Nirakar Sahoo

Cysteine 723 in the C-linker segment confers oxidative inhibition of hERG1 potassium channels

Excess reactive oxygen species (ROS) play a crucial role under pathophysiological conditions, such as ischaemia/reperfusion and diabetes, potentially contributing to cardiac arrhythmia. hERG1 (KCNH2) potassium channels terminate the cardiac action potential and malfunction can lead to long‐QT syndrome and fatal arrhythmia. To investigate the molecular mechanisms of hERG1 channel alteration by ROS, hERG1 and mutants thereof were expressed in HEK293 cells and studied with the whole‐cell patch‐clamp method. Even mild ROS stress induced by hyperglycaemia markedly decreased channel current. Intracellular H2O2 or cysteine‐specific modifiers also strongly inhibited channel activity and accelerated deactivation kinetics. Mutagenesis revealed that cysteine 723 (C723), a conserved residue in a structural element linking the C‐terminal domain to the channels gate, is critical for oxidative functional modification. Moreover, kinetics of channel closure strongly influences ROS‐induced modification, where rapid channel deactivation diminishes ROS sensitivity. Because of its fast deactivation kinetics, the N‐terminally truncated splice variant hERG1b possesses greater resistance to oxidative modification.

Oxidative Modulation of Voltage-Gated Potassium Channels

SIGNIFICANCE Voltage-gated K+ channels are a large family of K+-selective ion channel protein complexes that open on membrane depolarization. These K+ channels are expressed in diverse tissues and their function is vital for numerous physiological processes, in particular of neurons and muscle cells. Potentially reversible oxidative regulation of voltage-gated K+ channels by reactive species such as reactive oxygen species (ROS) represents a contributing mechanism of normal cellular plasticity and may play important roles in diverse pathologies including neurodegenerative diseases. RECENT ADVANCES Studies using various protocols of oxidative modification, site-directed mutagenesis, and structural and kinetic modeling provide a broader phenomenology and emerging mechanistic insights. CRITICAL ISSUES Physicochemical mechanisms of the functional consequences of oxidative modifications of voltage-gated K+ channels are only beginning to be revealed. In vivo documentation of oxidative modifications of specific amino-acid residues of various voltage-gated K+ channel proteins, including the target specificity issue, is largely absent. FUTURE DIRECTIONS High-resolution chemical and proteomic analysis of ion channel proteins with respect to oxidative modification combined with ongoing studies on channel structure and function will provide a better understanding of how the function of voltage-gated K+ channels is tuned by ROS and the corresponding reducing enzymes to meet cellular needs.

Analysis of Fe(III) Heme Binding to Cysteine-Containing Heme-Regulatory Motifs in Proteins

Regulatory heme binds to specific motifs in proteins and controls a variety of biochemical processes. Several of these proteins were recently shown to form complexes with ferric and/or ferrous heme via a cysteine residue as axial ligand. The objective of this study was to examine the heme-binding properties of a series of cysteine-containing peptides with focus on CP motif sequences. The peptides displayed different binding behavior upon Fe(III) heme application with characteristic wavelength shifts of the Soret band to 370 nm or 420-430 nm and in some cases to both wavelengths. Whereas for most of the peptides containing a cysteine only a shift to 420-430 nm was observed, CP-containing peptides exhibited a preference for a shift to 370 nm. Detailed structural investigation using Raman and NMR spectroscopy on selected representatives revealed different binding modes with respect to iron ion coordination, which reflected the results of the UV-vis studies. A predicted short sequence stretch derived from dipeptidyl peptidase 8 was additionally examined with respect to CP motif binding to heme on the peptide as well as on the protein level. The heme association was confirmed with the first solution structure of a CP-peptide-heme complex and, moreover, an inhibitory effect of Fe(III) heme on the enzymes activity. The relevance of both the use of model compounds to elucidate the molecular mechanism underlying regulatory heme binding and its potential for the investigation of regulatory heme control is discussed.

Heme impairs the ball-and-chain inactivation of potassium channels.

