Regulation of voltage-gated potassium channels by HHDPs

电压门控K+ (Kv)通道涉及多种生理过程，如神经元电信号和肌肉收缩。血红素及其分解产物（HHDPs），如一氧化碳（CO）、铁离子（Fe2+）和胆红素氧化终产物（box），被认为是信号分子，部分原因是它们能够直接影响离子通道。因此，了解HHDP-通道相互作用是推断HHDP在生理和病理生理条件下的后果的先决条件。在这里，我们研究了HHDPs影响两种电压门控K+通道亚型的分子机制：eag型通道（Kv10.1和Kv11.1）被细胞内血红素有效抑制，而通过特定a型通道（如Kv1.4）的K+电流，即那些具有快速通道失活的通道，被血红素强烈增强。我们将应用电生理和生化方法，研究HHDPs如何在功能和结构水平上干扰这些离子通道。利用系统诱变方法结合功能分析和结合研究，我们将确定Kv通道蛋白作为血红素及其分解产物传感器的结构模块，从而有助于对HHDPs如何干扰蛋白质的一般理解。考虑到血红素敏感的Kv通道与心律和神经元信号的相关性，我们进一步期望深入了解HHDPs如何参与调节这些综合生理功能。在与FOR 1738的其他合作伙伴的共同努力下，我们将设计和评估用于CO和Fe2+靶向管理的分子工具，旨在低分子量模拟血红素加氧酶活性并消除与现有CO释放分子相关的交叉反应性。基于已建立的形成血红素调控基序的蛋白质序列，我们将产生荧光分子血红素传感器，用于单细胞检测。

Voltage-gated K+ (Kv) channels are implicated in various physiological processes such as neuronal electrical signaling and muscle contraction. Heme and its breakdown products (HHDPs), such as carbon monoxide (CO), Fe2+, and bilirubin oxidation end products (BOXes), are being recognized as signaling molecules, in part because of their ability to directly affect ion channels. Understanding HHDP- channel interactions therefore is a prerequisite to infer about the consequences of HHDPs under physiological and pathophysiological conditions. Here we study the molecular mechanisms involved in HHDPs affecting two subtypes of voltage-gated K+ channels: EAG-type channels (Kv10.1 and Kv11.1) are potently inhibited by intracellular heme, while K+ current through select A-type channels (e.g. Kv1.4), i.e. those featuring rapid channel inactivation, is strongly augmented by heme. We will investigate how HHDPs interfere with these ion channels on a functional and structural level, applying electrophysiological and biochemical methods. Using systematic mutagenesis approaches combined with functional assays and binding studies, we will identify structural modules of Kv channel proteins serving as sensors for heme and its breakdown products and will therefore contribute to a general understanding of how HHDPs interfere with proteins. Given the relevance of heme-sensitive Kv channels for the cardiac rhythm and neuronal signaling, we furthermore expect to gain insight into how HHDPs take part in regulating such integrated physiological functions. In a joint effort with other partners of FOR 1738, we will devise and evaluate molecular tools for the targeted administration of CO and Fe2+, aiming for low molecular weight mimics of heme oxygenase activity and an elimination of cross-reactivity associated with existing CO-releasing molecules. Based on established protein sequences forming heme-regulatory motifs, we will generate fluorescent molecular heme sensors to be applied in single-cell assays.