Ion Channels In Excitable Cells Research Articles

Automated patch-clamp with automated analysis: extracting compound-specific, concentration-independent biophysical properties of inhibition for sodium channel inhibitors

When targeting voltage-gated ion channels of excitable cells, one needs to deal with a “moving target”. Ion channels keep changing their conformation, and drugs keep dynamically associate and dissociate, depending on the conformation. It is not affinity in itself, that is important, but how the dynamics of cellular activity relates to the dynamics of drug access/egress and binding/unbinding.

High-Throughput Analysis of Drug Safety Responses in Induced Pluripotent Stem Cell-Derived Cardiomyocytes Using Multielectrode Array.

Microelectrode array (MEA) isan electrophysiological instrument used to track activities of ion channels in excitable cells. Neurons and cardiomyocytes are seeded to form a cell monolayer on a field of sensors able to detect electrical signals, called extracellular field potentials (EFPs). This noninvasive tool allows researchers to investigate key parameters such as EFP amplitude, duration, and arrhythmias. MEA is progressively considered the gold standard for high-throughput in vitro electrophysiological evaluation, particularly for cardiac disease modeling and cardiac toxicity assessment.

Isoform-specific regulation of HCN4 channels by a family of endoplasmic reticulum proteins

Ion channels in excitable cells function in macromolecular complexes in which auxiliary proteins modulate the biophysical properties of the pore-forming subunits. Hyperpolarization-activated, cyclic nucleotide-sensitive HCN4 channels are critical determinants of membrane excitability in cells throughout the body, including thalamocortical neurons and cardiac pacemaker cells. We previously showed that the properties of HCN4 channels differ dramatically in different cell types, possibly due to the endogenous expression of auxiliary proteins. Here, we report the discovery of a family of endoplasmic reticulum (ER) transmembrane proteins that associate with and modulate HCN4. Lymphoid-restricted membrane protein (LRMP, Jaw1) and inositol trisphosphate receptor-associated guanylate kinase substrate (IRAG, Mrvi1, and Jaw1L) are homologous proteins with small ER luminal domains and large cytoplasmic domains. Despite their homology, LRMP and IRAG have distinct effects on HCN4. LRMP is a loss-of-function modulator that inhibits the canonical depolarizing shift in the voltage dependence of HCN4 in response to the binding of cAMP. In contrast, IRAG causes a gain of HCN4 function by depolarizing the basal voltage dependence in the absence of cAMP. The mechanisms of action of LRMP and IRAG are independent of trafficking and cAMP binding, and they are specific to the HCN4 isoform. We also found that IRAG is highly expressed in the mouse sinoatrial node where computer modeling predicts that its presence increases HCN4 current. Our results suggest important roles for LRMP and IRAG in the regulation of cellular excitability, as tools for advancing mechanistic understanding of HCN4 channel function, and as possible scaffolds for coordination of signaling pathways.

Paradoxical effects on voltage-gated Na+conductance in adrenal chromaffin cells by twin vs single high intensity nanosecond electric pulses.

We previously reported that a single 5 ns high intensity electric pulse (NEP) caused an E-field-dependent decrease in peak inward voltage-gated Na+ current (INa) in isolated bovine adrenal chromaffin cells. This study explored the effects of a pair of 5 ns pulses on INa recorded in the same cell type, and how varying the E-field amplitude and interval between the pulses altered its response. Regardless of the E-field strength (5 to 10 MV/m), twin NEPs having interpulse intervals ≥ than 5 s caused the inhibition of TTX-sensitive INa to approximately double relative to that produced by a single pulse. However, reducing the interval from 1 s to 10 ms between twin NEPs at E-fields of 5 and 8 MV/m but not 10 MV/m decreased the magnitude of the additive inhibitory effect by the second pulse in a pair on INa. The enhanced inhibitory effects of twin vs single NEPs on INa were not due to a shift in the voltage-dependence of steady-state activation and inactivation but were associated with a reduction in maximal Na+ conductance. Paradoxically, reducing the interval between twin NEPs at 5 or 8 MV/m but not 10 MV/m led to a progressive interval-dependent recovery of INa, which after 9 min exceeded the level of INa reached following the application of a single NEP. Disrupting lipid rafts by depleting membrane cholesterol with methyl-β-cyclodextrin enhanced the inhibitory effects of twin NEPs on INa and ablated the progressive recovery of this current at short twin pulse intervals, suggesting a complete dissociation of the inhibitory effects of twin NEPs on this current from their ability to stimulate its recovery. Our results suggest that in contrast to a single NEP, twin NEPs may influence membrane lipid rafts in a manner that enhances the trafficking of newly synthesized and/or recycling of endocytosed voltage-gated Na+ channels, thereby pointing to novel means to regulate ion channels in excitable cells.

