Types Of Ion Channels Research Articles

A New Transgenic Mouse Model for Studying the Neurotoxicity of Spermine Oxidase Dosage in the Response to Excitotoxic Injury

Spermine oxidase is a FAD-containing enzyme involved in polyamines catabolism, selectively oxidizing spermine to produce H2O2, spermidine, and 3-aminopropanal. Spermine oxidase is highly expressed in the mouse brain and plays a key role in regulating the levels of spermine, which is involved in protein synthesis, cell division and cell growth. Spermine is normally released by neurons at synaptic sites where it exerts a neuromodulatory function, by specifically interacting with different types of ion channels, and with ionotropic glutamate receptors. In order to get an insight into the neurobiological roles of spermine oxidase and spermine, we have deregulated spermine oxidase gene expression producing and characterizing the transgenic mouse model JoSMOrec, conditionally overexpressing the enzyme in the neocortex. We have investigated the effects of spermine oxidase overexpression in the mouse neocortex by transcript accumulation, immunohistochemical analysis, enzymatic assays and polyamine content in young and aged animals. Transgenic JoSMOrec mice showed in the neocortex a higher H2O2 production in respect to Wild-Type controls, indicating an increase of oxidative stress due to SMO overexpression. Moreover, the response of transgenic mice to excitotoxic brain injury, induced by kainic acid injection, was evaluated by analysing the behavioural phenotype, the immunodistribution of neural cell populations, and the ultrastructural features of neocortical neurons. Spermine oxidase overexpression and the consequently altered polyamine levels in the neocortex affects the cytoarchitecture in the adult and aging brain, as well as after neurotoxic insult. It resulted that the transgenic JoSMOrec mouse line is more sensitive to KA than Wild-Type mice, indicating an important role of spermine oxidase during excitotoxicity. These results provide novel evidences of the complex and critical functions carried out by spermine oxidase and spermine in the mammalian brain.

Automated Parallel Recordings of Topologically Identified Single Ion Channels

Although ion channels are attractive targets for drug discovery, the systematic screening of ion channel-targeted drugs remains challenging. To facilitate automated single ion-channel recordings for the analysis of drug interactions with the intra- and extracellular domain, we have developed a parallel recording methodology using artificial cell membranes. The use of stable lipid bilayer formation in droplet chamber arrays facilitated automated, parallel, single-channel recording from reconstituted native and mutated ion channels. Using this system, several types of ion channels, including mutated forms, were characterised by determining the protein orientation. In addition, we provide evidence that both intra- and extracellular amyloid-beta fragments directly inhibit the channel open probability of the hBK channel. This automated methodology provides a high-throughput drug screening system for the targeting of ion channels and a data-intensive analysis technique for studying ion channel gating mechanisms.

Receptors and mechanisms mediating the biphasic response evoked by bradykinin in rat colonic smooth muscle

In rat duodenum, bradykinin induces a relaxation followed by a contraction. Different types of ion channels and receptors as well as non-muscle cells have been suggested to be involved in this response. As it is unclear whether these changes are observed also in rat large intestine and the mechanisms which might underlie this response, the effect of bradykinin on rat colonic motility was tested. Isometric contractions were measured on full-thickness preparations or preparations, from which individual layers had been dissected. The expression of bradykinin receptors was analyzed by immunohistochemistry and RT-PCR. Isolated intestinal muscle cells were investigated with Ca(2+) -imaging techniques. Bradykinin caused a biphasic contractile response (initial relaxation followed by contraction) in rat colon, which was resistant against tetrodotoxin. The kinin-induced relaxation was inhibited by tetrapentylammonium chloride, a blocker of Ca(2+) -activated K(+) channels. Des-arg(9) -bradykinin did not induce any effect on the native colon, although after 5h in vitro preincubation, a contractile response was evoked by this B1 receptor agonist. The consecutive ablation of adherent layers of the intestinal wall strongly reduced the response to bradykinin in comparison with a control stimulus, i.e., carbachol, suggesting a contribution of non-muscle cells in the mediation of this response. Bradykinin induced a biphasic change in contractility in the rat colon. In the native intestine, only the B2 receptor is involved in this effect. Neighboring cell obviously sensitize the smooth muscle to the stimulation of these receptors.

