Salinity-induced ion flux patterns from the excised roots of <Emphasis Type="Italic">Arabidopsis sos</Emphasis> mutants

The SOS signal-transduction pathway is known to be important for ion homeostasis and salt tolerance in plants. However, there is a lack of in planta electrophysiological data about how the changes in signalling and ion transport activity are integrated at the cellular and tissue level. In this study, using the non-invasive ion flux MIFE technique, we compared net K⁺, H⁺ and Na⁺ fluxes from elongation and mature root zones of Arabidopsis wild type Columbia and sos mutants. Our results can be summarised as follows: (1) SOS mutations affect the function of the entire root, not just the root apex; (2) SOS signalling pathway is highly branched; (3) Na⁺ effects on SOS1 may by-pass the SOS2/SOS3 complex in the root apex; (4) SOS mutation affects H⁺ transport even in the absence of salt stress; (5) SOS1 mutation affects intracellular K⁺ homeostasis with a plasma membrane depolarisation-activated outward-rectifying K⁺ channel being a likely target; (6) H⁺ pump also may be a target of SOS signalling. We provide an improved model of SOS signalling and discuss physiological mechanisms underlying salt stress perception and signalling in plants. Our work shows that in planta studies are essential for understanding the functional genomics of plant salt tolerance.     

Membrane-delimited cell signaling complexes: Direct ion channel regulation by G Proteins

Summary Ion channels are signaling molecules and by them-selves perform no work. In this regard they are un like the usual membrane enzyme effectors for G proteins. The pathways of G protein receptor, G protein and ion channels are, therefore, purely infor mational in function. Because a single G protein may have several ion channels as effectors, the effects should be coordinated and this seems to be the case. Inhibition of Ca²⁺ current and stimulation of K⁺ currents would have a greater impact than either alone. Additional flexibility is provided by spontane ous noise in the complexes of G protein receptor, G protein, and ion channel. By having a non-zero setpoint, the range of control is extended and the responses become bi-directional.     

Antiarrhythmic effect of atorvastatin on autoimmune myocarditis is mediated by improving myocardial repolarization

3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors, or statins, are known to inhibit cholesterol biosynthesis and prevent inflammation and oxidative stress. To explore the effects of atorvastatin on inflammatory progression and major cardiac electrophysiological changes in myocarditis, we used an animal model of experimental autoimmune myocarditis (EAM). In this model, BALB/c mice were treated with atorvastatin and we evaluated the levels of inflammation markers and currents of ionic channels that contribute to the duration of action potential (APD) of ventricular myocytes. We demonstrated that atorvastatin treatment attenuated inflammatory infiltration and suppressed the increase in TNF-alpha and IFN-gamma levels in EAM mouse hearts. In the whole-cell patch-clamp experiment, ventricular cardiomyocyte APD was prolonged in EAM group, and atorvastatin blocked this change. We further found that atorvastatin attenuated the significant decrease in outward potassium currents in EAM myocytes. Our results suggested that atorvastatin may ameliorate EAM progression by reducing inflammatory cytokine level. Atorvastatin exerted the antiarrhythmic effects by selectively affecting cardiomyocyte ion channel activity and therefore improves myocardial repolarization.     

Modulation of plant ion channels by oxidizing and reducing agents

Ion channels are proteins forming hydrophilic pathways through the membranes of all living organisms. They play important roles in the electrogenic transport of ions and metabolites. Because of biophysical properties such as high selectivity for the permeant ion, high turnover rate, and modulation by physico-chemical parameters (e.g., membrane potential, calcium concentration), they are involved in several physiological processes in plant cells (e.g., maintenance of the turgor pressure, stomatal movements, and nutrient absorption by the roots). As plants cannot move, plant metabolism must be flexible and dynamic, to cope with environmental changes, to compete with other living species and to prevent pathogen invasion. An example of this flexibility and dynamic behavior is represented by their handling of the so-called reactive oxygen species, inevitable by-products of aerobic metabolism. Plants cope with these species on one side avoiding their toxic effects, on the other utilizing them as signalling molecules and as a means of defence against pathogens. In this review, we present the state-of-the-art of the modulation of plant ion channels by oxidizing and reducing agents.     

