Voltage-independent Ion Channel Research Articles

Neuronal nitric oxide synthase inhibition exacerbates atrial electrical remodeling via small-conductance Ca2+-activated K+ channel activation

Abstract Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): The Japan Society for the Promotion of Science KAKENHI Background The presence of atrial fibrillation (AF) is associated with electrical remodeling processes that promote a substrate for the maintenance of AF itself. Small conductance Ca2+-activated K+ (SK) channels; K+-selective and voltage-independent ion channels are key factors in the atrial electrical remodeling (2, 3). However, the mechanism of its activation remains unclear. A recent study showed that neuronal nitric oxide synthase (nNOS) expression and activity are reduced in AF patients and that nNOS depletion causes the abbreviation of action potential duration (APD), leading to increased AF inducibility in animal experiments (4). Decreased NO production, especially driven by nNOS inhibition, might play a key role in the atrial electrical remodeling, and the downstream alteration of SK channels might result from this process. Purpose We aimed to evaluate the potential of SK channel blocking to mitigate abnormal electrophysiological properties and the inducibility of atrial tachyarrhythmia (ATA) which was induced by nNOS depletion, and to describe the related mechanism. Methods Atrial tachyarrhythmia induction and optical mapping were performed in perfused rat hearts. nNOS was pharmacologically inhibited by S-methylthiocitrulline (SMTC, 100 nM). The influence of the SK channel was examined by a specific channel inhibitor, apamin (100 nM). APD, conduction velocity, and calcium transient (CaT) parameters (CaTD, rise time, time to 50% decay, and tau) were evaluated by voltage and calcium dual optical mapping. Dominant frequency was evaluated to analyze the wave dynamics of AF. Results SMTC increased the inducibility of ATA and apamin mitigated the nNOS inhibition-induced arrhythmogenicity (0% [0/7] vs 62% [8/13] vs 15% [2/13] in control, SMTC and SMTC + apamin). SMTC caused the abbreviation and enhanced spatial dispersion of APD, which were reversed by apamin. In contrast, conduction velocity was not affected by SMTC or apamin. Moreover, apamin reduced the dominant frequency of SMTC-induced ATA. In voltage and calcium optical mapping, STMC and apamin did not alter the parameters associated with CaT, however, SMTC caused the abbreviation of APD, which was reversed by apamin (APD80: 48.4 ± 2.7 msec in control group, 30.8 ± 1.5 msec in SMTC group, p <0.0001 vs. control, 41.8 ± 1.5 msec in SMTC + apamin, p = 0.01 vs. SMTC by One-way ANOVA and Tukey’s multiple-comparison test) (Figure A-C). Conclusion Acute nNOS inhibition abbreviated APD via activating SK channels. A specific SK channel blocker mitigated APD abbreviation without the alteration of CaT, implying an underlying mechanism of post translational modification of SK channels.

Altered ENaC is Associated With Aortic Baroreceptor Dysfunction in Chronic Heart Failure.

Abnormal baroreceptor function contributes to attenuated arterial baroreflex sensitivity in chronic heart failure (CHF). As a mechanosensor in mammalian nonepithelium, the epithelial sodium channel (ENaC) is an amiloride-sensitive and voltage-independent ion channel. The ENaC is thought to be a component of baroreceptor mechanosensitive ion channels in aortic baroreceptor cell bodies and nerve terminals. In this study, therefore, we measured the expression and activation of the ENaC in nodose neuronal cell bodies and aortic baroreceptor nerve terminals in sham and CHF rats. CHF was induced by surgical ligation of left coronary artery. The development of CHF was confirmed by hemodynamic and morphological characteristics. The aortic baroreceptor sensitivity was blunted in anesthetized CHF rats, compared with that in sham rats. The data from immunostaining and western blot analysis showed that the protein of β- and γ-ENaC subunits was expressed in nodose neuronal cell bodies and aortic baroreceptor nerve terminals, whereas the protein of α-ENaC subunit was undetectable. CHF reduced protein expression of β- and γ-ENaC subunits in nodose neuronal cell bodies and aortic baroreceptor nerve terminals. Additionally, the data recorded by the whole cell patch-clamp technique demonstrated that ENaC currents in aortic baroreceptor neurons were lower in CHF rats than that in sham rats. These results suggest that reduced protein expression of the ENaC decreases the ENaC activation, which could be involved in attenuation of the aortic baroreceptor sensitivity in the CHF state. Baroreceptors should be a potential therapeutic target for reducing mortality in CHF.