Cation-permeable vacuolar ion channels in the moss Physcomitrella patens: a patch-clamp study

Patch-clamp studies carried out on the tonoplast of the moss Physcomitrella patens point to existence of two types of cation-selective ion channels: slowly activated (SV channels), and fast-activated potassium-selective channels. Slowly and instantaneously saturating currents were observed in the whole-vacuole recordings made in the symmetrical KCl concentration and in the presence of Ca2+ on both sides of the tonoplast. The reversal potential obtained at the KCl gradient (10 mM on the cytoplasmic side and 100 mM in the vacuole lumen) was close to the reversal potential for K+ (E K), indicating K+ selectivity. Recordings in cytoplasm-out patches revealed two distinct channel populations differing in conductance: 91.6 ± 0.9 pS (n = 14) at −80 mV and 44.7 ± 0.7 pS (n = 14) at +80 mV. When NaCl was used instead of KCl, clear slow vacuolar SV channel activity was observed both in whole-vacuole and cytoplasm-out membrane patches. There were no instantaneously saturating currents, which points to impermeability of fast-activated potassium channels to Na+ and K+ selectivity. In the symmetrical concentration of NaCl on both sides of the tonoplast, currents have been measured exclusively at positive voltages indicating Na+ influx to the vacuole. Recordings with different concentrations of cytoplasmic and vacuolar Ca2+ revealed that SV channel activity was regulated by both cytoplasmic and vacuolar calcium. While cytoplasmic Ca2+ activated SV channels, vacuolar Ca2+ inhibited their activity. Dependence of fast-activated potassium channels on the cytoplasmic Ca2+ was also determined. These channels were active even without Ca2+ (2 mM EGTA in the cytosol and the vacuole lumen), although their open probability significantly increased at 0.1 μM Ca2+ on the cytoplasmic side. Apart from monovalent cations (K+ and Na+), SV channels were permeable to divalent cations (Ca2+ and Mg2+). Both monovalent and divalent cations passed through the channels in the same direction—from the cytoplasm to the vacuole. The identity of the vacuolar ion channels in Physcomitrella and ion channels already characterised in different plants is discussed.

Single ion channel recordings with CMOS-anchored lipid membranes.

We present single-ion-channel recordings performed with biomimetic lipid membranes which are directly attached to the surface of a complementary metal-oxide-semiconductor (CMOS) preamplifier chip. With this system we resolve single-channel currents from several types of bacterial ion channels, including fluctuations of a single alamethicin channel at a bandwidth of 1 MHz which represent the fastest single-ion-channel recordings reported to date. The platform is also used for high-resolution α-hemolysin nanopore recordings. These results illustrate the high signal fidelity, fine temporal resolution, small geometry, and multiplexed integration which can be achieved by leveraging integrated semiconductor platforms for advanced ion channel interfaces.

Selectivity Problems with Drugs Acting on Cardiac Na<sup>+</sup> and Ca<sup>2+</sup> Channels

With the increase of our knowledge on cardioactive agents it comes more and more clear that practically none of the currently used compounds shows absolute selectivity to one or another ion channel type. This is particularly true for Na(+) and Ca(2+) channel modulators, which are widely applied in the clinical practice and biomedical research. The best example might be probably the marine guanidine poison tetrodotoxin, which has long been considered as a selective Na(+) channel blocker, while recently it turned out to effectively inhibit cardiac Ca(2+) currents as well. In the present study the cross actions observed between the effects of various blockers of Na(+) channels (such as toxin inhibitors, class I antiarrhythmics and local anesthetics) and Ca(2+) channels (like phenylalkylamines, dihydropyridine compounds, diltiazem and mibefradil) are overviewed in light of the known details of the respective channel structures. Similarly, activators of Na(+) channels, including veratridine and batrachotoxin, are also compared. The binding of tetrodotoxin and saxitoxin to Cav1.2 and Nav1.5 channel proteins is presented by construction of theoretical models to reveal common structures in their pore forming regions to explain cross reactions. Since these four domain channels can be traced back to a common ancestor, a close similarity in their structure can well be demonstrated. Thus, the poor selectivity of agents acting on cardiac Na(+) and Ca(2+) channels is a consequence of evolution. As a conclusion, since the limited selectivity is an intrinsic property of drug receptors, it has to be taken into account when designing new cardioactive compounds for either medical therapy or experimental research in the future.

