Channel Activity (channel + activity)

Distribution by Scientific Domains
Distribution within Life Sciences

Kinds of Channel Activity

  • ca2+ channel activity
  • ion channel activity
  • k+ channel activity

  • Selected Abstracts

    Dependence of Hyperpolarisation-Activated Cyclic Nucleotide-Gated Channel Activity on Basal Cyclic Adenosine Monophosphate Production in Spontaneously Firing GH3 Cells

    K. Kretschmannova
    Abstract The hyperpolarisation-activated cyclic nucleotide-gated (HCN) channels play a distinct role in the control of membrane excitability in spontaneously active cardiac and neuronal cells. Here, we studied the expression and role of HCN channels in pacemaking activity, Ca2+ signalling, and prolactin secretion in GH3 immortalised pituitary cells. Reverse transcriptase-polymerase chain reaction analysis revealed the presence of mRNA transcripts for HCN2, HCN3 and HCN4 subunits in these cells. A hyperpolarisation of the membrane potential below ,,60 mV elicited a slowly activating voltage-dependent inward current (Ih) in the majority of tested cells, with a half-maximal activation voltage of ,89.9 4.2 mV and with a time constant of 1.4 0.2 s at ,120 mV. The bath application of 1 mM Cs+, a commonly used inorganic blocker of Ih, and 100 M ZD7288, a specific organic blocker of Ih, inhibited Ih by 90 4.1% and 84.3 1.8%, respectively. Receptor- and nonreceptor-mediated activation of adenylyl and soluble guanylyl cyclase and the addition of a membrane permeable cyclic adenosine monophosphate (cAMP) analogue, 8-Br-cAMP, did not affect Ih. Inhibition of basal adenylyl cyclase activity, but not basal soluble guanylyl cyclase activity, led to a reduction in the peak amplitude and a leftward shift in the activation curve of Ih by 23.7 mV. The inhibition of the current was reversed by stimulation of adenylyl cyclase with forskolin and by the addition of 8-Br-cAMP, but not 8-Br-cGMP. Application of Cs+ had no significant effect on the resting membrane potential or electrical activity, whereas ZD7288 exhibited complex and Ih -independent effects on spontaneous electrical activity, Ca2+ signalling, and prolactin release. These results indicate that HCN channels in GH3 cells are under tonic activation by basal level of cAMP and are not critical for spontaneous firing of action potentials. [source]

    Functional Characterisation of the Volume-Sensitive Anion Channel in Rat Pancreatic ,-Cells

    L. Best
    The whole-cell and perforated patch configurations of the patch-clamp technique were used to characterise the volume-sensitive anion channel in rat pancreatic ,-cells. The channel showed high permeability (P) relative to Cl, to extracellular monovalent organic anions (PSCN/PCll= 1.73, Pacetate/PCll= 0.39, Plactate/PCll= 0.38, Pacetoacetate/PCll= 0.32, Pglutamate/PCll= 0.28) but was less permeable to the divalent anion malate (Pmalate/PCll= 0.14). Channel activity was inhibited by a number of putative anion channel inhibitors, including extracellular ATP (10 mM), 1,9-dideoxyforskolin (100 ,M) and 4-OH tamoxifen (10 ,M). Inclusion of the catalytic subunit of protein kinase A in the pipette solution did not activate the volume-sensitive anion channel in non-swollen cells. Furthermore, addition of 8-bromoadenosine 3,,5,-cyclic monophosphate (8-BrcAMP) or forskolin failed to activate the channel in intact cells under perforated patch conditions. Addition of phorbol 12,13-dibutyrate (200 nM), either before or after cell swelling, also failed to affect channel activation. Our findings do not support the suggestion that the volume-sensitive anion channel in pancreatic ,-cells can be activated by protein kinase A. Furthermore, the ,-cell channel does not appear to be subject to regulation via protein kinase C. [source]