Cardiac disorders and mode of action of the Egyptian scorpion venom Androctonus bicolor on isolated toad’s heart

Scorpion venom is a complex mixture of components with various pharmacological and toxicological effects. It is characterized by the presence of a large number of toxins that specifically interact with ion channels of excitable cells. The Egyptian scorpion Androctonus bicolor belongs to the family of Buthidae and until now no information is available about the effect of its venom on cardiac muscles. Using an in vitro approach, cardiotoxicity and mode of action of A. bicolor venom on isolated toad’s heart were investigated. Direct application of scorpion venom (0.5μg/ml) into isolated toad’s heart induced a remarkable bradycardia concomitant with a protraction in the conduction time (P–R interval). In the meantime, a significant increase in the R-wave amplitude (ventricular contraction) was noticed after 5min of venom perfusion. Various cases of cardiac disorders were recorded such as sinus arrhythmias, ectopic beats and different degrees of heart block. Through using different autonomic and ion channel blockers, the possible mechanism of action of A. bicolor venom on isolated toad’s heart was revealed. The application of both atropine (4μg/ml) and verapamil (5μg/ml) could not alleviate the pronounced negative chronotropic and positive inotropic effects. Meanwhile, a significant decrease in the R-wave amplitude was observed after propranolol (5μg/ml) application. In conclusion, our findings indicate that the venom of A. bicolor directly influenced the cardiac electrical activity of toads through β-adrenergic receptors. The direct effect of this venom on cardiac tissues may significantly contribute in the development of several cardiotoxic effects following scorpion sting.

Novel perspectives in cancer therapy: Targeting ion channels.

By controlling ion fluxes at multiple time scales, ion channels shape rapid cell signals, such as action potential and synaptic transmission, as well as much slower processes, such as mitosis and cell migration. As is currently increasingly recognized, a variety of channel types are involved in cancer hallmarks, and regulate specific stages of neoplastic progression. Long-term in vitro work has established that inhibition of these ion channels impairs the growth of cancer cells. Recently, these studies have been followed up in vivo, hence revealing that ion channels constitute promising pharmacological targets in oncology. The channel proteins can be often accessed from the extracellular milieu, which allows use of lower drug doses and decrease untoward toxicity. However, because of the central physiological roles exerted by ion channels in excitable cells, other types of side effects may arise, the gravest of which is cardiac arrhythmia. A paradigmatic case is offered by Kv11.1 (hERG1) channels. HERG1 blockers attenuate the progression of both hematologic malignancies and solid tumors, but may also lead to the lengthening of the electrocardiographic QT interval, thus predisposing the patient to ventricular arrhythmias. These side effects can be avoided by specifically inhibiting the channel isoforms which are highly expressed in certain tumors, such as Kv11.1B and the neonatal forms of voltage-gated Na(+) channels. Preclinical studies are also being explored in breast and prostate cancer (targeting voltage-gated Na(+) channels), and gliomas (targeting CLC-3). Overall, the possible approaches to improve the efficacy and safety of ion channel targeting in oncology include: (1) the development of specific inhibitors for the channel subtypes expressed in specific tumors; (2) drug delivery into the tumor by using antibodies or nanotechnology-based approaches; (3) combination regimen therapy and (4) blocking specific conformational states of the ion channel. We believe that expanding this relatively neglected field of oncology research might lead to unforeseen therapeutic benefits for cancer patients.

Effects of monoterpenes on ion channels of excitable cells.