Modulation of nociceptive ion channels and receptors via protein-protein interactions: implications for pain relief

In the last 2 decades biomedical research has provided great insights into the molecular signatures underlying painful conditions. However, chronic pain still imposes substantial challenges to researchers, clinicians and patients alike. Under pathological conditions, pain therapeutics often lack efficacy and exhibit only minimal safety profiles, which can be largely attributed to the targeting of molecules with key physiological functions throughout the body. In light of these difficulties, the identification of molecules and associated protein complexes specifically involved in chronic pain states is of paramount importance for designing selective interventions. Ion channels and receptors represent primary targets, as they critically shape nociceptive signaling from the periphery to the brain. Moreover, their function requires tight control, which is usually implemented by protein-protein interactions (PPIs). Indeed, manipulation of such PPIs entails the modulation of ion channel activity with widespread implications for influencing nociceptive signaling in a more specific way. In this review, we highlight recent advances in modulating ion channels and receptors via their PPI networks in the pursuit of relieving chronic pain. Moreover, we critically discuss the potential of targeting PPIs for developing novel pain therapies exhibiting higher efficacy and improved safety profiles.     

Modulation of nociceptive ion channels and receptors via protein-protein interactions: implications for pain relief

In the last 2 decades biomedical research has provided great insights into the molecular signatures underlying painful conditions. However, chronic pain still imposes substantial challenges to researchers, clinicians and patients alike. Under pathological conditions, pain therapeutics often lack efficacy and exhibit only minimal safety profiles, which can be largely attributed to the targeting of molecules with key physiological functions throughout the body. In light of these difficulties, the identification of molecules and associated protein complexes specifically involved in chronic pain states is of paramount importance for designing selective interventions. Ion channels and receptors represent primary targets, as they critically shape nociceptive signaling from the periphery to the brain. Moreover, their function requires tight control, which is usually implemented by protein-protein interactions (PPIs). Indeed, manipulation of such PPIs entails the modulation of ion channel activity with widespread implications for influencing nociceptive signaling in a more specific way. In this review, we highlight recent advances in modulating ion channels and receptors via their PPI networks in the pursuit of relieving chronic pain. Moreover, we critically discuss the potential of targeting PPIs for developing novel pain therapies exhibiting higher efficacy and improved safety profiles.     

Channelling auxin action: modulation of ion transport by indole-3-acetic acid

The growth hormone auxin is a key regulator of plant cell division and elongation. Since plants lack muscles, processes involved in growth and movements rely on turgor formation, and thus on the transport of solutes and water. Modern electrophysiological techniques and molecular genetics have shed new light on the regulation of plant ion transporters in response to auxin. Guard cells, hypocotyls and coleoptiles have advanced to major model systems in studying auxin action. This review will therefore focus on the molecular mechanism by which auxin modulates ion transport and cell expansion in these model cell types.     

Allosteric regulation of pentameric ligand-gated ion channels: An emerging mechanistic perspective

Pentameric ligand-gated ion channels (pLGICs) play a central role in intercellular communications in the nervous system by converting the binding of a chemical messenger—a neurotransmitter—into an ion flux through the postsynaptic membrane. They are oligomeric assemblies that provide prototypical examples of allosterically regulated integral membrane proteins. Here, we present an overview of the most recent advances on the signal transduction mechanism based on the X-ray structures of both prokaryotic and invertebrate eukaryotic pLGICs and on atomistic Molecular Dynamics simulations. The present results suggest that ion gating involves a large structural reorganization of the molecule mediated by two distinct quaternary transitions, a global twisting and the blooming of the extracellular domain, which can be modulated by ligand binding at the topographically distinct orthosteric and allosteric sites. The emerging model of gating is consistent with a wealth of functional studies and will boost the development of novel pharmacological strategies.     