Recessive truncating NALCN mutation in infantile neuroaxonal dystrophy with facial dysmorphism

BackgroundInfantile neuroaxonal dystrophy (INAD) is a recessive disease that results in total neurological degeneration and death in childhood. PLA2G6 mutation is the underlying genetic defect, but rare genetic heterogeneity has...

Structural plasticity and dynamic selectivity of acid-sensing ion channel–spider toxin complexes

Acid-sensing ion channels (ASICs) are voltage-independent, amiloride-sensitive channels involved in diverse physiological processes ranging from nociception to taste. Despite the importance of ASICs in physiology, we know little about the mechanism of channel activation. Here we show that psalmotoxin activates non-selective and Na(+)-selective currents in chicken ASIC1a at pH 7.25 and 5.5, respectively. Crystal structures of ASIC1a-psalmotoxin complexes map the toxin binding site to the extracellular domain and show how toxin binding triggers an expansion of the extracellular vestibule and stabilization of the open channel pore. At pH 7.25 the pore is approximately 10 Å in diameter, whereas at pH 5.5 the pore is largely hydrophobic and elliptical in cross-section with dimensions of approximately 5 by 7 Å, consistent with a barrier mechanism for ion selectivity. These studies define mechanisms for activation of ASICs, illuminate the basis for dynamic ion selectivity and provide the blueprints for new therapeutic agents.

Regulation of Delta-Enac Ion Channels by the Neuronal-Specific Sgk1.1 Kinase

The epithelial sodium channel (ENaC) is a voltage-independent ion channel that plays a fundamental role in kidney transepithelial sodium transport and extracellular volume homeostasis. Previous work from our group and others have identified a novel ENaC subunit that is prominently expressed in neurons but not in kidney epithelia. The physiological role of delta-ENaC channels in neurons is unknown, but it could be involved in the regulation of membrane resting potential and hence of neuronal excitability. Kidney ENaC activity is increased by the serum and glucocorticoid-induced kinase 1 (SGK1). Recently, a new neuronal-specific isoform of SGK1, named SGK1.1, has been identified. We have tested whether SGK1.1 regulates delta-ENaC activity. Co-injection of SGK1.1 and delta-ENaC channels in Xenopus oocytes increased sodium current by two-fold. SGK1.1 increased delta-ENaC plasma membrane expression by 1.6-fold. In situ hybridization experiments confirmed the co-expression of delta ENaC and SGK1.1 in pyramidal neurons of the human cerebral cortex, indicating that this regulation could be physiologically relevant. In summary, we have identified a new regulator of delta-ENaC ion channels that could play a role in the control of neuronal resting potential and excitability.

Potentiation of acid-sensing ion channels by sulfhydryl compounds

The acid-sensing ion channels (ASICs) are voltage-independent ion channels activated by acidic extracellular pH. ASICs play a role in sensory transduction, behavior, and acidotoxic neuronal death, which occurs during stroke and ischemia. During these conditions, the extracellular concentration of sulfhydryl reducing agents increases. We used perforated patch-clamp technique to analyze the impact of sulfhydryls on H(+)-gated currents from Chinese hamster ovary (CHO) cells expressing human ASIC1a (hASIC1a). We found that hASIC1a currents activated by pH 6.5 were increased almost twofold by the sulfhydryl-containing reducing agents dithiothreitol (DTT) and glutathione. DTT shifted the pH-dose response of hASIC1a toward a more neutral pH (pH(0.5) from 6.54 to 6.69) and slowed channel desensitization. The effect of reducing agents on native mouse hippocampal neurons and transfected mouse ASIC1a was similar. We found that the effect of DTT on hASIC1a was mimicked by the metal chelator TPEN, and mutant hASIC1a channels with reduced TPEN potentiation showed reduced DTT potentiation. Furthermore, the addition of DTT in the presence of TPEN did not result in further increases in current amplitude. These results suggest that the effect of DTT on hASIC1a is due to relief of tonic inhibition by transition metal ions. We found that all ASICs examined remained potentiated following the removal of DTT. This effect was reversed by the oxidizing agent DTNB in hASIC1a, supporting the hypothesis that DTT also impacts ASICs via a redox-sensitive site. Thus sulfhydryl compounds potentiate H(+)-gated currents via two mechanisms, metal chelation and redox modulation of target amino acids.