Opposing Actions of Sevoflurane on GABAergic and Glycinergic Synaptic Inhibition in the Spinal Ventral Horn

BackgroundThe ventral horn is a major substrate in mediating the immobilizing properties of the volatile anesthetic sevoflurane in the spinal cord. In this neuronal network, action potential firing is controlled by GABAA and glycine receptors. Both types of ion channels are sensitive to volatile anesthetics, but their role in mediating anesthetic-induced inhibition of spinal locomotor networks is not fully understood.Methodology/Principal FindingsTo compare the effects of sevoflurane on GABAergic and glycinergic inhibitory postsynaptic currents (IPSCs) whole-cell voltage-clamp recordings from ventral horn interneurons were carried out in organotypic spinal cultures. At concentrations close to MAC (minimum alveolar concentration), decay times of both types of IPSCs were significantly prolonged. However, at 1.5 MAC equivalents, GABAergic IPSCs were decreased in amplitude and reduced in frequency. These effects counteracted the prolongation of the decay time, thereby decreasing the time-averaged GABAergic inhibition. In contrast, amplitudes and frequency of glycinergic IPSCs were not significantly altered by sevoflurane. Furthermore, selective GABAA and glycine receptor antagonists were tested for their potency to reverse sevoflurane-induced inhibition of spontaneous action potential firing in the ventral horn. These experiments confirmed a weak impact of GABAA receptors and a prominent role of glycine receptors at a high sevoflurane concentration.ConclusionsAt high concentrations, sevoflurane mediates neuronal inhibition in the spinal ventral horn primarily via glycine receptors, and less via GABAA receptors. Our results support the hypothesis that the impact of GABAA receptors in mediating the immobilizing properties of volatile anesthetics is less essential in comparison to glycine receptors.

Circulation Editors’ Picks

<i>Circulation</i> Editors’ Picks

The where and how of PIP regulation of cone photoreceptor CNG channels

In this issue, [Dai et al.][1] present an investigation of the biophysics of phosphatidylinositide (PIP) regulation of the cyclic-nucleotide gated (CNG) channels expressed in cone photoreceptors. Although PIPs facilitate or stabilize the activation of many other types of ion channels, they strongly

The insecticidal potential of venom peptides.

Pest insect species are a burden to humans as they destroy crops and serve as vectors for a wide range of diseases including malaria and dengue. Chemical insecticides are currently the dominant approach for combating these pests. However, the de-registration of key classes of chemical insecticides due to their perceived ecological and human health risks in combination with the development of insecticide resistance in many pest insect populations has created an urgent need for improved methods of insect pest control. The venoms of arthropod predators such as spiders and scorpions are a promising source of novel insecticidal peptides that often have different modes of action to extant chemical insecticides. These peptides have been optimized via a prey-predator arms race spanning hundreds of millions of years to target specific types of insect ion channels and receptors. Here we review the current literature on insecticidal venom peptides, with a particular focus on their structural and pharmacological diversity, and discuss their potential for deployment as insecticides.