Significance Heme, traditionally viewed as a stable protein cofactor such as in hemoglobin, also serves as an acute signaling molecule and is cytotoxic at high concentrations. Here, we show that free intracellular heme potently enhances A-type potassium channel function. Such channels determine action potential frequency in excitable cells, and their dysfunction often contributes to pathological hyperexcitability, such as in pain and epilepsy. Binding of free heme at nanomolar concentrations to the “ball-and-chain” N terminus of A-type potassium channels, which typically closes the channels, introduces a stable structure in the otherwise disordered region and allows for a greater efflux of potassium ions, thus reducing cellular excitability. Heme therefore could be a powerful negative-feedback regulator in brain and muscle function. Fine-tuned regulation of K+ channel inactivation enables excitable cells to adjust action potential firing. Fast inactivation present in some K+ channels is mediated by the distal N-terminal structure (ball) occluding the ion permeation pathway. Here we show that Kv1.4 K+ channels are potently regulated by intracellular free heme; heme binds to the N-terminal inactivation domain and thereby impairs the inactivation process, thus enhancing the K+ current with an apparent EC50 value of ∼20 nM. Functional studies on channel mutants and structural investigations on recombinant inactivation ball domain peptides encompassing the first 61 residues of Kv1.4 revealed a heme-responsive binding motif involving Cys13:His16 and a secondary histidine at position 35. Heme binding to the N-terminal inactivation domain induces a conformational constraint that prevents it from reaching its receptor site at the vestibule of the channel pore.

Kcnh1 voltage-gated potassium channels are essential for early zebrafish development

Background: Kcnh1 is a voltage-gated potassium channel that is primarily expressed in brain. Results: Knockdown of kcnh1 in zebrafish delays neural development and causes embryonic lethality. Conclusion: Kcnh1 is involved in cell proliferation during early zebrafish development. Significance: The finding that Kcnh1 has basic functions beyond neural signaling will help to elucidate its roles in physiology and cancer formation. The Kcnh1 gene encodes a voltage-gated potassium channel highly expressed in neurons and involved in tumor cell proliferation, yet its physiological roles remain unclear. We have used the zebrafish as a model to analyze Kcnh1 function in vitro and in vivo. We found that the kcnh1 gene is duplicated in teleost fish (i.e. kcnh1a and kcnh1b) and that both genes are maternally expressed during early development. In adult zebrafish, kcnh1a and kcnh1b have distinct expression patterns but share expression in brain and testis. Heterologous expression of both genes in Xenopus oocytes revealed a strong conservation of characteristic functional properties between human and fish channels, including a unique sensitivity to intracellular Ca2+/calmodulin and modulation of voltage-dependent gating by extracellular Mg2+. Using a morpholino antisense approach, we demonstrate a strong kcnh1 loss-of-function phenotype in developing zebrafish, characterized by growth retardation, delayed hindbrain formation, and embryonic lethality. This late phenotype was preceded by transcriptional up-regulation of known cell-cycle inhibitors (p21, p27, cdh2) and down-regulation of pro-proliferative factors, including cyclin D1, at 70% epiboly. These results reveal an unanticipated basic activity of kcnh1 that is crucial for early embryonic development and patterning.

Determination of Hemin‐Binding Characteristics of Proteins by a Combinatorial Peptide Library Approach

Studies of the binding of heme/hemin to proteins or peptides have recently intensified as it became evident that heme serves not only as a prosthetic group, but also as a regulator and effector molecule interacting with transmembrane and cytoplasmic proteins. The iron‐ion‐containing heme group can associate with these proteins in different ways, with the amino acids Cys, His, and Tyr allowing individual modes of binding. Strong coordinate‐covalent binding, such as in cytochrome c, is known, and reversible attachment is also discussed. Ligands for both types of binding have been reported independently, though sometimes with different affinities for similar sequences. We applied a combinatorial approach using the library (X)4(C/H/Y)(X)4 to characterize peptide ligands with considerable hemin binding capacities. Some of the library‐selected peptides were comparable in terms of hemin association independently of whether or not a cysteine residue was present in the sequence. Indeed, a preference for His‐based (≈39 %) and Tyr‐based (≈40 %) sequences over Cys‐based ones (≈21 %) was detected. The binding affinities for the library‐selected peptides, as determined by UV/Vis spectroscopy, were in the nanomolar range. Moreover, selected representatives efficiently competed for hemin binding with the human BK channel hSlo1, which is known to be regulated by heme through binding to its heme‐binding domain.