Members of the Arabidopsis AtTPK/KCO family form homomeric vacuolar channels in planta

The Arabidopsis thaliana K+ channel family of AtTPK/KCO proteins consists of six members including a 'single-pore' (Kir-type) and five 'tandem-pore' channels. AtTPK4 is currently the only ion channel of this family for which a function has been demonstrated in planta. The protein is located at the plasma membrane forming a voltage-independent K+ channel that is blocked by extracellular calcium ions. In contrast, AtTPK1 is a tonoplast-localized protein, that establishes a K+-selective, voltage-independent ion channel activated by cytosolic calcium when expressed in a heterologous system, i.e. yeast. Here, we provide evidence that other AtTPK/KCO channel subunits, i.e. AtTPK2, AtTPK3, AtTPK5 and AtKCO3, are also targeted to the vacuolar membrane, opening the possibility that they interact at the target membrane to form heteromeric ion channels. However, when testing the cellular expression patterns of AtTPK/KCO genes we observed distinct expression domains that overlap in only a few tissues of the Arabidopsis plant, making it unlikely that different channel subunits interact to form heteromeric channels. This conclusion was substantiated by in planta expression of combinations of selected tonoplast AtTPK/KCO proteins. Fluorescence resonance energy transfer assays indicate that protein interaction occurs between identical channel subunits (most efficiently between AtTPK1 or AtKCO3) but not between different channel subunits. The finding could be confirmed by bimolecular fluorescence complementation assays. We conclude that tonoplast-located AtTPK/KCO subunits form homomeric ion channels in vivo.

Serotonin-Induced Inhibition of K V Current

See related article, pages 931–938 Pulmonary vascular tone is largely determined by potassium and calcium currents in the smooth muscle cells (SMCs) of the small resistance arteries. Under normoxic circumstances an outward potassium current ( I K) passes through voltage-gated (Kv), calcium-activated (KCa), and background tandem-pore domain (K2P) potassium channels. The latter (coded by the KCNK family of genes) include the twik-related acid sensitive K+ channel TASK and TASK-like channels. This current keeps the resting membrane potential in the range of −50 to −60 mV and inhibits calcium entry through L-type calcium channels.1 Vasodilator substances such as nitric oxide and prostacyclin, released from the endothelium, increase I K, one of several actions that lead to further vasodilatation.2,3 Vasoconstrictor substances, such as endothelin, can inhibit I K4 and/or release calcium from the sarcoplasmic reticulum.5 In addition to vasoactive effects, an increase in cytosolic potassium inhibits smooth muscle cell apoptosis,6 and an increase in cytosolic calcium promotes cellular proliferation.7 Consequently, the inhibition of I K is important not only in causing membrane depolarization and calcium entry but also in stimulating vascular remodeling and in the development of chronic pulmonary hypertension. Agents that can cause pulmonary hypertension, such as the anorectic drugs and hypoxia, inhibit I K8,9 and also enhance calcium release from the sarcoplasmic reticulum, leading to subsequent repletion of calcium through store-operated channels.10,11,12 The smooth muscle cells in the resistance pulmonary arteries of patients with pulmonary arterial hypertension (PAH) exhibit both reduced I K and decreased expression of Kv channels,13 and also increased expression of the TRPC ( t ransient r eceptor p otential, c anonical) genes that code for store-operated and receptor-operated calcium channels.14 The decreased expression and function of the Kv channels may …

Inhibition of TRPM2 function by PARP inhibitors protects cells from oxidative stress‐induced death

TRPM2 is a member of the transient receptor potential (TRP) protein superfamily of calcium-permeable, voltage-independent ion channels expressed in nonexcitable cells. Activation of TRPM2 by oxidative stress results in calcium influx and susceptibility to cell death, whereas inhibition of TRPM2 function enhances cell survival. In the present edition of this journal, Fonfria et al. demonstrate a role for poly(ADP ribose) polymerase (PARP) as a mediator between oxidative stress and TRPM2 activation. They present evidence that inhibition of either PARP or TRPM2 protects cells from plasma membrane damage and cell death. The therapeutic implications of this important observation are discussed.