Monoterpenes are a structurally diverse group of phytochemicals and a major constituent of plant-derived 'essential oils'. Monoterpenes such as menthol, carvacrol, and eugenol have been utilized for therapeutical purposes and food additives for centuries and have been reported to have anti-inflammatory, antioxidant and analgesic actions. In recent years there has been increasing interest in understanding the pharmacological actions of these molecules. There is evidence indicating that monoterpenes can modulate the functional properties of several types of voltage and ligand-gated ion channels, suggesting that some of their pharmacological actions may be mediated by modulations of ion channel function. In this report, we review the literature concerning the interaction of monoterpenes with various ion channels.

An Analytical Method for the Quantification of hERG1 Channel Gene Expression in Human Colorectal Cancer

Cancer molecular investigation revealed a huge molecular heterogeneity between different types of cancers as well as among cancer patients affected by the same cancer type. This implies the necessity of a personalized approach for cancer diagnosis and therapy, on the basis of the development of standardized protocols to facilitate the application of molecular techniques in the clinical decision-making process. Ion channels encoding genes are acquiring increasing relevance in oncological translational studies, representing new candidates for molecular diagnostic and therapeutic purposes. Hence, the development of molecular protocols for the quantification of ion channels encoding genes in tumor specimens may have relevance for diagnostic and prognostic investigation. Two main hindrances must be overcome for these purposes: the use of formalin-fixed and paraffin-embedded samples for gene expression analysis and the physiological expression of ion channels in excitable cells, potentially present in the tumor sample. We here propose a method for hERG1 gene quantification in colorectal cancer samples in both cryopreserved and formalin-fixed and paraffin-embedded samples. An analytical method was developed to estimate hERG1 gene expression exclusively in epithelial cancer cells. Indeed, we found that the hERG1 gene was expressed at significant levels by myofibroblasts present in the tumor stroma. This method was based on the normalization on a smooth muscle-myofibroblast-specific gene, MYH11, with no need of microdissection. By applying this method, hERG1 expression turned out to correlate with VEGF-A expression, confirming previous immunohistochemical data.

The Electric Fingerprint of Membrane Voltage Sensor Domains

Voltage-sensor domains (VSDs) are electrically-charged constructs controlling the voltage-dependent activity of ion channels in excitable cells. Four packed transmembrane (TM) helices, S1 through S4, form the domain in which S4 contains 4 to 7 positively charged basic amino acids, mostly arginines. VSDs operate essentially by transferring the S4 charges across the transmembrane electric field (E), giving rise to the observable Q, the so-called “gating charge”. Mostly supported by structure-function studies on voltage-gated potassium (Kv) channels, a focused E has been identified as one key electric property of the VSD machinery. The recent increasing availability of other VSD-containing ion channel structures, including the x-ray structures for the NavAb and NavRh voltage-gated Na+ channels, provides us with the opportunity to extend the structure-based investigation of the domain electrostatic properties over a larger set of distinct conformations and isoforms. Using all-atom MD simulations in combination with electrostatic calculations, founded on an energetic formalism, we show that, over the entire set of available VSD structures, a specific hydration of the voltage sensor focuses E over a narrow TM region across the domain, at the vicinity of the so-called catalytic center. Furthermore, its focalization and shape is largely preserved over distinct conformations of the construct. Our results support that a focused and conformation-independent TM field is a robust electric feature of the VSD machinery, despite sequence variations or local structural modifications of the domain. This electric fingerprint seems to favor a highly conserved sensing mechanism for VSDs over the large family of voltage-gated cation channels.

Conus Venom Peptide Pharmacology

Conopeptides are a diverse group of recently evolved venom peptides used for prey capture and/or defense. Each species of cone snails produces in excess of 1000 conopeptides, with those pharmacologically characterized (≈ 0.1%) targeting a diverse range of membrane proteins typically with high potency and specificity. The majority of conopeptides inhibit voltage- or ligand-gated ion channels, providing valuable research tools for the dissection of the role played by specific ion channels in excitable cells. It is noteworthy that many of these targets are found to be expressed in pain pathways, with several conopeptides having entered the clinic as potential treatments for pain [e.g., pyroglutamate1-MrIA (Xen2174)] and one now marketed for intrathecal treatment of severe pain [ziconotide (Prialt)]. This review discusses the diversity, pharmacology, structure-activity relationships, and therapeutic potential of cone snail venom peptide families acting at voltage-gated ion channels (ω-, μ-, μO-, δ-, ι-, and κ-conotoxins), ligand-gated ion channels (α-conotoxins, σ-conotoxin, ikot-ikot, and conantokins), G-protein-coupled receptors (ρ-conopeptides, conopressins, and contulakins), and neurotransmitter transporters (χ-conopeptides), with expanded discussion on the clinical potential of sodium and calcium channel inhibitors and α-conotoxins. Expanding the discovery of new bioactives using proteomic/transcriptomic approaches combined with high-throughput platforms and better defining conopeptide structure-activity relationships using relevant membrane protein crystal structures are expected to grow the already significant impact conopeptides have had as both research probes and leads to new therapies.

