Regulation of voltage-gated potassium channels by HHDPs

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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.