Project description:Ciguatoxins (CTX) and brevetoxins (BTX) are polycyclic ethereal compounds biosynthesized by the worldwide distributed planktonic and epibenthic dinoflagellates of Gambierdiscus and Karenia genera, correspondingly. Ciguatera, evoked by CTXs, is a type of ichthyosarcotoxism, which involves a variety of gastrointestinal and neurological symptoms, while BTXs cause so-called neurotoxic shellfish poisoning. Both types of toxins are reviewed together because of similar mechanisms of their action. These are the only molecules known to activate voltage-sensitive Na⁺-channels in mammals through a specific interaction with site 5 of its α-subunit and may compete for it, which results in an increase in neuronal excitability, neurotransmitter release and impairment of synaptic vesicle recycling. Most marine ciguatoxins potentiate Nav channels, but a considerable number of them, such as gambierol and maitotoxin, have been shown to affect another ion channel. Although the extrinsic function of these toxins is probably associated with the function of a feeding deterrent, it was suggested that their intrinsic function is coupled with the regulation of photosynthesis via light-harvesting complex II and thioredoxin. Antagonistic effects of BTXs and brevenal may provide evidence of their participation as positive and negative regulators of this mechanism.
Project description:Ion currents associated with channel proteins in the presence of membrane potential are ubiquitous in cellular and organelle membranes. When an ion current occurs through a channel protein, Joule heating should occur. However, this Joule heating seems to have been largely overlooked in biology. Here we show theoretical investigation of Joule heating involving channel proteins in biological processes. We used electrochemical potential to derive the Joule's law for an ion current through an ion transport protein in the presence of membrane potential, and we suggest that heat production and absorption can occur. Simulation of temperature distribution around a single channel protein with the Joule heating revealed that the temperature increase was as small as <10-3 K, although an ensemble of channel proteins was suggested to exhibit a noticeable temperature increase. Thereby, we theoretically investigated the Joule heating of systems containing ensembles of channel proteins. Nerve is known to undergo rapid heat production followed by heat absorption during the action potential, and our simulation of Joule heating for a squid giant axon combined with the Hodgkin-Huxley model successfully reproduced the feature of the heat. Furthermore, we extended the theory of Joule heating to uncoupling protein 1 (UCP1), a solute carrier family transporter, which is important to the non-shivering thermogenesis in brown adipose tissue mitochondria (BATM). Our calculations showed that the Joule heat involving UCP1 was comparable to the literature calorimetry data of BATM. Joule heating of ion transport proteins is likely to be one of important mechanisms of cellular thermogenesis.
Project description:Molecular dynamics (MD) simulations allow researchers to investigate the behavior of desired biological targets at ever-decreasing costs with ever-increasing precision. Among the biological macromolecules, ion channels are remarkable transmembrane proteins, capable of performing special biological processes and revealing a complex regulatory matrix, including modulation by small molecules, either endogenous or exogenous. Recently, given the developments in ion channel structure determination and accessibility of bio-computational techniques, MD and related tools are becoming increasingly popular in the intense research area regarding ligand-channel interactions. This review synthesizes and presents the most important fields of MD involvement in investigating channel-molecule interactions, including, but not limited to, deciphering the binding modes of ligands to their ion channel targets and the mechanisms through which chemical compounds exert their effect on channel function. Special attention is devoted to the importance of more elaborate methods, such as free energy calculations, while principles regarding drug design and discovery are highlighted. Several technical aspects involving the creation and simulation of channel-molecule MD systems (ligand parameterization, proper membrane setup, system building, etc.) are also presented.
Project description:Protein S-palmitoylation, the reversible thioester linkage of a 16-carbon palmitate lipid to an intracellular cysteine residue, is rapidly emerging as a fundamental, dynamic, and widespread post-translational mechanism to control the properties and function of ligand- and voltage-gated ion channels. Palmitoylation controls multiple stages in the ion channel life cycle, from maturation to trafficking and regulation. An emerging concept is that palmitoylation is an important determinant of channel regulation by other signaling pathways. The elucidation of enzymes controlling palmitoylation and developments in proteomics tools now promise to revolutionize our understanding of this fundamental post-translational mechanism in regulating ion channel physiology.
Project description:Kcv, a 94-aa protein encoded by Paramecium bursaria chlorella virus 1, is the smallest known protein to form a functional potassium ion channel and basically corresponds to the "pore module" of potassium channels. Both viral replication and channel activity are inhibited by the ion channel blockers barium and amantadine but not by cesium. Genes encoding Kcv-like proteins were isolated from 40 additional chlorella viruses. Differences in 16 of the 94 amino acids were detected, producing six Kcv-like proteins with amino acid substitutions occurring in most of the functional domains of the protein (N terminus, transmembrane 1, pore helix, selectivity filter, and transmembrane 2). The six proteins form functional potassium selective channels in Xenopus oocytes with different properties including altered current kinetics and inhibition by cesium. The amino acid changes together with the different properties observed in the six Kcv-like channels will be used to guide site-directed mutations, either singularly or in combination, to identify key amino acids that confer specific properties to Kcv.