    Dysregulation of human bestrophin-1 by ceramide-induced dephosphorylation

    Qinghuan Xiao
    Best vitelliform macular dystrophy is an inherited autosomal dominant, juvenile onset form of macular degeneration caused by mutations in a chloride ion channel, human bestrophin-1 (hBest1). Mutations in Best1 have also been linked to several other forms of retinopathy. In addition to mutations, hBest1 dysfunction might come about by disruption of other processes that regulate Best1 function. Here we show that hBest1 chloride channel activity is regulated by ceramide and phosphorylation. We have identified a protein kinase C (PKC) phosphorylation site (serine 358) in hBest1 that is important for sustained channel function. Channel activity is maintained by PKC activators, protein phosphatase inhibitors, or pseudo-phosphorylation by substitution of glutamic acid for serine 358. When ceramide levels are elevated by exogenous addition of ceramide to the bath, by addition of bacterial sphingomyelinase, or by hypertonic stress, S358 is rapidly dephosphorylated. The dephosphorylation is mediated by protein phosphatase 2A. Hypertonic stress-induced dephosphorylation is blocked by a dihydroceramide, an inactive form of ceramide, and manumycin, an inhibitor of neutral sphingomyelinase. Our results support a model in which ceramide accumulation during early stages of retinopathy inhibits hBest1 function, leading to abnormal fluid transport across the retina, and enhanced inflammation. [source]

    Functional NR2B- and NR2D-containing NMDA receptor channels in rat substantia nigra dopaminergic neurones

    Susan Jones
    NMDA receptors regulate burst firing of dopaminergic neurones in the substantia nigra pars compacta (SNc) and may contribute to excitotoxic cell death in Parkinson's disease (PD). In order to investigate the subunit composition of functional NMDA receptors in identified rat SNc dopaminergic neurones, we have analysed the properties of individual NMDA receptor channels in outside-out patches. NMDA (100 nm) activated channels corresponding to four chord conductances of 18, 30, 41 and 54 pS. Direct transitions were observed between all conductance levels. Between 18 pS and 41 pS conductance levels, direct transitions were asymmetric, consistent with the presence of NR2D-containing NMDA receptors. Channel activity in response to 100 nm or 200 ,m NMDA was not affected by zinc or TPEN (N,N,N,,N,-tetrakis-[2-pyridylmethyl]-ethylenediamine), indicating that SNc dopaminergic neurones do not contain functional NR2A subunits. The effect of the NR2B antagonist ifenprodil was complex: 1 ,m ifenprodil reduced open probability, while 10 ,m reduced channel open time but had no effect on open probability of channels activated by 100 nm NMDA. When the concentration of NMDA was increased to 200 ,m, ifenprodil (10 ,m) produced the expected reduction in open probability. These results indicate that NR2B subunits are present in SNc dopaminergic neurones. Taken together, these findings indicate that NR2D and NR2B subunits form functional NMDA receptor channels in SNc dopaminergic neurones, and suggest that they may form a triheteromeric NMDA receptor composed of NR1/NR2B/NR2D subunits. [source]

    Xanthine-analog, KMUP-2, enhances cyclic GMP and K+ channel activities in rabbit aorta and corpus cavernosum with associated penile erection

    Rong-Jyh Lin
    Abstract The pharmacological properties of KMUP-2 were examined in isolated rabbit aorta and corpus cavernosum smooth muscle (CCSM). KMUP-2 caused relaxations that were attenuated by removed endothelium, high K+, and pretreatment with the soluble guanylate cyclase (sGC) inhibitors methylene blue (10 ,M) and ODQ (1 ,M), a NOS inhibitor, L-NAME (100 ,M), a K+ channel blocker TEA (10 mM), a KATP channel blocker glibenclamide (1 ,M), a voltage-dependent K+ channel blocker 4-AP (100 ,M), and the Ca2+ -dependent K+ channel blockers apamin (1 ,M) and charybdotoxin (ChTX, 0.1 ,M). The relaxant responses of KMUP-2 (0.01, 0.05, 0.1 ,M) together with a PDE inhibitor, IBMX (0.5 ,M), had additive effects on rabbit aorta and CCSM. Additionally, KMUP-2 (100 ,M) also affected cGMP metabolism, due to its inhibiting activity on PDE in human platelets. KMUP-2 (0.1,100 ,M) further induced an increase of intracellular cGMP levels in the primary cultured rabbit aortic and CCSM cells. These increases in cGMP content were abolished in the presence of methylene blue (100 ,M) and ODQ (10 ,M). Obviously, the relaxant effects of KMUP-2 on rabbit isolated tissues are more sensitive in CCSM than in aorta. Moreover, KMUP-2 also stimulated NO/sGC/cGMP pathway and subsequent elevation of cGMP by blockade of PDE and enhanced opening of K+ channels in rabbit aorta and CCSM. KMUP-2 (0.2, 0.4, 0.6 mg/kg), similar to KMUP-1 and sildenafil, caused increases of intracavernous pressure (ICP) and duration of tumescene (DT) in a dose-dependent manner. It is concluded that both the increases of cGMP and the opening activity of K+ channels play prominent roles in KMUP-2-induced aortic smooth muscle and CCSM relaxation and increases of ICP in rabbits. Drug Dev. Res. 55:162,172, 2002. 2002 Wiley-Liss, Inc. [source]

