Modulation of potassium channels inhibits bunyavirus infection

Bunyaviruses are considered to be emerging pathogens facilitated by the segmented nature of their genome that allows reassortment between different species to generate novel viruses with altered pathogenicity. Bunyaviruses are transmitted via a diverse range of arthropod vectors, as well as rodents, and have established a global disease range with massive importance in healthcare, animal welfare and economics. There are no vaccines or anti-viral therapies available to treat human bunyavirus infections and so development of new anti-viral strategies is urgently required. Bunyamwera virus (BUNV; genus Orthobunyavirus) is the model bunyavirus, sharing aspects of its molecular and cellular biology with all Bunyaviridae family members. Here, we show for the first time that BUNV activates and requires cellular potassium (K+) channels to infect cells. Time of addition assays using K+ channel modulating agents demonstrated that K+ channel function is critical to events shortly after virus entry but prior to viral RNA synthesis/replication. A similar K+ channel dependence was identified for other bunyaviruses namely Schmallenberg virus (Orthobunyavirus) as well as the more distantly related Hazara virus (Nairovirus). Using a rational pharmacological screening regimen, twin-pore domain K+ channels (K2P) were identified as the K+ channel family mediating BUNV K+ channel dependence. As several K2P channel modulators are currently in clinical use, our work suggests they may represent a new and safe drug class for the treatment of potentially lethal bunyavirus disease.

Eag 1, Eag 2 and Kcnn3 gene brain expression of isolated reared rats

The Eag1 and Eag2, voltage-dependent potassium channels, and the small-conductance calcium-activated potassium channel (Kcnn3) are highly expressed in limbic regions of the brain, where their function is still unknown. Eag1 co-localizes with tyrosine hydroxilase enzyme in the substantia nigra and ventral tegmental area. Kcnn3 deficiency leads to enhanced serotonergic and dopaminergic neurotransmission accompanied by distinct alterations in emotional behaviors. As exposure to stress is able to change the expression and function of several ion channels, suggesting that they might be involved in the consequences of stress, we aimed at investigating Eag 1, Eag2 and Kcnn3 mRNA expression in the brains of rats submitted to isolation rearing. As the long-lasting alterations in emotional and behavioral regulation after stress have been related to changes in serotonergic neurotransmission, expressions of serotonin Htr1a and Htr2a receptors in male Wistar rats` brain were also investigated. Rats were reared in isolation or in groups of five for nine weeks after weaning. Isolated and socially reared rats were tested for exploratory activity in the open field test for 5 min and brains were processed for reverse-transcription coupled to quantitative polymerase chain reaction (qRT-PCR). Isolated reared rats showed decreased exploratory activity in the open field. Compared to socially reared rats, isolated rats showed reduced Htr2a mRNA expression in the striatum and brainstem and reduced Eag2 mRNA expression in all examined regions except cerebellum. To our knowledge, this is the first work to show that isolation rearing can change Eag2 gene expression in the brain. The involvement of this channel in stress-related behaviors is discussed.

Tryptophan hydroxylase is modulated by L-type calcium channels in the rat pineal gland

Calcium is an important second messenger in the rat pineal gland, as well as cAMP. They both contribute to melatonin synthesis mediated by the three main enzymes of the melatonin synthesis pathway: tryptophan hydroxylase, arylalkylamine N-acetyltransferase and hydroxyindole-O-methyltransferase. The cytosolic calcium is elevated in pinealocytes following alpha(1)-adrenergic stimulation, through IP3-and membrane calcium channels activation. Nifedipine, an L-type calcium channel blocker, reduces melatonin synthesis in rat pineal glands in vitro. With the purpose of investigating the mechanisms involved in melatonin synthesis regulation by the L-type calcium channel, we studied the effects of nifedipine on noradrenergic stimulated cultured rat pineal glands. Tryptophan hydroxylase, arylalkylamine N-acetyltransferase and hydroxyindole-O-methyltransferase activities were quantified by radiometric assays and 5-hydroxytryptophan, serotonin, N-acetylserotonin and melatonin contents were quantified by HPLC with electrochemical detection. The data showed that calcium influx blockaded by nifedipine caused a decrease in tryptophan hydroxylase activity, but did not change either arylalkylamine N-acetyltransferase or hydroxyindole-O-methyltransferase activities. Moreover, there was a reduction of 5-hydroxytryptophan, serotonin, N-acetylserotonin and melatonin intracellular content, as well as a reduction of scrotonin and melatonin secretion. Thus, it seems that the calcium influx through L-type high voltage-activated calcium channels is essential for the full activation of tryptophan hydroxylase leading to melatonin synthesis in the pineal gland. (c) 2007 Elsevier Inc. All rights reserved.

