Department of Pharmacology and Toxicology, VCU Program in Enteric Neuromuscular Sciences, Virginia Commonwealth University, Richmond, VA 23298, USA.
Br J Pharmacol. 2010 Mar;159(6):1226-35. doi: 10.1111/j.1476-5381.2009.00599.x. Epub 2010 Jan 27.
Excitation-transcriptional coupling involves communication between plasma membrane ion channels and gene expression in the nucleus. Calcium influx through L-type Ca(2+) channels induces phosphorylation of the transcription factor, cyclic-AMP response element binding protein (CREB) and downstream activation of the cyclic-AMP response element (CRE) promoter regions. Tyrosine nitration of Ca(2+) channels attenuates interactions with c-Src kinase, decreasing Ca(2+) channel currents and smooth muscle contraction during colonic inflammation. In this study we examined the effect of tyrosine nitration and colonic inflammation on Ca(2+) channel mediated phosphorylation of CREB and CRE activation.
CREB and phospho-CREB were detected by Western blots and CRE activation measured by dual luciferase assay. Chinese hamster ovary (CHO) cells were transfected with hCa(v)1.2b and hCa(v)1.2b c-terminal mutants. Colonic inflammation was induced by intracolonic instillation of 2,4,6 trinitrobenzene sulphonic acid in mouse colon.
In hCa(v)1.2b transfected CHO cells and in native colonic smooth muscle, depolarization with 80 mM KCl induced CREB phosphorylation (pCREB). Treatment with peroxynitrite inhibited KCl-induced pCREB. Following experimental colitis, KCl-induced CREB phosphorylation was abolished in smooth muscle, concomitant with tyrosine nitration of Ca(2+) channels. Depolarization increased CRE activation in hCa(v)1.2b CHO cells by 2.35 fold which was blocked by nifedipine and by protein nitration of Ca(2+) channels with peroxynitrite. The Src-kinase inhibitor, PP2, blocked depolarization-induced CRE activation. Mutation of the C-terminus tyrosine residue, Y2134F, but not Y1861F, blocked CRE activation.
Post-translational modification of Ca(2+) channels due to tyrosine nitration modified excitation-transcriptional coupling in colonic inflammation.
兴奋转录偶联涉及质膜离子通道与核内基因表达之间的通讯。L 型钙通道的钙内流诱导转录因子环磷腺苷反应元件结合蛋白(CREB)的磷酸化,以及环磷腺苷反应元件(CRE)启动子区域的下游激活。钙通道的酪氨酸硝化会减弱与 c-Src 激酶的相互作用,从而减少在结肠炎症期间钙通道电流和平滑肌收缩。在这项研究中,我们研究了酪氨酸硝化和结肠炎症对钙通道介导的 CREB 磷酸化和 CRE 激活的影响。
通过 Western blot 检测 CREB 和磷酸化 CREB,通过双荧光素酶测定法测量 CRE 激活。用 hCa(v)1.2b 和 hCa(v)1.2b C 末端突变体转染中国仓鼠卵巢(CHO)细胞。通过在小鼠结肠内注入 2,4,6-三硝基苯磺酸诱导结肠炎症。
在 hCa(v)1.2b 转染的 CHO 细胞和天然结肠平滑肌中,用 80 mM KCl 去极化诱导 CREB 磷酸化(pCREB)。过氧亚硝酸盐处理抑制 KCl 诱导的 pCREB。在实验性结肠炎后,平滑肌中 KCl 诱导的 CREB 磷酸化被消除,同时钙通道发生酪氨酸硝化。去极化使 hCa(v)1.2b CHO 细胞中的 CRE 激活增加了 2.35 倍,该作用被硝苯地平阻断,并被过氧亚硝酸盐的钙通道蛋白硝化阻断。Src 激酶抑制剂 PP2 阻断了去极化诱导的 CRE 激活。C 末端酪氨酸残基 Y2134F 的突变,但不是 Y1861F 的突变,阻断了 CRE 的激活。
由于酪氨酸硝化引起的钙通道的翻译后修饰改变了结肠炎症中的兴奋转录偶联。