Osmotic tolerance and intracellular ion concentrations of bovine sperm are affected by cryopreservation

In this study, the effects of cryopreservation on osmoregulation and ion homeostasis in bovine sperm were studied. We determined: (1) the osmotic tolerance limits and cell volume response upon exposure to anisotonic conditions, (2) the intracellular pH and potassium concentration, and (3) expression and localization of proteins encoding for potassium and chloride ion channels. A flow cytometric approach was used for simultaneous assessment of cell volume and viability of propidium iodide stained sperm in anisotonic media. Osmotic tolerance was found to be decreased after cryopreservation, especially in the 120 to 60 mOsm/kg osmotic range. The critical osmolality at which half of the sperm population survived increased from 55 to 89 mOsm/kg. The osmotic cell volume response for viable sperm was similar before and after cryopreservation, with an osmotic inactive volume of about 70%. The intracellular pH, determined by recording changes in carboxyfluorescein fluorescence of sperm in media with different pH before and after addition of digitonin, decreased from 6.28 in diluted sperm to 6.16 after cryopreservation. The intracellular potassium concentration, determined using the potassium ionophore nigericin and incubation in media with various potassium concentrations, increased from 154 mM to 183 mM before and after cryopreservation, respectively. The levels of the chloride and potassium ion channel proteins chloride channel 3 protein (CLC-3) and two pore domain potassium channel 2 protein (TASK-2), as detected using Western blot analysis, were not affected by cryopreservation. Immunolocalization studies showed that CLC-3 is present in the acrosome and midpiece as well as in the upper and lower tail. In conclusion, cryopreserved sperm exhibit reduced tolerance to hypotonic stress, a decreased intracellular pH, and increased intracellular potassium level.     

Catalytic competence of the Ras-GEF domain of hSos1 requires intra-REM domain interactions mediated by phenylalanine 577

The Ras-specific guanine nucleotide exchange region of hSos1 consists of two consecutive domains: the catalytic core (residues 742-1024) contains all residues binding to Ras, including the catalytic hairpin, and an upstream REM domain (residues 553-741), so called because it contains an evolutionary conserved Ras Exchange Motif (REM). We functionally define the boundaries of the REM domain through a combination of in vivo and in vitro assays. We show that an intra-REM domain interaction, mediated by phenylalanine 577, is required to allow interaction of the REM domain with the catalytic core, constraining it in the active conformation.     

Regulation of aldosterone production by ion channels: From basal secretion to primary aldosteronism

Aldosterone is produced by zona glomerulosa (ZG) cells of the adrenal cortex and plays a key role in balancing water and electrolytes levels. Autonomous overproduction of aldosterone leads to primary aldosteronism (PA), which is the most common form of secondary endocrine hypertension. Recently, significant progress has been made towards understanding the genetic basis of PA, where increasing clinical evidence suggests that mutations in ion channels appear to be the major cause of aldosterone-producing adenomas. In this review, we focused on potassium and calcium channels that regulate aldosterone secretion, and their roles in the pathology of PA. Because potassium and calcium channels are differentially expressed in ZG cells in different species of mammals, the limitations of published studies are also discussed.     

Indirect multi-trophic interactions mediated by induced plant resistance: impact of caterpillar feeding on aphid parasitoids

Cotton produces insecticidal terpenoids that are induced by tissue-feeding herbivores. Damage by Heliothis virescens caterpillars increases the terpenoid content, which reduces the abundance of aphids. This effect is not evident in Bt-transgenic cotton, which is resistant to H. virescens. We determined whether induction of terpenoids by caterpillars influences the host quality of Aphis gossypii for the parasitoid Lysiphlebus testaceipes and whether this interaction is influenced by Bt cotton. The exposure of parasitoids to terpenoids was determined by quantifying terpenoids in the aphids. We detected several terpenoids in aphids and found a positive relationship between their concentrations in plants and aphids. When L. testaceipes was allowed to parasitize aphids on Bt and non-Bt cotton that was infested or uninfested with H. virescens, fewer parasitoid mummies were found on infested non-Bt than on Bt cotton. Important parasitoid life-table parameters, however, were not influenced by induced resistance following H. virescens infestation, or the Bt trait. Our study provides an example of a tritrophic indirect interaction web, where organisms are indirectly linked through changes in plant metabolites.     