Project description:The dysfunction of voltage-gated ion channels contributes to the pathology of ischemic stroke. In this study, we developed rat models of transient ischemic attack (TIA) and reversible ischemic neurological deficit (RIND) that was induced via the injection of artificial embolic particles during full consciousness, that allow us to monitor the neurologic deficit and positron emission tomography (PET) scans in real-time. We then evaluated the infarction volume of brain tissue was confirmed by 2,3,5-triphenyl tetrazolium chloride (TTC) staining, and gene expressions were evaluated by quantitative real-time PCR (qPCR). We found that rats with TIA or RIND exhibited neurological deficits as determined by negative TTC and PET findings. However, the expression of voltage-gated sodium channels in the hippocampus was significantly up-regulated in the qPCR array study. Furthermore, an altered expression of sodium channel β-subunits and potassium channels, were observed in RIND compared to TIA groups. In conclusion, to our knowledge, this is the first report of the successful evaluation of voltage-gated ion channel gene expression in TIA and RIND animal models. This model will aid future studies in investigating pathophysiological mechanisms, and in developing new therapeutic compounds for the treatment of TIA and RIND.
Project description:The homotrimeric P2X7 purinergic receptor has sparked interest because of its capacity to sense adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide (NAD) released from cells and to induce calcium signaling and cell death. Here, we examine the response of arginine mutants of P2X7 to soluble and covalently bound ligands. High concentrations of ecto-ATP gate P2X7 by acting as a soluble ligand and low concentrations of ecto-NAD gate P2X7 following ADP-ribosylation at R125 catalyzed by toxin-related ecto-ADP-ribosyltransferase ART2.2. R125 lies on a prominent cysteine-rich finger at the interface of adjacent receptor subunits, and ADP-ribosylation at this site likely places the common adenine nucleotide moiety into the ligand-binding pocket of P2X7.
Project description:Protein S-acylation, the reversible covalent fatty-acid modification of cysteine residues, has emerged as a dynamic posttranslational modification (PTM) that controls the diversity, life cycle, and physiological function of numerous ligand- and voltage-gated ion channels. S-acylation is enzymatically mediated by a diverse family of acyltransferases (zDHHCs) and is reversed by acylthioesterases. However, for most ion channels, the dynamics and subcellular localization at which S-acylation and deacylation cycles occur are not known. S-acylation can control the two fundamental determinants of ion channel function: (1) the number of channels resident in a membrane and (2) the activity of the channel at the membrane. It controls the former by regulating channel trafficking and the latter by controlling channel kinetics and modulation by other PTMs. Ion channel function may be modulated by S-acylation of both pore-forming and regulatory subunits as well as through control of adapter, signaling, and scaffolding proteins in ion channel complexes. Importantly, cross-talk of S-acylation with other PTMs of both cysteine residues by themselves and neighboring sites of phosphorylation is an emerging concept in the control of ion channel physiology. In this review, I discuss the fundamentals of protein S-acylation and the tools available to investigate ion channel S-acylation. The mechanisms and role of S-acylation in controlling diverse stages of the ion channel life cycle and its effect on ion channel function are highlighted. Finally, I discuss future goals and challenges for the field to understand both the mechanistic basis for S-acylation control of ion channels and the functional consequence and implications for understanding the physiological function of ion channel S-acylation in health and disease.
Project description:Safety is one of the most important and critical issues in drug development. Many drugs were abandoned in clinical trials and retracted from the market because of unknown side effects. Cardiotoxicity is one of the most common reasons for drug retraction due to its potential side effects, i.e., inducing either tachycardia, bradycardia or arrhythmia. The zebrafish model could be used to screen drug libraries with potential cardiotoxicity in a high-throughput manner. In addition, the fundamental principles of replacement, reduction, and refinement of laboratory animal usage, 3R, could be achieved by using zebrafish as an alternative to animal models. In this study, we used a simple ImageJ-based method to evaluate and screen 70 ion channel ligands and successfully identify six compounds with strong cardiotoxicity in vivo. Next, we conducted an in silico-based molecular docking simulation to elucidate five identified compounds that might interact with domain III or domain IV of the Danio rerio L-type calcium channel (LTCC), a known pharmaceutically important target for arrhythmia. In conclusion, in this study, we provide a web lab and dry lab combinatorial approach to perform in vivo cardiotoxicity drug screening and in silico mechanistic studies.
Project description:Voltage-gated potassium channels (Kv) are targets for drugs of large chemical diversity. Although hydrophobic cations block Kv channels with Hill coefficients of 1, uncharged electron-rich (cationophilic) molecules often display Hill coefficients of 2. The mechanism of the latter block is unknown. Using a combination of computational and experimental approaches, we mapped the receptor for the immunosuppressant PAP-1 (5-(4-phenoxybutoxy)psoralen), a high-affinity blocker of Kv1.3 channels in lymphocytes. Ligand-docking using Monte Carlo minimizations suggested a model in which two cationophilic PAP-1 molecules coordinate a K(+) ion in the pore with their coumarin moieties, whereas the hydrophobic phenoxyalkoxy side chains extend into the intrasubunit interfaces between helices S5 and S6. We tested the model by generating 58 point mutants involving residues in and around the predicted receptor and then determined their biophysical properties and sensitivity to PAP-1 by whole-cell patch-clamp. The model correctly predicted the key PAP-1-sensing residues in the outer helix, the P-loop, and the inner helix and explained the Hill coefficient of 2 by demonstrating that the Kv1.3 pore can accommodate two or even four PAP-1 molecules. The model further explained the voltage-dependence of block by PAP-1 and its thousand-fold selectivity for Kv1.3 over non-Kv1 channels. The 23- to 125-fold selectivity of PAP-1 for Kv1.3 over other Kv1 channels is probably due to its preferential affinity to the C-type inactivated state, in which cessation of K(+) flux stabilizes the tripartite PAP-1:K(+):PAP-1 complex in the pore. Our study provides a new concept for potassium channel block by cationophilic ligands.