Sodium Channels as Targets for Volatile Anesthetics

The molecular mechanisms of modern inhaled anesthetics are still poorly understood although they are widely used in clinical settings. Considerable evidence supports effects on membrane proteins including ligand- and voltage-gated ion channels of excitable cells. Na+ channels are crucial to action potential initiation and propagation, and represent potential targets for volatile anesthetic effects on central nervous system depression. Inhibition of presynaptic Na+ channels leads to reduced neurotransmitter release at the synapse and could therefore contribute to the mechanisms by which volatile anesthetics produce their characteristic end points: amnesia, unconsciousness, and immobility. Early studies on crayfish and squid giant axon showed inhibition of Na+ currents by volatile anesthetics at high concentrations. Subsequent studies using native neuronal preparations and heterologous expression systems with various mammalian Na+ channel isoforms implicated inhibition of presynaptic Na+ channels in anesthetic actions at clinical concentrations. Volatile anesthetics reduce peak Na+ current (INa) and shift the voltage of half-maximal steady-state inactivation (h∞) toward more negative potentials, thus stabilizing the fast-inactivated state. Furthermore recovery from fast-inactivation is slowed, together with enhanced use-dependent block during pulse train protocols. These effects can depress presynaptic excitability, depolarization and Ca2+ entry, and ultimately reduce transmitter release. This reduction in transmitter release is more potent for glutamatergic compared to GABAergic terminals. Involvement of Na+ channel inhibition in mediating the immobility caused by volatile anesthetics has been demonstrated in animal studies, in which intrathecal infusion of the Na+ channel blocker tetrodotoxin increases volatile anesthetic potency, whereas infusion of the Na+ channels agonist veratridine reduces anesthetic potency. These studies indicate that inhibition of presynaptic Na+ channels by volatile anesthetics is involved in mediating some of their effects.

Persistent membranous cross correlations due to the multiplicity of gates in ion channels

Ion channels in excitable cells reveal spontaneous intermittent opening and closing. As the membrane area reduces, this stochasticity enables spontaneous firing and elevates the cell's ability to fire at weaker stimuli. A multiple number of gates are accommodated in each individual ion channel. Here we investigate the possible impact of that gate multiplicity on the cell's function specifically when the membrane area is of limited size. It is shown that a non-trivially persistent correlation then takes place between the transmembrane voltage fluctuations (also between the fluctuations in the gating variables) and the component of open channel fluctuations attributed to the above gate multiplicity. This cross correlation persistency is found to be playing a major augmentative role in the elevation of the cell's excitability and spontaneous firing; without the persistency, the cell would be much less excitable. The cross correlation persistency is also found to enhance spike coherence. The stochastic Hodgkin-Huxley equations, put forward by Fox and Lu, are addressed in the context of their recognized failure to produce accurate enough statistics of spike generation. Our results indicate that the major source of that inaccuracy is the incapability of the stochastic Hodgkin-Huxley description to reflect the above cross correlation persistency.

Errors in the measurement of voltage-activated ion channels in cell-attached patch-clamp recordings

Patch-clamp recording techniques have revolutionized understanding of the function and sub-cellular location of ion channels in excitable cells. The cell-attached patch-clamp configuration represents the method of choice to describe the endogenous properties of voltage-activated ion channels in the axonal, somatic and dendritic membrane of neurons, without disturbance of the intracellular milieu. Here, we directly examine the errors associated with cell-attached patch-clamp measurement of ensemble ion channel activity. We find for a number of classes of voltage-activated channels, recorded from the soma and dendrites of neurons in acute brain-slices and isolated cells, that the amplitude and kinetics of ensemble ion channel activity recorded in cell-attached patches is significantly distorted by transmembrane voltage changes generated by the flow of current through the activated ion channels. We outline simple error–correction procedures that allow a more accurate description of the density and properties of voltage-activated channels to be incorporated into computational models of neurons.