    Myocytes from congenital myotonic dystrophy display abnormal Na+ channel activities

    MUSCLE AND NERVE, Issue 4 2005
    Annalisa Bernareggi PhD
    Abstract Na+ currents were measured in myocytes from a fetus with congenital myotonic dystrophy type 1 (DM1) using the patch-clamp whole-cell technique. Steady-state activation and inactivation properties of Na+ channels were not substantially different between these cells and age-matched control cells. However, a decrease in Na+ channel density and a faster rate of recovery from inactivation were found in myocytes from congenital DM1 suggesting that changes in functional Na+ channels may affect cell excitability of muscle cells of patients with this disorder. Muscle Nerve, 2005 [source]

    Photosensory Functions of Channelrhodopsins in Native Algal Cells,

    Oleg A. Sineshchekov
    Photomotility responses in flagellate alga are mediated by two types of sensory rhodopsins (A and B). Upon photoexcitation they trigger a cascade of transmembrane currents which provide sensory transduction of light stimuli. Both types of algal sensory rhodopsins demonstrate light-gated ion channel activities when heterologously expressed in animal cells, and therefore they have been given the alternative names channelrhodopsin 1 and 2. In recent publications their channel activity has been assumed to initiate the transduction chain in the native algal cells. Here we present data showing that: (1) the modes of action of both types of sensory rhodopsins are different in native cells such as Chlamydomonas reinhardtii than in heterologous expression systems, and also differ between the two types of rhodopsins; (2) the primary function of Type B sensory rhodopsin (channelrhodopsin-2) is biochemical activation of secondary Ca2+ -channels with evidence for amplification and a diffusible messenger, sufficient for mediating phototaxis and photophobic responses; (3) Type A sensory rhodopsin (channelrhodopsin-1) mediates avoidance responses by direct channel activity under high light intensities and exhibits low-efficiency amplification. These dual functions of algal sensory rhodopsins enable the highly sophisticated photobehavior of algal cells. [source]

    Effect of Testosterone on Potassium Channel Opening in Human Corporal Smooth Muscle Cells

    Deok Hyun Han MD
    ABSTRACT Introduction., In humans, the role of testosterone in sexual functions, including sexual desire, nocturnal penile erections, and ejaculatory volume, has been relatively well established. However, the effects of testosterone on intrapenile structure in humans remains controversial. Aim., We assessed the direct effects of testosterone on potassium channels in human corporal smooth muscle cells, in an effort to understand the mechanisms inherent to the testosterone-induced relaxation of corporal smooth muscle cells at the cellular and molecular levels. Methods., We conducted electrophysiologic studies using cultured human corporal smooth muscle cells. We evaluated the effects of testosterone on potassium channels,BKCa and KATP channels,by determining the whole-cell currents and single-channel activities. For the electrophysiologic recordings, whole-cell and cell-attached configuration patch-clamp techniques were utilized. Main Outcome Measures., Changes in whole-cell currents and channel activities of BKCa and KATP channels by testosterone. Results., Testosterone (200 nM) significantly increased the single-channel activity of calcium-activated potassium (BKCa) channels and whole-cell K+ currents by 443.4 83.4% (at +60 mV; N = 11, P < 0.05), and this effect was abolished by tetraethylammonium (TEA) (1 mM), a BKCa channel blocker. The whole-cell inward K+ currents of the KATP channels were also increased by 226.5 49.3% (at ,100 mV; N = 7, P < 0.05). In the presence of a combination of vardenafil (10 nM) and testosterone (200 nM), the BKCa channel was activated to a significantly higher degree than was induced by testosterone alone. Conclusions., The results of patch-clamp studies provided direct molecular evidence that testosterone stimulates the activity of BKCa channels and KATP channels. An understanding of the signaling mechanisms that couple testosterone receptor activation to potassium channel stimulation will provide us with an insight into the cellular processes underlying the vasorelaxant effects of testosterone. Han DH, Chae MR, Jung JH, So I, Park JK, and Lee SW. Effect of testosterone on potassium channel opening in human corporal smooth muscle cells. J Sex Med 2008;5:822,832. [source]