Caveolin-1 Regulates Kir2.1 Channel activity through Interaction with Cholesterol

A growing number of studies show that different types of ion channels are regulated by the level of membrane cholesterol and localize in caveolae. It has been proposed that cholesterol-induced regulation of ion channels might be attributed to partitioning into caveolae and association with caveolin-1 (Cav-1). We tested, therefore, whether Cav-1 is required for cholesterol sensitivity of Kir2.1 channels. Our observations show that Cav-1 co-precipitates with Kir2.1 channels and that co-expression of Kir2 channels with Cav-1 in HEK293 cells, a cell line with very low levels of endogenous caveolin, results in suppression of Kir2 activity indicating that Cav-1 is a negative regulator of Kir2 function. These observations are confirmed by comparing Kir currents in BMDMs isolated from Cav1-/- and WT animals. However, in spite of low caveolin level in HEK293 cells, Kir2.1 expressed in HEK293 cells maintain their cholesterol sensitivity. Furthermore, we also show that Kir currents in Bone-Marrow Derived Macrophages (BMDMs) isolated from Cav1-/- knockout mice are also cholesterol sensitive. Thus, these studies indicate that caveolin-1 and/or intact caveolae are not required for cholesterol sensitivity of Kir channels. Moreover, a single point mutation of Kir2.1, L222I that abrogates the sensitivity of the channels to cholesterol also abolishes their sensitivity to Cav-1 suggesting that the two modulators regulate Kir2.1 channels via a common mechanism.

Calcium Transients in Muscle Fibers Expressing Voltage-Sensitive Phosphoinositide Phosphatases

Phosphoinositides play a role in a variety of cellular signaling processes; PtdIns(4,5)P2 regulates the function of several types of ion channels in the plasma membrane and is the source of important second messengers. We tested the possible role of changes in the PtdIns(4,5)P2 level in E-C coupling and Ca2+ homeostasis in mouse muscle fibers under voltage-clamp conditions. Measurements of indo-1 Ca2+ transients in fibers injected with PtdIns(4,5)P2 revealed no significant changes in the voltage-dependence and maximum value for peak Ca2+ release. We then studied the consequences of the activation of a voltage-sensitive PtdIns-phosphatase (VSP). Expression of either Ci-VSP or Dr-VSP was achieved by in vivo electroporation. Confocal images of N-terminally EGFP-tagged Dr-VSP revealed a double-banded pattern of expression consistent with the triadic region and membrane current measurements from a depolarized holding potential showed the presence of a charge movement component consistent with the voltage-sensitive domain active in the t-tubule membrane. Rhod-2 Ca2+ transients generated by single depolarizing pulses within the voltage-dependent range of activation of Ca2+ release appeared unaffected by the presence of either VSP. In order to strongly activate the VSPs, fibers were depolarized by 10 successive 200 ms-long pulses from −80 mV to +80 mV; Ca2+ transients were compared to the ones elicited by an analogous series of pulses to +10 mV: on average, in control and Ci-VSP-positive fibers, the value for peak Ca2+ transient in response to the 10th pulse to +80 mV was 94 ± 7 % (n=9) and 72 ± 6 % (n=7) the value in response to the corresponding pulse to +10 mV, respectively. The significant depression in Ci-VSP expressing fibers may be indicative that depletion of t-tubule PtdIns(4,5)P2 can affect voltage-activated Ca2+ release. The work was supported by AFM, CNRS and Universite Claude Bernard.

Post-translational regulation of P2X receptor channels: modulation by phospholipids

P2X receptor channels mediate fast excitatory signaling by ATP and play major roles in sensory transduction, neuro-immune communication and inflammatory response. P2X receptors constitute a gene family of calcium-permeable ATP-gated cation channels therefore the regulation of P2X signaling is critical for both membrane potential and intracellular calcium homeostasis. Phosphoinositides (PIPn) are anionic signaling phospholipids that act as functional regulators of many types of ion channels. Direct PIPn binding was demonstrated for several ligand- or voltage-gated ion channels, however no generic motif emerged to accurately predict lipid-protein binding sites. This review presents what is currently known about the modulation of the different P2X subtypes by phospholipids and about critical determinants underlying their sensitivity to PIPn levels in the plasma membrane. All functional mammalian P2X subtypes tested, with the notable exception of P2X5, have been shown to be positively modulated by PIPn, i.e., homomeric P2X1, P2X2, P2X3, P2X4, and P2X7, as well as heteromeric P2X1/5 and P2X2/3 receptors. Based on various results reported on the aforementioned subtypes including mutagenesis of the prototypical PIPn-sensitive P2X4 and PIPn-insensitive P2X5 receptor subtypes, an increasing amount of functional, biochemical and structural evidence converges on the modulatory role of a short polybasic domain located in the proximal C-terminus of P2X subunits. This linear motif, semi-conserved in the P2X family, seems necessary and sufficient for encoding direct modulation of ATP-gated channels by PIPn. Furthermore, the physiological impact of the regulation of ionotropic purinergic responses by phospholipids on pain pathways was recently revealed in the context of native crosstalks between phospholipase C (PLC)-linked metabotropic receptors and P2X receptor channels in dorsal root ganglion sensory neurons and microglia.