Morphine peripheral analgesia depends on activation of the PI3K gamma/AKT/nNOS/NO/K(ATP) signaling pathway

Morphine is one of the most prescribed and effective drugs used for the treatment of acute and chronic pain conditions. In addition to its central effects, morphine can also produce peripheral analgesia. However, the mechanisms underlying this peripheral action of morphine have not yet been fully elucidated. Here, we show that the peripheral antinociceptive effect of morphine is lost in neuronal nitric-oxide synthase null mice and that morphine induces the production of nitric oxide in primary nociceptive neurons. The activation of the nitric-oxide pathway by morphine was dependent on an initial stimulation of PI3K gamma/AKT protein kinase B (AKT) and culminated in increasedactivation of K(ATP) channels. In the latter, this intracellular signaling pathway might cause a hyperpolarization of nociceptive neurons, and it is fundamental for the direct blockade of inflammatory pain by morphine. This understanding offers new targets for analgesic drug development.

Effects of a high fat diet on liver mitochondria: increased ATP-sensitive K(+) channel activity and reactive oxygen species generation

High fat diets are extensively associated with health complications within the spectrum of the metabolic syndrome. Some of the most prevalent of these pathologies, often observed early in the development of high-fat dietary complications, are non-alcoholic fatty liver diseases. Mitochondrial bioenergetics and redox state changes are also widely associated with alterations within the metabolic syndrome. We investigated the mitochondrial effects of a high fat diet leading to non-alcoholic fatty liver disease in mice. We found that the diet does not substantially alter respiratory rates, ADP/O ratios or membrane potentials of isolated liver mitochondria. However, H(2)O(2) release using different substrates and ATP-sensitive K(+) transport activities are increased in mitochondria from animals on high fat diets. The increase in H(2)O(2) release rates was observed with different respiratory substrates and was not altered by modulators of mitochondrial ATP-sensitive K(+) channels, indicating it was not related to an observed increase in K(+) transport. Altogether, we demonstrate that mitochondria from animals with diet-induced steatosis do not present significant bioenergetic changes, but display altered ion transport and increased oxidant generation. This is the first evidence, to our knowledge, that ATP-sensitive K(+) transport in mitochondria can be modulated by diet.

The locus coeruleus and central chemosensitivity

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Signaling path of the action of AVP on distal K+ secretion