    Isolated plant nuclei as mechanical and thermal sensors involved in calcium signalling

    THE PLANT JOURNAL, Issue 1 2004
    Tou Cheu Xiong
    Summary Calcium signals in the nucleus elicit downstream effects that are distinct from those of cytosolic calcium signals. In the present work, we have evaluated the ability of plant nuclei to sense stimuli directly and to convert them into calcium changes. We show that individual mechanical stimulation of isolated nuclei elicits a single calcium transient at acidic pHs, whereas a series of stimulations leads to oscillations whose frequency reflects that of the stimuli. Conversely, at alkaline pHs, nuclei respond to temperature but not to stretch. The stretch- and the temperature-activated processes differ by their sensitivity to pharmacological drugs known to affect ion channel activities in animal cells. Our data demonstrate that isolated nuclei are able to gauge physical parameters of their environment. This might have a profound influence on the functioning of calcium-dependent processes known to control a large array of molecular events in the nucleus. [source]

    KMUP-1 activates BKCa channels in basilar artery myocytes via cyclic nucleotide-dependent protein kinases

    Bin-Nan Wu
    This study investigated whether KMUP-1, a synthetic xanthine-based derivative, augments the delayed-rectifier potassium (KDR)- or large-conductance Ca2+ -activated potassium (BKCa) channel activity in rat basilar arteries through protein kinase-dependent and -independent mechanisms. Cerebral smooth muscle cells were enzymatically dissociated from rat basilar arteries. Conventional whole cell, perforated and inside-out patch-clamp electrophysiology was used to monitor K+ - and Ca2+ channel activities. KMUP-1 (1 ,M) had no effect on the KDR current but dramatically enhanced BKCa channel activity. This increased BKCa current activity was abolished by charybdotoxin (100 nM) and iberiotoxin (100 nM). Like KMUP-1, the membrane-permeable analogs of cGMP (8-Br-cGMP) and cAMP (8-Br-cAMP) enhanced the BKCa current. BKCa current activation by KMUP-1 was markedly inhibited by a soluble guanylate cyclase inhibitor (ODQ 10 ,M), an adenylate cyclase inhibitor (SQ 22536 10 ,M), competitive antagonists of cGMP and cAMP (Rp-cGMP, 100 ,M and Rp-cAMP, 100 ,M), and cGMP- and cAMP-dependent protein kinase inhibitors (KT5823, 300 nM and KT5720, 300 nM). Voltage-dependent L-type Ca2+ current was significantly suppressed by KMUP-1 (1 ,M), and nearly abolished by a calcium channel blocker (nifedipine, 1 ,M). In conclusion, KMUP-1 stimulates BKCa currents by enhancing the activity of cGMP-dependent protein kinase, and in part this is due to increasing cAMP-dependent protein kinase. Physiologically, this activation would result in the closure of voltage-dependent calcium channels and the relaxation of cerebral arteries. British Journal of Pharmacology (2005) 146, 862,871. doi:10.1038/sj.bjp.0706387 [source]

    Multiple effects of mefenamic acid on K+ currents in smooth muscle cells from pig proximal urethra

    N Teramoto
    The effects of mefenamic acid on both membrane potential and K+ currents in pig urethral myocytes were investigated using patch-clamp techniques (conventional whole-cell, cell-attached, outside-out and inside-out configuration). In the current-clamp mode, mefenamic acid caused a concentration-dependent hyperpolarization, which was inhibited by preapplication of 1 ,M glibenclamide. In the voltage-clamp mode, mefenamic acid induced an outward current that was blocked by glibenclamide even in the presence of iberiotoxin (IbTX, 300 nM) at ,50 mV. ATP-sensitive K+ channels (KATP channels) could be activated in the same patch by mefenamic acid and levcromakalim, with the same unitary amplitude and the similar opening gating at ,50 mV in cell-attached configuration. In outside-out recording, external application of mefenamic acid activated intracellular Ca2+ -activated IbTX-sensitive large-conductance K+ channels (BKCa channels). Mefenamic acid (30 ,M) activated spontaneous transient outward currents (STOCs). In contrast, mefenamic acid (100 ,M) increased sustained outward currents, diminishing the activity of STOCs. Over the whole voltage range, mefenamic acid caused opposite effects on the membrane currents in the absence and presence of 5 ,M glibenclamide. In the presence of 10 mM 4-aminopyridine (4-AP), mefenamic acid only increased the outward currents. These results indicate that mefenamic acid increases the channel activities of two distinct types of K+ channels (i.e. BKCa channels and KATP channels) and decreased 4-AP-sensitive K+ channels in pig urethral myocytes. British Journal of Pharmacology (2003) 140, 1341,1350. doi:10.1038/sj.bjp.0705524 [source]

    3133: Planar patch-clamping in human corneal endothelial cells: a new tool for clinical application?

