Proteomic Analysis of the Functional Inward Rectifier Potassium Channel (Kir) 2.1 Reveals Several Novel Phosphorylation Sites.
Proteomic Analysis of the Functional Inward Rectifier Potassium Channel (Kir) 2.1 Reveals Several Novel Phosphorylation Sites.
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对功能性内向整流钾通道(KIR)2.1的蛋白质组学分析揭示了几个新的磷酸化位点。
DOI：
10.1021/acs.biochem.1c00555
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发表时间：
2021-11-09
期刊：
影响因子：
2.9
通讯作者：
Eckhardt, Lee L.
中科院分区：
文献类型：
--
作者：
Brown, Kyle A.;Anderson, Corey;Reilly, Louise;Sondhi, Kunal;Ge, Ying;Eckhardt, Lee L.
膜蛋白代表了一个大家族的蛋白质，执行重要的生理作用，并代表关键的药物靶标。尽管其重要性，生物分析方法，旨在全面表征翻译后修饰（PTM）的膜蛋白相比，其他类别的蛋白质，部分是因为其固有的低表达和疏水性仍然具有挑战性。内向整流钾通道（Kir）2.1是一种膜蛋白，对维持静息膜电位和心脏动作电位的3相复极化至关重要。这一通道对心脏生理学的重要性通过几种猝死综合征、Andersen−Tawil和短QT综合征的认识而得到强调，这些综合征与Kir2.1功能突变的丧失或获得有关，通常由β-肾上腺素能张力的变化触发。因此，了解该通道的PTM（特别是β-肾上腺素能紧张驱动的磷酸化）对于心律失常的预防非常重要。在这里，我们开发了一种蛋白质组学方法，整合自上而下（完整的蛋白质）和自下而上（酶消化后）的蛋白质组学分析，以表征重组野生型和突变型Kir2.1的PTM，成功地绘制了五个新的磷酸化位点，并确认了第六个位点。我们的研究为未来的工作提供了一个框架，以评估PTM在调节Kir2.1功能中的作用。
Membrane proteins represent a large family of proteins that perform vital physiological roles and represent key drug targets. Despite their importance, bioanalytical methods aiming to comprehensively characterize the post-translational modification (PTM) of membrane proteins remain challenging compared to other classes of proteins in part because of their inherent low expression and hydrophobicity. The inward rectifier potassium channel (Kir) 2.1, an integral membrane protein, is critical for the maintenance of the resting membrane potential and phase-3 repolarization of the cardiac action potential in the heart. The importance of this channel to cardiac physiology is highlighted by the recognition of several sudden arrhythmic death syndromes, Andersen−Tawil and short QT syndromes, which are associated with loss or gain of function mutations in Kir2.1, often triggered by changes in the β-adrenergic tone. Therefore, understanding the PTMs of this channel (particularly β-adrenergic tone-driven phosphorylation) is important for arrhythmia prevention. Here, we developed a proteomic method, integrating both top-down (intact protein) and bottom-up (after enzymatic digestion) proteomic analyses, to characterize the PTMs of recombinant wild-type and mutant Kir2.1, successfully mapping five novel sites of phosphorylation and confirming a sixth site. Our study provides a framework for future work to assess the role of PTMs in regulating Kir2.1 functions.
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