Pore formation by beta-2-microglobulin: A mechanism for the pathogenesis of dialysis associated amyloidosis

Beta-2 microglobulin (β2M, molecular weight 10,000) is a 99 residue immune system protein which is part of the MHC Class I complex whose role is to present antigens to T cells. β2M serum levels rise dramatically in renal failure, and a syndrome called “dialysis associated amyloidosis” occurs with time in a majority of hemodialysis patients who exhibit β2M amyloid deposits in joints, bone and other organs1. β2McAn also induce Ca++ efflux from calvariae, collagenase production, and bone resorption2-4. We report here that β2M formed relatively nonselective, long-lived, voltage independent ion channels in planar phospholipid bilayer membranes at physiologically relevant concentrations. The channels were inhibited by Congo red and blocked by zinc suggesting that they exist in an aggregated beta sheet state as is common with other amyloid fibril forming peptides. Multiple single channel conductances were seen suggesting that various oligomers of β2M may be capable of forming channel structures. We suggest that β2M channel formation may account for some of the pathophysiologic effects seen in dialysis associated amyloidosis. These findings lend further weight to the “channel hypothesis” of amyloid pathogenesis.

Dorsal unpaired median neurones in the insect central nervous system: towards a better understanding of the ionic mechanisms underlying spontaneous electrical activity.

The efferent dorsal unpaired median (DUM) neurones, which include octopaminergic neurones, are among the most intensively studied neurones in the insect central nervous system. They differ from other insect neurones in generating endogenous spontaneous overshooting action potentials. The second half of the 1980s is certain to be considered a turning point in the study of the ion channels underlying the electrical activity of DUM neurones. Recent advances made using the patch-clamp technique have stimulated an increasing interest in the understanding of the biophysical properties of both voltage-dependent and voltage-independent ion channels. Patch-clamp studies of DUM neurones in cell culture demonstrate that these neurones express a wide variety of ion channels. At least five different types of K(+) channel have been identified: inward rectifier, delayed rectifier and A-like channels as well as Ca(2+)- and Na(+)-activated K(+) channels. Moreover, besides voltage-dependent Na(+) and Ca(2+)-sensitive Cl(-) channels, DUM neurones also express four types of Ca(2+) channel distinguished on the basis of their kinetics, voltage range of activation and pharmacological profile. Finally, two distinct resting Ca(2+) and Na(+) channels have been shown to be involved in maintaining the membrane potential and in regulating the firing pattern. In this review, we have also attempted critically to evaluate these existing ion channels with regard to their specific functions in the generation of the different phases of the spontaneous electrical activity of the DUM neurone.

Alzheimer amyloid aβ1-42 channels: Effects of solvent, pH, and congo red

Substantial genetic and biochemical evidence implicates amyloid peptides (Abeta) in the etiology of Alzheimer's Disease (AD). Recent evidence indicates that Abeta1-42 is the predominant species in the hallmark senile amyloid plaque of AD. Furthermore, Abeta1-42 forms aggregates inside lysosomes of cultured neurons leading to lysosomal disruption and cell death. We report here that Abeta1-42 forms slightly cation selective, voltage-independent ion channels with multiple conductance levels at neurotoxic concentrations in planar bilayer membranes. The channels show substantial irregularity of activity, and the size of conductances and the length of open lifetimes depend on solvent history. Formation of channels requires anionic lipids, is enhanced in acidic solutions, and is inhibited by Congo Red. These properties suggest that the channels are formed by aggregates of Abeta1-42. In addition, the channels are reversibly blocked by zinc in a voltage-independent manner. The properties of these channels would likely render them neurotoxic to relevant neurons in vivo. These results are consistent with the channel hypothesis of Abeta neurotoxicity.