    Purpose Identification of apoptotic or damaged human corneal endothelial cells (HCECs) is limited to morphological evaluation such as phase contrast microscopy and vital staining. The molecular mechanisms of corneal endothelial cell loss are not fully understood. Special investigations in cellular signalling and ion channel research are necessary to elucidate the mechanisms of corneal cell loss. In this context, it is known that this cell loss is often caused by apoptosis in oxidative stress. Methods Automated planar patch-clamp has become common in drug development and safety programs because it enables efficient and systematic testing of compounds against ion channels during voltage-clamp. A particularly successful automated approach is based on planar patch-clamp chips and this is the basis for the technology used here. Routine intracellular or extracellular perfusion opens possibilities for studying the regulation and pharmacology of ion channels. Previously, these studies were available only to highly skilled and dedicated experimenters. Results Notable, definite ion channel activities could be demonstrated by conventional as well as by planar patch-clamp in HCECs for the first time. In particular, temperature-sensing transient receptor potential (TRP)-like non-selective cation channel currents as well as capsaicin-sensitive ion channel currents could be detected. The expression of TRPV1-3 ion channels in HCEC could also be confirmed by RT-PCR, Western blot analysis and fluorescence cell imaging. Conclusion The administration of this novel measuring technology opens new perspectives in the investigation of the physiology of HCEC. The findings may have direct clinical implication (eye banking procedures, keratoplasty). [source]

    Single mechano-gated channels activated by mechanical deformation of acutely isolated cardiac fibroblasts from rats

    ACTA PHYSIOLOGICA, Issue 3 2010
    A. Kamkin
    Abstract Aim:, Mechanosensitive conductances were reported in cardiac fibroblasts, but the properties of single channels mediating their mechanosensitivity remain uncharacterized. The aim of this work was to investigate single mechano-gated channels (MGCs) activated by mechanical deformations of cardiac fibroblasts. Methods:, Currents through single MGCs and mechanosensitive whole-cell currents were recorded from isolated rat atrial fibroblasts using the cell-attached and whole-cell patch-clamp configurations respectively. Defined mechanical stress was applied via the patch pipette used for the whole-cell recordings. Results:, Under resting conditions occasional short openings of two types of single MGCs with conductances of 43 and 87 pS were observed. Both types of channels displayed a linear current,voltage relationship with the reversal potential around 0 mV. Small (1 ,m) mechanical deformations affected neither single nor whole-cell mechano-gated currents. Cell compressions (2, 3 and 4 ,m) augmented the whole-cell currents and increased the frequency and duration of single channel openings. Cell stretches (2, 3 and 4 ,m) inactivated the whole-cell currents and abolished the activity of single MGCs. Gd3+ (8 ,m) blocked the whole-cell currents within 5 min. No single channel activity was observed in the cell-attached mode when Gd3+ was added to the intrapipette solution. Cytochalasin D and colchicine (100 ,m each) completely blocked both the whole-cell and single channel currents. Conclusions:, These findings show that rat atrial fibroblasts express two types of MGCs whose activity is governed by cell deformation. We conclude that fibroblasts can sense the direction of applied stress and contribute to mechano-electrical coupling in the heart. [source]

    Cysteine-rich secretory proteins are not exclusively expressed in the male reproductive tract

    Thulasimala Reddy
    The Cysteine-RIch Secretory Proteins (CRISPs) are abundantly produced in the male reproductive tract of mammals and within the venom of reptiles and have been shown to regulate ion channel activity. CRISPs, along with the Antigen-5 proteins and the Pathogenesis related-1 (Pr-1) proteins, form the CAP superfamily of proteins. Analyses of EST expression databases are increasingly suggesting that mammalian CRISPs are expressed more widely than in the reproductive tract. We, therefore, conducted a reverse transcription PCR expression profile and immunohistochemical analyses of 16 mouse tissues to define the sites of production of each of the four murine CRISPs. These data showed that each of the CRISPs have distinct and sometimes overlapping expression profiles, typically associated with the male and female reproductive tract, the secretory epithelia of exocrine glands, and immune tissues including the spleen and thymus. These investigations raise the potential for a role for CRISPs in general mammalian physiology. Developmental Dynamics 237:3313,3323, 2008. 2008 Wiley-Liss, Inc. [source]