Background. Previous studies from our laboratory have shown that luminal perfusion with arginine vasopressin (AVP) stimulates distal tubule secretory potassium flux (J(K)) via V1 receptors (Am J Physiol 278: F809- F816, 2000). In the present work, we investigate the cell signaling mechanism of this process.Methods. In vivo stationary microperfusion was performed in rat cortical distal tubules and luminal K was measured using double K+ resin/reference microelectrodes.Results. In control conditions, J(K) was 0.71 +/- 0.05 nmol. cm(-2).second(-1); this process was inhibited (14%) by 10(-5) mol/L 8-bromo-cyclic adenosine monophosphate (cAMP), and increased by 35% with 10(-8) mol/L phorbol ester [phorbol 12-myristate 13-acetate (PMA), which activates protein kinase C (PKC)]. During luminal perfusion with 10(-11) mol/L AVP, J(K) increased to 0.88 +/- 0.08 nmol. cm(-2).seconds(-1). In the presence of 10(-11) mol/L AVP, J(K) was not affected by 10(-4) mol/L H89, a blocker of protein kinase A (PKA), but was inhibited (45%) by 10(-5) mol/L staurosporine, an inhibitor of PKC, and by 41% during perfusion with 5 x 10(-5) mol/L of the cell Ca2+ chelator bis (2-aminophenoxy) ethane-tetraacetic acid (BAPTA). In order to study the role of Ca2+-dependent K channels in the luminal hormonal action, the tubules were perfused with 5 mmol/L tetraethylammonium chloride (TEA) or 10(-7) mol/L iberiotoxin, in the presence of AVP, and JK was significantly reduced by both agents. Iberiotoxin reduced AVP-stimulated J(K) by 36.4%, and AVP-independent J(K) (after blocking V1 receptors) by only 16%.Conclusion. The results suggest that the luminal V1-receptor effect of AVP on J(K) was mediated by the phospholipase C (PLC)/ Ca2+/PKC signaling path and not by adenylate cyclase/cAMP/PKA, therefore probably acting on maxi-potassium channels.

AMINO-ACID-SEQUENCE OF TSTX-V, AN ALPHA-TOXIN FROM TITYUS-SERRULATUS SCORPION-VENOM, AND ITS EFFECT ON K+ PERMEABILITY OF BETA-CELLS FROM ISOLATED RAT ISLETS OF LANGERHANS

Highly purified Tityustoxin V (TsTX-V), an alpha-toxin isolated from the venom of the Brazilian scorpion Tityus serrulatus, was obtained by ion exchange chromatography on carboxymethylcellulose-52. It was shown to be homogeneous by reverse phase high performance liquid chromatography, N-terminal sequencing (first 39 residues) of the reduced and alkylated protein and by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate and tricine. Following enzymatic digestion, the complete amino acid sequence (64 residues) was determined. The sequence showed higher homology with the toxins from the venoms of the North African than with those of the North and South American scorpions. Using the rate of Rb-86(+) release from depolarized rat pancreatic beta-cells as a measure of K+ permeability changes, TsTX-V (5.6 mu g/ml) was found to increase by 2.0-2.4-fold the rate of marker outflow in the presence of 8.3 mM glucose. This effect was persistent and slowly reversible, showing similarity to that induced by 100 mu-M veratridine, an agent that increases the open period of Na+ channels, delaying their inactivation. It is suggested that, by extending the depolarized period, TsTX-V indirectly affects beta-cell voltage-dependent K+ channels, thus increasing K+ permeability.

KCNQ Channels Determine Serotonergic Modulation of Ventral Surface Chemoreceptors and Respiratory Drive

Chemosensitive neurons in the retrotrapezoid nucleus (RTN) regulate breathing in response to CO2/H+ changes. Their activity is also sensitive to neuromodulatory inputs from multiple respiratory centers, and thus they serve as a key nexus of respiratory control. However, molecular mechanisms that control their activity and susceptibility to neuromodulation are unknown. Here, we show in vitro and in vivo that KCNQ channels are critical determinants of RTN neural activity. In particular, we find that pharmacological block of KCNQ channels (XE991, 10 mu M) increased basal activity and CO2 responsiveness of RTN neurons in rat brain slices, whereas KCNQ channel activation (retigabine, 2-40 mu M) silenced these neurons. Interestingly, we also find that KCNQ and apamin-sensitive SK channels act synergistically to regulate firing rate of RTN chemoreceptors; simultaneous blockade of both channels led to a increase in CO2 responsiveness. Furthermore, we also show that KCNQ channels but not SK channels are downstream effectors of serotonin modulation of RTN activity in vitro. In contrast, inhibition of KCNQ channel did not prevent modulation of RTN activity by Substance P or thyrotropin-releasing hormone, previously identified neuromodulators of RTN chemoreception. Importantly, we also show that KCNQ channels are critical for RTN activity in vivo. Inhibition of KCNQ channels lowered the CO2 threshold for phrenic nerve discharge in anesthetized rats and decreased the ventilatory response to serotonin in awake and anesthetized animals. Given that serotonergic dysfunction may contribute to respiratory failure, our findings suggest KCNQ channels as a new therapeutic avenue for respiratory complications associated with multiple neurological disorders.