New dinuclear copper(II) and zinc(II) complexes for the investigation of sugar–metal ion interactions

We have studied the binding interactions of biologically important carbohydrates (d-glucose, d-xylose and d-mannose) with the newly synthesized five-coordinate dinuclear copper(II) complex, [Cu₂(hpnbpda)(μ-OAc)] (1) and zinc(II) complex, [Zn₂(hpnbpda)(μ-OAc)] (2) [H₃hpnbpda = N,N′-bis(2-pyridylmethyl)-2-hydroxy-1,3-propanediamine-N,N′-diacetic acid] in aqueous alkaline solution. The complexes 1 and 2 are fully characterized both in solid and solution using different analytical techniques. A geometrical optimization was made of the ligand H₃hpnbpda and the complexes 1 and 2 by molecular mechanics (MM+) method in order to establish the stable conformations. All carbohydrates bind to the metal complexes in a 1:1 molar ratio. The binding events have been investigated by a combined approach of FTIR, UV–vis and ¹³C NMR spectroscopic techniques. UV–vis spectra indicate a significant blue shift of the absorption maximum of complex 1 during carbohydrate coordination highlighting the sugar binding ability of complex 1. The apparent binding constants of the substrate-bound copper(II) complexes have been determined from the UV–vis titration experiments. The binding ability and mode of binding of these sugar substrates with complex 2 are indicated by their characteristic coordination induced shift (CIS) values in ¹³C NMR spectra for carbon atoms C1, C2, and C3 of sugar substrates.     

Regulation of slow calcium channels of myocardial cells and vascular smooth muscle cells by cyclic nucleotides and phosphorylation

The slow Ca²⁺ channels (L-type) of the heart are stimulated by cAMP. Elevation of cAMP produces a very rapid increase in number of slow channels available for voltage activation during excitation. The probability of a Ca²⁺ channel opening and the mean open time of the channel are increased. Therefore, any agent that increases the cAMP level of the myocardial cell will tend to potentiate I_Ca, Ca²⁺ influx, and contraction. The action of cAMP is mediated by PK-A and phosphorylation of the slow Ca²⁺ channel protein or an associated regulatory protein (stimulatory type). The myocardial slow Ca²⁺ channels are also rogulated by cGMP, in a manner that is opposite orantagonistic to that of cAMP. We have demonstrated this at both the macroscople level (whole-cell voltage clamp) and the single-channel level. The effect of cGMP is mediated by PK-G and phosphorylation of a protein, as for example, a regulatory protein (inhibitory-type) associated with the Ca²⁺ channel. Introduction of PK-G intracellularly causes a relatively rapid inhibition of I_Ca(L) in both chick and rat heart cells. Such inhibition occurs for both the basal and stimulated I_Ca(L). In addition, the cGMP/PK-G system was reported to stimulate a phosphatase that dephosphorylates the Ca²⁺ channel. In addition to the slower indirect pathway—exerted via cAMP/PK-A—there is a faster more-direct pathway for I_Ca(L) stimulation by the -adrenergic receptor. This latter pathway involves direct modulation of the channel activity by the alpha subunit (_s*) of the G_s-protein. In vascular smooth muscle cells the two pathways (direct and indirect) also appear to be present, although the indirect pathway producesinhibition of I_Ca(L). PK-C and calmodulin-PK also may play roles in regulation of the myocardial slow Ca²⁺ channels. Both of these protein kinases stimulate the activity of these channels. Thus, it appears that the slow Ca²⁺ channel is a complex structure, including perhaps several associated regulatory proteins, which can be regulated by a number of factors intrinsic and extrinsic to the cell, and thereby control can be exercised over the force of contraction of the heart.This review-type article was prepared by modifying an article published in a book by Sperelakiset al., 1994.     