Contribution of Potassium Channels and Calcium-Activated Chloride Channels to Neuronal Signaling

In a long-term collaboration with Yuh Nung Jan, we have taken the approach of starting our ion channel studies with molecular identification so that we can study one type of ion channel at a time. Recent studies of the contribution of potassium channels and calcium-activated chloride channels to neuronal signaling will be presented.

Acetaldehyde-ethanol interactions on calcium-activated potassium (BK) channels in pituitary tumor (GH3) cells

Background: In the central nervous system ethanol (EtOH) is metabolized to acetaldehyde (ACA) primarily by the oxidative enzyme catalase. Evidence suggests that ACA is responsible for at least some of the effects on the brain that have been attributed to EtOH. Various types of ion channels which are involved in electrical signaling are targets of EtOH like maxi calcium-activated potassium (BK) channels. BK channels exhibit various functions like action potential repolarization, blood pressure regulation, hormone secretion, or transmitter release. In most neuronal and neuroendocrine preparations at physiological intracellular calcium levels, EtOH increases BK channel activity. The simultaneous presence of ACA and EtOH reflects the physiological situation after drinking and may result in synergistic as well as antagonistic actions compared to a single application of either drug. The action of ACA on electrical activity has yet not been fully established.Methods: GH3 pituitary tumor cells were used for outside-out and inside-out patch-clamp recordings of BK activity in excised patches. Unitary current amplitude, open probability and channel mean open time of BK channels were measured.Results: Extracellular EtOH raised BK channel activity. In the presence of intracellular ACA this increment of BK activity was suppressed in a dose- as well as calcium-dependent manner. Mean channel open time was significantly reduced by internal ACA, whereas BK channel amplitudes were not affected. The EtOH counteracting effect of ACA was found to depend on succession of application. EtOH was prevented from activating BK channels by pre-exposure of membrane patches to ACA. In contrast BK activation by a hypotonic solution was not affected by internal ACA.Conclusions: Our data suggest an inhibitory impact of ACA on BK activation by EtOH. ACA appears to interact specifically with EtOH at BK channels since intracellular ACA had no effect when BK channels were activated by hypotonicity.

The role of ion channels and transporters in cell proliferation and cancer

The role of ion channels and transporters in cell proliferation and cancer

K+ channelepsy: progress in the neurobiology of potassium channels and epilepsy

K+ channels are important determinants of seizure susceptibility. These membrane proteins, encoded by more than 70 genes, make the largest group of ion channels that fine-tune the electrical activity of neuronal and non-neuronal cells in the brain. Their ubiquity and extremely high genetic and functional diversity, unmatched by any other ion channel type, place K+ channels as primary targets of genetic variations or perturbations in K+-dependent homeostasis, even in the absence of a primary channel defect. It is therefore not surprising that numerous inherited or acquired K+ channels dysfunctions have been associated with several neurologic syndromes, including epilepsy, which often generate confusion in the classification of the associated diseases. Therefore, we propose to name the K+ channels defects underlying distinct epilepsies as “K+ channelepsies,” and introduce a new nomenclature (e.g., Kx.y-channelepsy), following the widely used K+ channel classification, which could be also adopted to easily identify other channelopathies involving Na+ (e.g., Navx.y-phenotype), Ca2+ (e.g., Cavx.y-phenotype), and Cl− channels. Furthermore, we discuss novel genetic defects in K+ channels and associated proteins that underlie distinct epileptic phenotypes in humans, and analyze critically the recent progress in the neurobiology of this disease that has also been provided by investigations on valuable animal models of epilepsy. The abundant and varied lines of evidence discussed here strongly foster assessments for variations in genes encoding for K+ channels and associated proteins in patients with idiopathic epilepsy, provide new avenues for future investigations, and highlight these proteins as critical pharmacological targets.