Loss-of-function mutations in the KCNJ8-encoded Kir6.1 K(ATP) channel and sudden infant death syndrome.

BACKGROUND Approximately 10% of sudden infant death syndrome (SIDS) may stem from cardiac channelopathies. The KCNJ8-encoded Kir6.1 (K(ATP)) channel critically regulates vascular tone and cardiac adaptive response to systemic metabolic stressors, including sepsis. KCNJ8-deficient mice are prone to premature sudden death, particularly with infection. We determined the spectrum, prevalence, and function of KCNJ8 mutations in a large SIDS cohort. METHODS AND RESULTS Using polymerase chain reaction, denaturing high-performance liquid chromatography, and DNA sequencing, comprehensive open reading frame/splice-site mutational analysis of KCNJ8 was performed on genomic DNA isolated from necropsy tissue on 292 unrelated SIDS cases (178 males, 204 white; age, 2.9±1.9 months). KCNJ8 mutations were coexpressed heterologously with SUR2A in COS-1 cells and characterized using whole-cell patch-clamp. Two novel KCNJ8 mutations were identified. A 5-month-old white male had an in-frame deletion (E332del) and a 2-month-old black female had a missense mutation (V346I). Both mutations localized to Kir6.1's C-terminus, involved conserved residues and were absent in 400 and 200 ethnic-matched reference alleles respectively. Both cases were negative for mutations in established channelopathic genes. Compared with WT, the pinacidil-activated K(ATP) current was decreased 45% to 68% for Kir6.1-E332del and 40% to 57% for V346I between -20 mV and 40 mV. CONCLUSIONS Molecular and functional evidence implicated loss-of-function KCNJ8 mutations as a novel pathogenic mechanism in SIDS, possibly by predisposition of a maladaptive cardiac response to systemic metabolic stressors akin to the mouse models of KCNJ8 deficiency.