Voltage-sensing phosphatase reveals temporal regulation of TRPC3/C6/C7 channels by membrane phosphoinositides

TRPC3/C6/C7 channels, a subgroup of classical/canonical TRP channels, are activated by diacylglycerol produced via activation of phospholipase C (PLC)-coupled receptors. Recognition of the physiological importance of these channels has been steadily growing, but the mechanism by which they are regulated remains largely unknown. We recently used a membrane-resident danio rerio voltage-sensing phosphatase (DrVSP) to study TRPC3/C6/C7 regulation and found that the channel activity was controlled by PtdIns(4,5)P 2-DAG signaling in a self-limiting manner (Imai Y et al., the Journal of Physiology, 2012). In this addendum, we present the advantages of using DrVSP as a molecular tool to study PtdIns(4,5)P 2 regulation. DrVSP should be readily applicable for studying phosphoinositide metabolism-linked channel regulation as well as lipid dynamics. Furthermore, in comparison to other modes of self-limiting ion channel regulation, the regulation of TRPC3/C6/C7 channels seems highly susceptible to activation signal strength, which could potentially affect both open duration and the time to peak activation and inactivation. Dysfunction of such self-limiting regulation may contribute to the pathology of the cardiovascular system, gastrointestinal tract and brain, as these channels are broadly distributed and affected by numerous neurohormonal agonists.     

Voltage-sensing phosphatase reveals temporal regulation of TRPC3/C6/C7 channels by membrane phosphoinositides

TRPC3/C6/C7 channels, a subgroup of classical/canonical TRP channels, are activated by diacylglycerol produced via activation of phospholipase C (PLC)-coupled receptors. Recognition of the physiological importance of these channels has been steadily growing, but the mechanism by which they are regulated remains largely unknown. We recently used a membrane-resident danio rerio voltage-sensing phosphatase (DrVSP) to study TRPC3/C6/C7 regulation and found that the channel activity was controlled by PtdIns(4,5)P₂-DAG signaling in a self-limiting manner (Imai Y et al., the Journal of Physiology, 2012). In this addendum, we present the advantages of using DrVSP as a molecular tool to study PtdIns(4,5)P₂ regulation. DrVSP should be readily applicable for studying phosphoinositide metabolism-linked channel regulation as well as lipid dynamics. Furthermore, in comparison to other modes of self-limiting ion channel regulation, the regulation of TRPC3/C6/C7 channels seems highly susceptible to activation signal strength, which could potentially affect both open duration and the time to peak activation and inactivation. Dysfunction of such self-limiting regulation may contribute to the pathology of the cardiovascular system, gastrointestinal tract and brain, as these channels are broadly distributed and affected by numerous neurohormonal agonists.     

Regulation of ligand binding to cardiac muscarinic receptors by ammonium ion and guanine nucleotides

Guanine nucleotides and Na⁺ are known to regulate ligand binding to cardiac muscarinic receptors, which are netagively couple to the adenylate cyclase system. In the present study, we found that NH₄⁺ was more potent than Na⁺ or other monovalent cations in regulating the affinity of the muscarinic receptor for agonists and antagonists. The effect of NH₄⁺ (or Na⁺) on the binding of the antagonist [³H]quinuclidinyl benzilate (QNB) to muscarinic receptors in homogenates of embryonic chick hearts depended on the assay buffer used. NH₄⁺ increased K_d in phosphate buffer or histidine and increased B_max in Tris. NH_f4⁺ (0.1 M) increased the IC₅₀ value for actylcholine inhibition of [³H]QNB binding 20-fold compared to 3–4-fold with 0.1 M Na⁺ or K⁺. Furthermore, NH₄⁺ could substitute for and was more potent than Na⁺ in producing synergistic effects with Gpp[NH]p to reduce the affinity of the receptor of acetylcholine. Tris depressed these effects. Gpp[NH]p plus 0.4 M NH₄Cl totally converted the receptor population to a low affinity agonist state and increased the IC₅₀ for acetylcholine by more than 2000-fold. Two conclusions can be made from the present results. First, NH₄⁺ appears to be the most potent effector yet studied of the monovalent cation site of the muscarinic receptor system. Second, the use of Tris in muscarinic receptor ligand binding assays will produce anomalous results concerning the properties of both agonist antagonist binding to the receptor.