    NaV1.6a is required for normal activation of motor circuits normally excited by tactile stimulation

    Sean E. Low
    Abstract A screen for zebrafish motor mutants identified two noncomplementing alleles of a recessive mutation that were named non-active (navmi89 and navmi130). nav embryos displayed diminished spontaneous and touch-evoked escape behaviors during the first 3 days of development. Genetic mapping identified the gene encoding NaV1.6a (scn8aa) as a potential candidate for nav. Subsequent cloning of scn8aa from the two alleles of nav uncovered two missense mutations in NaV1.6a that eliminated channel activity when assayed heterologously. Furthermore, the injection of RNA encoding wild-type scn8aa rescued the nav mutant phenotype indicating that scn8aa was the causative gene of nav. In-vivo electrophysiological analysis of the touch-evoked escape circuit indicated that voltage-dependent inward current was decreased in mechanosensory neurons in mutants, but they were able to fire action potentials. Furthermore, tactile stimulation of mutants activated some neurons downstream of mechanosensory neurons but failed to activate the swim locomotor circuit in accord with the behavioral response of initial escape contractions but no swimming. Thus, mutant mechanosensory neurons appeared to respond to tactile stimulation but failed to initiate swimming. Interestingly fictive swimming could be initiated pharmacologically suggesting that a swim circuit was present in mutants. These results suggested that NaV1.6a was required for touch-induced activation of the swim locomotor network. 2010 Wiley Periodicals, Inc. Develop Neurobiol 70:508,522, 2010 [source]

    Effects of sulfonylureas on mitochondrial ATP-sensitive K+ channels in cardiac myocytes: implications for sulfonylurea controversy

    Toshiaki Sato
    Abstract Background Mitochondrial ATP-sensitive K+ (mitoKATP) channel plays a key role in cardioprotection. Hence, a sulfonylurea that does not block mitoKATP channels would be desirable to avoid damage to the heart. Accordingly, we examined the effects of sulfonylureas on the mitoKATP channel and mitochondrial Ca2+ overload. Methods Flavoprotein fluorescence in rabbit ventricular myocytes was measured to assay mitoKATP channel activity. The mitochondrial Ca2+ concentration was measured by loading cells with rhod-2. Results The mitoKATP channel opener diazoxide (100 M) reversibly increased flavoprotein oxidation to 31.8 4.3% (n = 5) of the maximum value induced by 2,4-dinitrophenol. Glimepiride (10 M) alone did not oxidize the flavoprotein, and the oxidative effect of diazoxide was unaffected by glimepiride (35.4 3.2%, n = 5). Similarly, the diazoxide-induced flavoprotein oxidation was unaffected both by gliclazide (10 M) and by tolbutamide (100 M). Exposure to ouabain (1 mM) for 30 min produced mitochondrial Ca2+ overload, and the intensity of rhod-2 fluorescence increased to 197.4 7.2% of baseline (n = 11). Treatment with diazoxide significantly reduced the ouabain-induced mitochondrial Ca2+ overload (149.6 5.1%, n = 11, p < 0.05 versus ouabain alone), and the effect was antagonized by the mitoKATP channel blocker 5-hydroxydecanoate (189.8 27.8%, n = 5) and glibenclamide (193.1 7.7%, n = 8). On the contrary, cardioprotective effect of diazoxide was not abolished by glimepiride (141.8 7.8%, n = 6), gliclazide (139.0 9.4%, n = 5), and tolbutamide (141.1 4.5%, n = 7). Conclusions Our results indicate that glimepiride, gliclazide, and tolbutamide have no effect on mitoKATP channel, and do not abolish the cardioprotective effects of diazoxide. Therefore, these sulfonylureas, unlike glibenclamide, do not interfere with the cellular pathways that confer cardioprotection. Copyright 2006 John Wiley & Sons, Ltd. [source]