Cellular prostheses: functional abiotic nanosystems to probe, manipulate, and endow function in live cells

A class of nanoscale (∼1–10 nm) structures designed to probe, manipulate, or endow function by direct interfacing with live cells is considered. Such a concept of cellular-level prostheses is illustrated via the example of light-activated nanoscale photodiodes capable of creating local electric fields that modulate existing voltage-gated ion channels in excitable cells. The dynamics of the membrane potential modulation by such photovoltaic functional abiotic nanosystems (PV-FANs) is modeled through an appropriate equivalent circuit. The feasibility of exceeding the typical ∼10 mV depolarization threshold for activating the action potentials is examined. In view of the continuing advances in the ability to design, synthesize, and characterize abiotic nanoscale systems that can provide desired function, several approaches to the implementation of PV-FANs are discussed. The FANs as “cellular prostheses” can provide a variety of functions in response to different stimuli and represent a paradigm-changing opportunity at the frontiers of nanomedicine. From the Clinical Editor A class of nanoscale (~1-10nm) structures designed to probe, manipulate, or endow live cell functions is demonstrated in this work. More specifically, light-activated nanoscale photodiodes were found capable of creating local electric fields that modulate existing voltage gated ion channels in excitable cells, thus allowing the generation of action potentials in excitable cells via external light stimulus in a controlled fashion.

CaMKII locally encodes L-type channel activity to signal to nuclear CREB in excitation–transcription coupling

Communication between cell surface proteins and the nucleus is integral to many cellular adaptations. In the case of ion channels in excitable cells, the dynamics of signaling to the nucleus are particularly important because the natural stimulus, surface membrane depolarization, is rapidly pulsatile. To better understand excitation–transcription coupling we characterized the dependence of cAMP response element–binding protein phosphorylation, a critical step in neuronal plasticity, on the level and duration of membrane depolarization. We find that signaling strength is steeply dependent on depolarization, with sensitivity far greater than hitherto recognized. In contrast, graded blockade of the Ca2+ channel pore has a remarkably mild effect, although some Ca2+ entry is absolutely required. Our data indicate that Ca2+/CaM-dependent protein kinase II acting near the channel couples local Ca2+ rises to signal transduction, encoding the frequency of Ca2+ channel openings rather than integrated Ca2+ flux—a form of digital logic.

Association of Kv1.5 and Kv1.3 Contributes to the Major Voltage-dependent K+ Channel in Macrophages

Voltage-dependent K(+) (Kv) currents in macrophages are mainly mediated by Kv1.3, but biophysical properties indicate that the channel composition could be different from that of T-lymphocytes. K(+) currents in mouse bone marrow-derived and Raw-264.7 macrophages are sensitive to Kv1.3 blockers, but unlike T-cells, macrophages express Kv1.5. Because Shaker subunits (Kv1) may form heterotetrameric complexes, we investigated whether Kv1.5 has a function in Kv currents in macrophages. Kv1.3 and Kv1.5 co-localize at the membrane, and half-activation voltages and pharmacology indicate that K(+) currents may be accounted for by various Kv complexes in macrophages. Co-expression of Kv1.3 and Kv1.5 in human embryonic kidney 293 cells showed that the presence of Kv1.5 leads to a positive shift in K(+) current half-activation voltages and that, like Kv1.3, Kv1.3/Kv1.5 heteromers are sensitive to r-margatoxin. In addition, both proteins co-immunoprecipitate and co-localize. Fluorescence resonance energy transfer studies further demonstrated that Kv1.5 and Kv1.3 form heterotetramers. Electrophysiological and pharmacological studies of different ratios of Kv1.3 and Kv1.5 co-expressed in Xenopus oocytes suggest that various hybrids might be responsible for K(+) currents in macrophages. Tumor necrosis factor-alpha-induced activation of macrophages increased Kv1.3 with no changes in Kv.1.5, which is consistent with a hyperpolarized shift in half-activation voltage and a lower IC(50) for margatoxin. Taken together, our results demonstrate that Kv1.5 co-associates with Kv1.3, generating functional heterotetramers in macrophages. Changes in the oligomeric composition of functional Kv channels would give rise to different biophysical and pharmacological properties, which could determine specific cellular responses.