Use of Escin as a Perforating Agent on the IonWorks Quattro Automated Electrophysiology Platform

The automated electrophysiology platform IonWorks has facilitated the medium-throughput study of ion channel biology and pharmacology. Electrical and chemical access to the cell is by perforated patch, afforded by amphotericin. Permeation of the amphotericin pore is limited to monovalent cations. We describe here the use of the saponin escin as an alternative perforating agent. With respect to the number and robustness of seals formed across a variety of cell and ion channel types, the performance of escin is equal to that of amphotericin. Escin also permits the permeation of larger molecules through its pore. These include nucleotides, important intracellular modulators of ion channel activity that can be used to prevent ion channel rundown of, for instance, CaV1.2. Furthermore, pharmacologic agents such as QX314 can also permeate and be used for mechanistic studies. Escin, in combination with IonWorks, increases the scope of ion channel screening and can facilitate the assay of previously difficult-to-assay targets.

Three-dimensional tracking and analysis of ion channel signals across dendritic arbors

Most neuron types possess elaborate dendritic arbors that receive and integrate excitatory and inhibitory inputs from numerous other neurons to give rise to cell-type specific firing patterns. The computational properties of these dendrites are therefore crucial for neuronal information processing, and are strongly determined by the expression of many types of voltage-gated ion channels in their membrane. The dendritic distribution patterns of these ion channels are characteristic for each ion channel type, are dependent on the neuronal identity, and can be modified in a plastic or pathophysiological manner. We present a method that enables us to semi-automatically map and quantify in 3D the expression levels of specific ion channel types across the entire dendritic arbor. To achieve this, standard immunohistochemistry was combined with reconstruction and quantification procedures for the localization and relative distribution of ion channels with respect to dendritic morphology. This method can, in principle, be applied to any fluorescent signal, including fluorescently tagged membrane proteins, RNAs, or intracellular signaling molecules.

Endothelial cell and model membranes respond to shear stress by rapidly decreasing the order of their lipid phases

Endothelial cells (ECs) sense shear stress and transduce blood flow information into functional responses that play important roles in vascular homeostasis and pathophysiology. A unique feature of shear-stress-sensing is the involvement of many different types of membrane-bound molecules, including receptors, ion channels and adhesion proteins, but the mechanisms remain unknown. Because cell membrane properties affect the activities of membrane-bound proteins, shear stress might activate various membrane-bound molecules by altering the physical properties of EC membranes. To determine how shear stress influences the cell membrane, cultured human pulmonary artery ECs were exposed to shear stress and examined for changes in membrane lipid order and fluidity by Laurdan two-photon imaging and FRAP measurements. Upon shear stress stimulation, the lipid order of EC membranes rapidly decreased in an intensity-dependent manner, and caveolar membrane domains changed from the liquid-ordered state to the liquid-disordered state. Notably, a similar decrease in lipid order occurred when the artificial membranes of giant unilamellar vesicles were exposed to shear stress, suggesting that this is a physical phenomenon. Membrane fluidity increased over the entire EC membranes in response to shear stress. Addition of cholesterol to ECs abolished the effects of shear stress on membrane lipid order and fluidity and markedly suppressed ATP release, which is a well-known EC response to shear stress and is involved in shear-stress Ca(2+) signaling. These findings indicate that EC membranes directly respond to shear stress by rapidly decreasing their lipid phase order and increasing their fluidity; these changes could be linked to shear-stress-sensing and response mechanisms.