    Sodium channel SCN1A and epilepsy: Mutations and mechanisms

    EPILEPSIA, Issue 9 2010
    Andrew Escayg
    Summary Mutations in a number of genes encoding voltage-gated sodium channels cause a variety of epilepsy syndromes in humans, including genetic (generalized) epilepsy with febrile seizures plus (GEFS+) and Dravet syndrome (DS, severe myoclonic epilepsy of infancy). Most of these mutations are in the SCN1A gene, and all are dominantly inherited. Most of the mutations that cause DS result in loss of function, whereas all of the known mutations that cause GEFS+ are missense, presumably altering channel activity. Family members with the same GEFS+ mutation often display a wide range of seizure types and severities, and at least part of this variability likely results from variation in other genes. Many different biophysical effects of SCN1A -GEFS+ mutations have been observed in heterologous expression systems, consistent with both gain and loss of channel activity. However, results from mouse models suggest that the primary effect of both GEFS+ and DS mutations is to decrease the activity of GABAergic inhibitory neurons. Decreased activity of the inhibitory circuitry is thus likely to be a major factor contributing to seizure generation in patients with GEFS+ and DS, and may be a general consequence of SCN1A mutations. [source]

    NCS-1 differentially regulates growth cone and somata calcium channels in Lymnaea neurons

    Kwokyin Hui
    Abstract Local voltage-gated calcium channels, which regulate intracellular Ca2+ levels by allowing Ca2+ influx, play an important role in guiding and shaping growth cones, and in regulating the outgrowth and branching of neurites. Therefore, elucidating the mechanisms that regulate the biophysical properties of whole-cell calcium currents in the growth cones and somata of growing neurons is important to improving our understanding of neuronal development and regeneration. In this study, taking advantage of the large size of the pedal A (PeA) neurons in Lymnaea stagnalis, we compared the biophysical properties of somata and growth cone whole-cell calcium channel currents using Ba2+ and Ca2+ as current carriers. We found that somata and growth cone currents exhibit similar high-voltage activation properties. However, Ba2+ and Ca2+ currents in growth cones and somata are differentially affected by a dominant-negative peptide containing the C-terminal amino acid sequence of neuronal calcium sensor-1 (NCS-1). The peptide selectively reduces the peak and sustained components of current densities and the slope conductance in growth cones, and shifts the reversal potential of the growth cone currents to more hyperpolarized voltages. In contrast, the peptide had no significant effect on the somata calcium channels. Thus, we conclude that NCS-1 differentially modulates Ca2+ currents in the somata and growth cones of regenerating neurons, and may serve as a key regulator to facilitate the growth cone calcium channel activity. [source]

    The Drosophila cacts2 mutation reduces presynaptic Ca2+ entry and defines an important element in Cav2.1 channel inactivation

    G. T. Macleod
    Abstract Voltage-gated Ca2+ channels in nerve terminals open in response to action potentials and admit Ca2+, the trigger for neurotransmitter release. The cacophony gene encodes the primary presynaptic voltage-gated Ca2+ channel in Drosophila motor-nerve terminals. The cacts2 mutant allele of cacophony is associated with paralysis and reduced neurotransmission at non-permissive temperatures but the basis for the neurotransmission deficit has not been established. The cacts2 mutation occurs in the cytoplasmic carboxyl tail of the ,1 -subunit, not within the pore-forming trans-membrane domains, making it difficult to predict the mutation's impact. We applied a Ca2+ -imaging technique at motor-nerve terminals of mutant larvae to test the hypothesis that the neurotransmission deficit is a result of impaired Ca2+ entry. Presynaptic Ca2+ signals evoked by single and multiple action potentials showed a temperature-dependent reduction. The amplitude of the reduction was sufficient to account for the neurotransmission deficit, indicating that the site of the cacts2 mutation plays a role in Ca2+ channel activity. As the mutation occurs in a motif conserved in mammalian high-voltage-activated Ca2+ channels, we used a heterologous expression system to probe the effect of this mutation on channel function. The mutation was introduced into rat Cav2.1 channels expressed in human embryonic kidney cells. Patch-clamp analysis of mutant channels at the physiological temperature of 37 C showed much faster inactivation rates than for wild-type channels, demonstrating that the integrity of this motif is critical for normal Cav2.1 channel inactivation. [source]

    Characterization of the single-channel properties of NMDA receptors in laminae I and II of the dorsal horn of neonatal rat spinal cord