Letter Regarding Article by Darbar et al, “Unmasking of Brugada Syndrome by Lithium”

To the Editor: It was with great interest that we read the study by Darbar and colleagues in a recent issue of Circulation .1 The authors reported on 2 patients who developed the Brugada ECG pattern after administration of lithium, a commonly used drug not previously reported to block cardiac Na+ channels. Surprisingly, Darbar and colleagues found that LiCl caused a concentration-dependent block of peak INa, with an IC50 of 6.8±0.4 μmol/L, a level 100 times below the therapeutic range (&1 mmol/L). They concluded that lithium is a potent blocker of cardiac Na+ channels and may unmask patients with the Brugada syndrome. It is well known from numerous studies (conducted during the last 3 decades) that Li+ ions permeate Na+ channels, as well as do Na+ ions, in a variety of excitable cell …

Photochemical tools for remote control of ion channels in excitable cells

Various strategies have been developed recently for imparting light sensitivity onto normally insensitive cells. These include expression of natural photosensitive proteins, photolysis of caged agonists of native cell surface receptors and photoswitching of isomerizable tethered ligands that act on specially engineered ion channels and receptor targets. The development of chemical tools for optically stimulating or inhibiting signaling proteins has particular relevance for the nervous system, where precise, noninvasive control is an experimental and medical necessity.

How to read clinical journals: XII. How you too can profit from pharmaceutical advertisements

Conopeptides are a diverse group of recently evolved venom peptides used for prey capture and/or defense. Each species of cone snails produces in excess of 1000 conopeptides, with those pharmacologically characterized (∼0.1%) targeting a diverse range of membrane proteins typically with high potency and specificity. The majority of conopeptides inhibit voltage- or ligand-gated ion channels, providing valuable research tools for the dissection of the role played by specific ion channels in excitable cells. It is noteworthy that many of these targets are found to be expressed in pain pathways, with several conopeptides having entered the clinic as potential treatments for pain [e.g., pyroglutamate1-MrIA (Xen2174)] and one now marketed for intrathecal treatment of severe pain [ziconotide (Prialt)]. This review discusses the diversity, pharmacology, structure-activity relationships, and therapeutic potential of cone snail venom peptide families acting at voltage-gated ion channels (ω-, μ-, μO-, δ-, ι-, and κ-conotoxins), ligand-gated ion channels (α-conotoxins, σ-conotoxin, ikot-ikot, and conantokins), G-protein-coupled receptors (ρ-conopeptides, conopressins, and contulakins), and neurotransmitter transporters (χ-conopeptides), with expanded discussion on the clinical potential of sodium and calcium channel inhibitors and α-conotoxins. Expanding the discovery of new bioactives using proteomic/transcriptomic approaches combined with high-throughput platforms and better defining conopeptide structure-activity relationships using relevant membrane protein crystal structures are expected to grow the already significant impact conopeptides have had as both research probes and leads to new therapies.

Diversity of folds in animal toxins acting on ion channels.

Animal toxins acting on ion channels of excitable cells are principally highly potent short peptides that are present in limited amounts in the venoms of various unrelated species, such as scorpions, snakes, sea anemones, spiders, insects, marine cone snails and worms. These toxins have been used extensively as invaluable biochemical and pharmacological tools to characterize and discriminate between the various ion channel types that differ in ionic selectivity, structure and/or cell function. Alongside the huge molecular and functional diversity of ion channels, a no less impressive structural diversity of animal toxins has been indicated by the discovery of an increasing number of polypeptide folds that are able to target these ion channels. Indeed, it appears that these peptide toxins have evolved over time on the basis of clearly distinct architectural motifs, in order to adapt to different ion channel modulating strategies (pore blockers compared with gating modifiers). Herein, we provide an up-to-date overview of the various types of fold from animal toxins that act on ion channels selective for K+, Na+, Ca2+ or Cl- ions, with special emphasis on disulphide bridge frameworks and structural motifs associated with these peptide folds.