Alzheimer amyloid aβ1–42 channels: Effects of solvent, pH, and congo red

Substantial genetic and biochemical evidence implicates amyloid peptides (A|fg) in the etiology of Alzheimer's Disease (AD). Recent evidence indicates that A|fg1–42 is the predominant species in the hallmark senile amyloid plaque of AD. Furthermore, A|fg1–42 forms aggregates inside lysosomes of cultured neurons leading to lysosomal disruption and cell death. We report here that A|fg1–42 forms slightly cation selective, voltage-independent ion channels with multiple conductance levels at neurotoxic concentrations in planar bilayer membranes. The channels show substantial irregularity of activity, and the size of conductances and the length of open lifetimes depend on solvent history. Formation of channels requires anionic lipids, is enhanced in acidic solutions, and is inhibited by Congo Red. These properties suggest that the channels are formed by aggregates of A|fg1–42. In addition, the channels are reversibly blocked by zinc in a voltage-independent manner. The properties of these channels would likely render them neurotoxic to relevant neurons in vivo. These results are consistent with the channel hypothesis of A|fg neurotoxicity. J. Neurosci. Res. 57:467–478, 1999. © 1999 Wiley-Liss, Inc.

Mechanisms of erythropoietin signal transduction: involvement of calcium channels.

Erythropoietin (Epo) induces a dose-dependent increase in intracellular free calcium concentration ([Cai]) in single human BFU-E derived erythroblasts, which is specific for stage of differentiation and mediated through a voltage-independent ion channel permeable to calcium. This minireview examines the regulation of calcium channels by Epo during normal erythroid differentiation, as a model to delineate the immediate signaling events which follow interaction of Epo with its receptor.

Ion channels in human erythroblasts. Modulation by erythropoietin.

To investigate the mechanism of intracellular Ca2+ ([Cai]) increase in human burst-forming unit-erythroid-derived erythroblasts by erythropoietin, we measured [Cai] with digital video imaging, cellular phosphoinositides with high performance liquid chromatography, and plasma membrane potential and currents with whole cell patch clamp. Chelation of extracellular free Ca2+ abolished [Cai] increase induced by erythropoietin. In addition, the levels of inositol-1,4,5-trisphosphate did not increase in erythropoietin-treated erythroblasts. These results indicate that in erythropoietin-stimulated cells, Ca2+ influx rather than intracellular Ca2+ mobilization was responsible for [Cai] rise. Both Ni2+ and moderately high doses of nifedipine blocked [Cai] increase, suggesting involvement of ion channels. Resting membrane potential in human erythroblasts was -10.9 +/- 1.0 mV and was not affected by erythropoietin, suggesting erythropoietin modulated a voltage-independent ion channel permeable to Ca2+. No voltage-dependent ion channel but a Ca(2+)-activated K+ channel was detected in human erythroblasts. The magnitude of erythropoietin-induced [Cai] increase, however, was insufficient to open Ca(2+)-activated K+ channels. Our data suggest erythropoietin modulated a voltage-independent ion channel permeable to Ca2+, resulting in sustained increases in [Cai].

Molecular structure of the chick cerebellar kainate-binding subunit of a putative glutamate receptor

Kainate receptors mediate some of the excitatory transactions carried out in the central nervous system by the neurotransmitter glutamate. They are involved in neurotoxicity, possibly in neurodegenerative disorders and it has been suggested that they have a role in long-term potentiation. Kainate receptors are present both on neuronal and glial cell membranes where they regulate the gating of a voltage-independent ion channel. Nothing is known about their molecular structure. Taking advantage of the unusually high abundance of 3H-kainate binding sites in the chick cerebellum, we have isolated an oligomeric protein that displays a pharmacological profile similar to that of a kainate receptor, and have demonstrated, using the monoclonal antibody IX-50, that this protein is composed of a single polypeptide of Mr 49,000 which harbours the specific kainate recognition site. The structure of this kainate binding protein (KBP) is also of interest because of its exclusive cerebellar localization on Bergmann glial membrane in close proximity to established glutamatergic synapses. We now report the isolation of the complementary DNA containing the complete coding region of the kainate binding protein. The predicted structure of the mature protein has four putative transmembrane domains with a topology analogous to that found in the superfamily of ligand-gated ion channels. This raises the possibility, that kainate binding protein may form part of an ion channel and may be a subunit of a kainate subtype of glutamate receptor.