Current inhibition of human EAG1 potassium channels by the Ca2+ binding protein S100B

MINT‐7988123: CaM (uniprotkb:P62158) and EAG1 alpha (uniprotkb:O95259) physically interact (MI:0915) by competition binding (MI:0405)MINT‐7988019, MINT‐7988052: EAG1 alpha (uniprotkb:O95259) binds (MI:0407) to s100B (uniprotkb:P02638) by pull down (MI:0096)MINT‐7988074: EAG1 alpha (uniprotkb:O95259) and s100B (uniprotkb:P02638) physically interact (MI:0915) by competition binding (MI:0405)MINT‐7988100:CaM (uniprotkb:P62158) and EAG1 alpha (uniprotkb:O95259) bind (MI:0407) by fluorescence correlation spectroscopy (MI:0052).

CO-independent modification of K+ channels by tricarbonyldichlororuthenium(II) dimer (CORM-2)

Abstract Although toxic when inhaled in high concentrations, the gas carbon monoxide (CO) is endogenously produced in mammals, and various beneficial effects are reported. For potential medicinal applications and studying the molecular processes underlying the pharmacological action of CO, so‐called CO‐releasing molecules (CORMs), such as tricabonyldichlororuthenium(II) dimer (CORM‐2), have been developed and widely used. Yet, it is not readily discriminated whether an observed effect of a CORM is caused by the released CO gas, the CORM itself, or any of its intermediate or final breakdown products. Focusing on Ca2+‐ and voltage‐dependent K+ channels (KCa1.1) and voltage‐gated K+ channels (Kv1.5, Kv11.1) relevant for cardiac safety pharmacology, we demonstrate that, in most cases, the functional impacts of CORM‐2 on these channels are not mediated by CO. Instead, when dissolved in aqueous solutions, CORM‐2 has the propensity of forming Ru(CO)2 adducts, preferentially to histidine residues, as demonstrated with synthetic peptides using mass‐spectrometry analysis. For KCa1.1 channels we show that H365 and H394 in the cytosolic gating ring structure are affected by CORM‐2. For Kv11.1 channels (hERG1) the extracellularly accessible histidines H578 and H587 are CORM‐2 targets. The strong CO‐independent action of CORM‐2 on Kv11.1 and Kv1.5 channels can be completely abolished when CORM‐2 is applied in the presence of an excess of free histidine or human serum albumin; cysteine and methionine are further potential targets. Off‐site effects similar to those reported here for CORM‐2 are found for CORM‐3, another ruthenium‐based CORM, but are diminished when using iron‐based CORM‐S1 and absent for manganese‐based CORM‐EDE1. Graphical abstract Figure. No caption available.

Ca 2+ /calmodulin regulates Kvβ1.1-mediated inactivation of voltage-gated K + channels

A-type K+ channels open on membrane depolarization and undergo subsequent rapid inactivation such that they are ideally suited for fine-tuning the electrical signaling in neurons and muscle cells. Channel inactivation mostly follows the so-called ball-and-chain mechanism, in which the N-terminal structures of either the K+ channel’s α or β subunits occlude the channel pore entry facing the cytosol. Inactivation of Kv1.1 and Kv1.4 channels induced by Kvβ1.1 subunits is profoundly decelerated in response to a rise in the intracellular Ca2+ concentration, thus making the affected channel complexes negative feedback regulators to limit neuronal overexcitation. With electrophysiological and biochemical experiments we show that the Ca2+ dependence is gained by binding of calmodulin to the “chain” segment of Kvβ1.1 thereby compromising the mobility of the inactivation particle. Furthermore, inactivation regulation via Ca2+/calmodulin does not interfere with the β subunit’s enzymatic activity as an NADPH-dependent oxidoreductase, thus rendering the Kvβ1.1 subunit a multifunctional receptor that integrates cytosolic signals to be transduced to altered electrical cellular activity.