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Cellular and molecular mechanism underlying the modulation of neuronal excitability by heme and heme degradation products

Subject Area Molecular Biology and Physiology of Neurons and Glial Cells
Anatomy and Physiology
Biophysics
Term from 2018 to 2022
Project identifier Deutsche Forschungsgemeinschaft (DFG) - Project number 392037398
 
Final Report Year 2022

Final Report Abstract

Heme (Fe2+-protoporphyrin IX) is an important prosthetic group in hemoproteins with wellinvestigated physiological functions. Much less is known about the abundance and function of non-covalently bound heme in the cytosol and in the extracellular space. The same applies to heme degradation products, such as carbon monoxide (CO), Fe2+, biliverdin, and finally bilirubin oxidation end products (BOXes). This project aimed at investigating the impacts of heme and its degradation products (HHPDs) on neuronal cells with particular focus on ion channels. The most important results of this project fall into three groups: modulation of BKCa-channel function by CO and Fe2+, regulation of voltage-gated K+ (KV) channels by intracellular heme or hemin, and the influence of extracellular hemin on voltage-gated Na+ (NaV) channels. BKCa channels, which are required for inducing relaxation of smooth muscle cells and which contribute to the adaptation of electrical activity in neurons, are activated by CO. Here it could be shown that the degree of CO-mediated activation depends on the composition of the BKCa channel complex of a and b subunits, with b1 and b4 strongly enhancing the potency of CO to increase the channel’s open probability. However, the impact of CO is neither dependent on the presence of the so-called STREX exon of the a subunit, nor is it affected by deliberately altered intracellular heme levels. Moreover, intracellular Fe2+, which like CO is a first-line HHDP, is an even stronger activator of BKCa channels than CO. Kv10.1 channels (hEAG1) are primarily expressed in neuronal cells but also upregulated in various cancer entities. Intracellular hemin is a very potent inhibitor of Kv10.1: hemin suppresses the channel opening with a half-maximal concentration of about 4 nM under ambient conditions. Applying mutagenesis and various biochemical binding assays, we showed that hemin binds to a protein structure that connects the last transmembrane segment (S6) with the cytosolic cyclic nucleotide binding homology domain. Given the high affinity of hemin, this molecule may serve as a lead structure for the development of K10.1-specific antagonists. Besides the loss-of-function impact of hemin on Kv10.1 channels, the binding of heme or hemin to so-called A-type K+ channels results in a gain-of-function effect by impeding the mechanism of channel inactivation. This also applies to channel inactivation induced by select Kvb subunits. During an acute state of hemolysis extracellular hemin is expected to occur at much higher concentrations than intracellular hemin. According to our knowledge and own experiments, extracellular hemin only has a marginal impact on those ion channels previously reported to be subject to regulation by intracellular hemin (e.g., Kv10.1, BKCa). Therefore, and because NaV channels are responsible for the initiation of action potentials in excitable cells, we systematically studied the impact of extracellular hemin on NaV channels. While neuronal NaV channels are not particularly sensitive to hemin, the cardiac isoform NaV1.5 is potently inhibited by extracellular hemin but to by heme. The functional impact of hemin strongly depends on the channel’s activity state: the inhibitory influence is largely eliminated by pulse protocols leading to channel activation. The interaction of hemin with NaV1.5 features a reverse use dependence. This is an opposite phenomenon to what is observed for local anesthetics, which progressively block NaV channels with increasing channel activity. With channel mutagenesis and by performing electrophysiological recordings we showed that hemin specifically interacts with the voltage sensor of the NaV1.5 channel domain II. Hemin therefore acts on cardiac NaV channels similar to what thus far only has been described for voltage-sensor toxin peptides.

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