Environmental Health Sciences, University of Michigan School of Public Health, Ann Arbor, MI 48109-5632, USA.
Circ Res. 2012 Sep 14;111(7):842-53. doi: 10.1161/CIRCRESAHA.111.263525. Epub 2012 Jul 27.
Kv1.5 (KCNA5) is expressed in the heart, where it underlies the I(Kur) current that controls atrial repolarization, and in the pulmonary vasculature, where it regulates vessel contractility in response to changes in oxygen tension. Atrial fibrillation and hypoxic pulmonary hypertension are characterized by downregulation of Kv1.5 protein expression, as well as with oxidative stress. Formation of sulfenic acid on cysteine residues of proteins is an important, dynamic mechanism for protein regulation under oxidative stress. Kv1.5 is widely reported to be redox-sensitive, and the channel possesses 6 potentially redox-sensitive intracellular cysteines. We therefore hypothesized that sulfenic acid modification of the channel itself may regulate Kv1.5 in response to oxidative stress.
To investigate how oxidative stress, via redox-sensitive modification of the channel with sulfenic acid, regulates trafficking and expression of Kv1.5.
Labeling studies with the sulfenic acid-specific probe DAz and horseradish peroxidase-streptavidin Western blotting demonstrated a global increase in sulfenic acid-modified proteins in human patients with atrial fibrillation, as well as sulfenic acid modification to Kv1.5 in the heart. Further studies showed that Kv1.5 is modified with sulfenic acid on a single COOH-terminal cysteine (C581), and the level of sulfenic acid increases in response to oxidant exposure. Using live-cell immunofluorescence and whole-cell voltage-clamping, we found that modification of this cysteine is necessary and sufficient to reduce channel surface expression, promote its internalization, and block channel recycling back to the cell surface. Moreover, Western blotting demonstrated that sulfenic acid modification is a trigger for channel degradation under prolonged oxidative stress.
Sulfenic acid modification to proteins, which is elevated in diseased human heart, regulates Kv1.5 channel surface expression and stability under oxidative stress and diverts channel from a recycling pathway to degradation. This provides a molecular mechanism linking oxidative stress and downregulation of channel expression observed in cardiovascular diseases.
Kv1.5（KCNA5）在心脏中表达，它构成了控制心房复极化的 I(Kur)电流，在肺血管中，它调节血管收缩性以响应氧张力的变化。心房颤动和低氧性肺动脉高压的特征是 Kv1.5 蛋白表达下调，以及氧化应激。蛋白质半胱氨酸残基上的亚磺酸形成是氧化应激下蛋白质调节的重要、动态机制。Kv1.5 被广泛报道为氧化还原敏感，并且该通道具有 6 个潜在的氧化还原敏感的细胞内半胱氨酸。因此，我们假设通道本身的亚磺酸修饰可能会调节 Kv1.5 以响应氧化应激。
研究氧化应激如何通过亚磺酸对通道的氧化还原敏感修饰来调节 Kv1.5 的运输和表达。
用亚磺酸特异性探针 DAz 和辣根过氧化物酶-链霉亲和素 Western 印迹的标记研究表明，在患有心房颤动的人类患者中，整体上增加了亚磺酸修饰的蛋白质，以及心脏中的 Kv1.5 亚磺酸修饰。进一步的研究表明，Kv1.5 在单个 COOH 末端半胱氨酸（C581）上被亚磺酸修饰，并且亚磺酸水平增加是对氧化剂暴露的反应。使用活细胞免疫荧光和全细胞电压钳位，我们发现修饰这个半胱氨酸是减少通道表面表达、促进其内化以及阻止通道回收到细胞表面所必需和充分的。此外，Western 印迹表明，亚磺酸修饰是通道在长期氧化应激下降解的触发因素。
在患病的人类心脏中升高的蛋白质亚磺酸修饰调节 Kv1.5 通道表面表达和稳定性在氧化应激下，并将通道从再循环途径转移到降解途径。这为氧化应激与心血管疾病中观察到的通道表达下调之间的联系提供了分子机制。