The electrophysiology of glomerular mesangial cells

Glomerular mesangial cells (MC) are renal vascular cells that regulate the surface area of glomerular capillaries and thus, partly control glomerular filtration rate. Clarification of the signal transduction pathways and ionic mechanisms modulating MC tone are critical to understanding the physiology and pathophysiology of these cells, and the integrative role these cells play in fluid and electrolyte homeostasis. The patch clamp technique and an assay of cell concentration were used to electrophysiologically and pharmacologically analyze the ion channels of the plasmalemmal of human glomerular MC maintained in tissue culture. Moreover, the signal transduction pathways modulating channels involved in relaxation were investigated. Three distinct K$\sp+$-selective channels were identified: two low conductance channels (9 and 65pS) maintained MC at rest, while a larger conductance (206pS) K$\sp+$ channel was quiescent at rest. This latter channel was pharmacologically and biophysically similar to the large, Ca$\sp{2+}$-activated K$\sp+$ channel (BK$\rm\sb{Ca}$) identified in smooth muscle. BK$\rm\sb{Ca}$ played an essential role in relaxation of MC. In cell-attached patches, the open probability (P$\rm\sb{o}$) of BK$\rm\sb{Ca}$ increased from a basal level of $<$0.05 to 0.22 in response to AII (100nM)-induced mobilization of cytosolic Ca$\sp{2+}$. Activation in response to contractile signals (membrane depolarization and Ca$\sp{2+}$ mobilization) suggests that BK$\rm\sb{Ca}$ acts as a low gain feedback regulator of contraction. Atrial natriuretic factor (ANF; 1.0$\mu$M) and nitroprusside (NP; 0.1mM), via the second messenger, cGMP, increase the feedback gain of BK$\rm\sb{Ca}$. In cell-attached patches bathed with physiological saline, these agents transiently activated BK$\rm\sb{Ca}$ from a basal $\rm P\sb{o}<0.05$ to peak responses near 0.50. As membrane potential hyperpolarizes towards $\rm E\sb{K}$ (2-3 minutes), BK$\rm\sb{Ca}$ inactivates. Upon depolarizing V$\rm\sb{m}$ with 140 mM KCl, db-cGMP (10$\mu$M) activated BK$\rm\sb{Ca}$ to a sustained P$\rm\sb{o}$ = 0.51. Addition of AII in the presence of cGMP further increased P$\rm\sb{o}$ to 0.82. Activation of BK$\rm\sb{Ca}$ by cGMP occured via an endogenous cGMP-dependent protein kinase (PKG): in excised, inside-out patches, PKG in the presence of Mg-ATP (0.1mM) and cGMP increased P$\rm\sb{o}$ from 0.07 to 0.39. In contrast, neither PKC nor PKA influenced BK$\rm\sb{Ca}$. Endogenous okadaic acid-sensitive protein phosphatase suppressed BK$\rm\sb{Ca}$ activity. Binning the change in P$\rm\sb{o}\ (\Delta P\sb{o}$) of BK$\rm\sb{Ca}$ in response to PKG (n = 69) established two distinct populations of channels: one that responded ($\cong$67%, $\rm\Delta P\sb{o} = 0.45 \pm 0.03$) and one that was unresponsive ($\Delta\rm P\sb{o} = 0.00 \pm 0.01$) to PKG. Activation of BK$\rm\sb{Ca}$ by PKG resulted from a decrease in the Ca$\sp{2+}$- and voltage-activation thresholds independent of sensitivities. In conclusion, mesangial BK$\rm\sb{Ca}$ channels sense both electrical and chemical signals of contraction and act as feedback regulators by repolarizing the plasma membrane. ANF and NO, via cGMP, stimulate endogenous PKG, which subsequently decreases the activation threshold of BK$\rm\sb{Ca}$ to increase the gain of this feedback regulatory signal. ^

A heteromeric potassium channel involved in the modulation of the plasma membrane potential is essential for the survival of African trypanosomes.

Discovery of novel drug targets may lead to improved treatment of trypanosomiasis. We characterize here 2 gene products of Trypanosoma brucei that are essential for the growth of bloodstream form (BSF) parasites, as shown by RNA interference (RNAi)-mediated down-regulation of the individual mRNAs. The primary sequences of the 2 proteins--protein encoded by gene Tb927.1.4450 (TbK1) and protein encoded by gene Tb927.9.4820 (TbK2)--indicate that both belong to the family of putative, Ca(2+)-activated potassium channels. The proteins were expressed in Xenopus laevis oocytes and their functions investigated by use of electrophysiological techniques. Only combined expression of TbK1 and TbK2 results in the formation of sizeable currents, indicating that these proteins probably assemble into a heteromeric ion channel. The current mediated by this channel shows little time and voltage dependence and displays a permeability ratio of K(+)/Na(+) of >20. The known potassium channel blocker barium inhibits this channel with a half-maximal inhibitory concentration (IC50) of 98 ± 15 μM. The membrane potential of trypanosomes was measured with a fluorescent dye. Individual RNAi-mediated down-regulation of TbK1 or TbK2 eliminates a potassium conductance in the plasma membrane of BSF. Thus, this heteromeric potassium channel is involved in the modulation of the plasma membrane potential and represents a novel drug target in T. brucei.

