1080/15419060600631474.
Gap-junctional coupling among neurons is subject to regulation by a number of neurotransmitters including nitric oxide. We studied the mechanisms by which NO regulates coupling in cells expressing Cx35, a connexin expressed in neurons throughout the central nervous system. NO donors caused potent uncoupling of HeLa cells stably transfected with Cx35. This effect was mimicked by Bay 21-4272, an activator of guanylyl cyclase. A pharmacological analysis indicated that NO-induced uncoupling involved both PKG-dependent and PKG-independent pathways. PKA was involved in both pathways, suggesting that PKG-dependent uncoupling may be indirect. In vitro, PKG phosphorylated Cx35 at three sites: Ser110, Ser276, and Ser289. A mutational analysis indicated that phosphorylation on Ser110 and Ser276, sites previously shown also to be phosphorylated by PKA, had a significant influence on regulation. Ser289 phosphorylation had very limited effects. We conclude that NO can regulate coupling through Cx35 and that regulation is indirect in HeLa cells.
神经元之间的缝隙连接耦合受到包括一氧化氮在内的多种神经递质的调节。我们研究了一氧化氮调节表达Cx35（一种在整个中枢神经系统的神经元中表达的连接蛋白）的细胞中耦合的机制。一氧化氮供体导致稳定转染Cx35的HeLa细胞发生有效的解偶联。这种效应被鸟苷酸环化酶激活剂Bay 21-4272模拟。药理学分析表明，一氧化氮诱导的解偶联涉及依赖蛋白激酶G（PKG）和不依赖PKG的途径。蛋白激酶A（PKA）参与了这两条途径，这表明依赖PKG的解偶联可能是间接的。在体外，PKG在三个位点使Cx35磷酸化：丝氨酸110、丝氨酸276和丝氨酸289。突变分析表明，丝氨酸110和丝氨酸276的磷酸化（先前也显示可被PKA磷酸化的位点）对调节有显著影响。丝氨酸289磷酸化的影响非常有限。我们得出结论，一氧化氮可以通过Cx35调节耦合，并且在HeLa细胞中这种调节是间接的。