Coordinate regulation of gonadotropin-releasing hormone neuronal firing patterns by cytosolic calcium and store depletion

Elevation of cytosolic free Ca2+ concentration ([Ca2+]i) in excitable cells often acts as a negative feedback signal on firing of action potentials and the associated voltage-gated Ca2+ influx. Increased [Ca2+]i stimulates Ca2+-sensitive K+ channels (IK-Ca), and this, in turn, hyperpolarizes the cell and inhibits Ca2+ influx. However, in some cells expressing IK-Ca the elevation in [Ca2+]i by depletion of intracellular stores facilitates voltage-gated Ca2+ influx. This phenomenon was studied in hypothalamic GT1 neuronal cells during store depletion caused by activation of gonadotropin-releasing hormone (GnRH) receptors and inhibition of endoplasmic reticulum (Ca2+)ATPase with thapsigargin. GnRH induced a rapid spike increase in [Ca2+]i accompanied by transient hyperpolarization, followed by a sustained [Ca2+]i plateau during which the depolarized cells fired with higher frequency. The transient hyperpolarization was caused by the initial spike in [Ca2+]i and was mediated by apamin-sensitive IK-Ca channels, which also were operative during the subsequent depolarization phase. Agonist-induced depolarization and increased firing were independent of [Ca2+]i and were not mediated by inhibition of K+ current, but by facilitation of a voltage-insensitive, Ca2+-conducting inward current. Store depletion by thapsigargin also activated this inward depolarizing current and increased the firing frequency. Thus, the pattern of firing in GT1 neurons is regulated coordinately by apamin-sensitive SK current and store depletion-activated Ca2+ current. This dual control of pacemaker activity facilitates voltage-gated Ca2+ influx at elevated [Ca2+]i levels, but also protects cells from Ca2+ overload. This process may also provide a general mechanism for the integration of voltage-gated Ca2+ influx into receptor-controlled Ca2+ mobilization.

A human intermediate conductance calcium-activated potassium channel

An intermediate conductance calcium-activated potassium channel, hIK1, was cloned from human pancreas. The predicted amino acid sequence is related to, but distinct from, the small conductance calcium-activated potassium channel subfamily, which is ≈50% conserved. hIK1 mRNA was detected in peripheral tissues but not in brain. Expression of hIK1 in Xenopus oocytes gave rise to inwardly rectifying potassium currents, which were activated by submicromolar concentrations of intracellular calcium (K0.5 = 0.3 μM). Although the K0.5 for calcium was similar to that of small conductance calcium-activated potassium channels, the slope factor derived from the Hill equation was significantly reduced (1.7 vs. 3.5). Single-channel current amplitudes reflected the macroscopic inward rectification and revealed a conductance level of 39 pS in the inward direction. hIK1 currents were reversibly blocked by charybdotoxin (Ki = 2.5 nM) and clotrimazole (Ki = 24.8 nM) but were minimally affected by apamin (100 nM), iberiotoxin (50 nM), or ketoconazole (10 μM). These biophysical and pharmacological properties are consistent with native intermediate conductance calcium-activated potassium channels, including the erythrocyte Gardos channel.

Stimulation of CD95 (Fas) blocks T lymphocyte calcium channels through sphingomyelinase and sphingolipids

Calcium influx through store-operated calcium release-activated calcium channels (CRAC) is required for T cell activation, cytokine synthesis, and proliferation. The CD95 (Apo-1/Fas) receptor plays a role in self-tolerance and tumor immune escape, and it mediates apoptosis in activated T cells. In this paper we show that CD95-stimulation blocks CRAC and Ca2+ influx in lymphocytes through the activation of acidic sphingomyelinase (ASM) and ceramide release. The block of Ca2+ entry is lacking in CD95-defective lpr lymphocytes as well as in ASM-defective cells and can be restored by retransfection of ASM. C2 ceramide, C6 ceramide, and sphingosine block CRAC reversibly, whereas the inactive dihydroceramide has no effect. CD95-stimulation or the addition of ceramide prevents store-operated Ca2+ influx, activation of the transcriptional regulator NFAT, and IL-2 synthesis. The block of CRAC by sphingomyelinase metabolites adds a function to the repertoire of the CD95 receptor inhibiting T cell activation signals.