    G. Mark Green
    Abstract The single-channel properties of native NMDA receptors in laminae I and II of the dorsal horn of the neonatal rat spinal cord were studied using outside-out patch-clamp techniques. These receptors were found to have several features that distinguish them from native NMDA receptors elsewhere in the CNS. Single-channel currents activated by NMDA (100 nm) and glycine (10 m) exhibited five distinct amplitude components with slope-conductance values of 19.9 0.8, 32.9 0.6, 42.2 1.1, 53.0 1.0 and 68.7 1.5 pS. Direct transitions were observed between all conductance levels but transitions between 69-pS openings and 20-, 33- and 42-pS openings were rare. There was no significant difference in the frequency of direct transitions from 42- to 20-pS compared to 20- to 42-pS transitions. The Kb (0 mV) for Mg2+ was 89 m. The Mg2+ unblocking rate constant was similar to other reported values. However, the Mg2+ blocking rate constant was larger than other reported values, suggesting an unusually high sensitivity to Mg2+. The NR2B subunit-selective antagonist, ifenprodil, had no significant effect on overall channel activity but significantly decreased the mean open time of 53-pS openings. These results suggest neonatal laminae I and II NMDA receptors are not simply composed of NR1 and NR2B subunits or NR1 and NR2D subunits. It is possible that these properties are due to an as yet uninvestigated combination of two NR2 subunits with the NR1 subunit or a combination of NR3A, NR2 and NR1 subunits. [source]

    A modulatory role for protein phosphatase 2B (calcineurin) in the regulation of Ca2+ entry

    J. Russell Burley
    Abstract The Ca2+/calmodulin-dependent protein phosphatase 2B (PP2B) also known as calcineurin (CN) has been implicated in the Ca2+ -dependent inactivation of Ca2+ channels in several cell types. To study the role of calcineurin in the regulation of Ca2+ -channel activity, phosphatase expression was altered in NG108-15 cells by transfection of sense and antisense plasmid constructs carrying the catalytic subunit of human PP2B,3. Relative to mock-transfected (wild-type) controls, cells overexpressing calcineurin showed dramatically reduced high-voltage-activated Ca2+ currents which were recoverable by the inclusion of 1 ,m FK506 in the patch pipette. Conversely, in cells with reduced calcineurin expression, high-voltage-activated Ca2+ currents were larger relative to controls. Additionally in these cells, low-voltage-activated currents were significantly reduced. Analysis of high-voltage-activated Ca2+ currents revealed that the kinetics of inactivation were significantly accelerated in cells overexpressing calcineurin. Following the delivery of a train of depolarizing pulses in experiments designed to produce large-scale Ca2+ influx across the cell membrane, Ca2+ -dependent inactivation of high-voltage-activated Ca2+ currents was increased in sense cells, and this increase could be reduced by intracellular application of 1 m m BAPTA or 1 ,m FK506. These data support a role of calcineurin in the negative feedback regulation of Ca2+ entry through voltage-operated Ca2+ channels. [source]

    Involvement of Calmodulin in Glucagon-Like Peptide 1(7-36) Amide-Induced Inhibition of the ATP-Sensitive K+ Channel in Mouse Pancreatic ,-Cells

    W. G. Ding
    The present investigation was designed to examine whether calmodulin is involved in the inhibition of the ATP-sensitive K+ (KATP) channel by glucagon-like peptide 1(7-36) amide (GLP-1) in mouse pancreatic ,-cells. Membrane potential, single channel and whole-cell currents through the KATP channels, and intracellular free Ca2+ concentration ([Ca2+]i) were measured in single mouse pancreatic ,-cells. Whole-cell patch-clamp experiments with amphotericin-perforated patches revealed that membrane conductance at around the resting potential is predominantly supplied by the KATP channels in mouse pancreatic ,-cells. The addition of 20 nM GLP-1 in the presence of 5 mM glucose significantly reduced the membrane KATP conductance, accompanied by membrane depolarization and the generation of electrical activity. A calmodulin inhibitor N -(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide (W-7, 20 ,M) completely reversed the inhibitory actions of GLP-1 on the membrane KATP conductance and resultant membrane depolarization. Cell-attached patch recordings confirmed the inhibition of the KATP channel activity by 20 nM GLP-1 and its restoration by 20 ,M W-7 or 10 ,M calmidazolium at the single channel level. Bath application of 20 ,M W-7 also consistently abolished the GLP-1-evoked increase in [Ca2+]i in the presence of 5 mM glucose. These results strongly suggest that the mechanisms by which GLP-1 inhibits the KATP channel activity accompanied by the initiation of electrical activity in mouse pancreatic ,-cells include a calmodulin-dependent mechanism in addition to the well-documented activation of the cyclic AMP-protein kinase A system. [source]

    Existence of a tightly regulated water channel in Saccharomyces cerevisiae

    FEBS JOURNAL, Issue 2 2001
    Valrie Meyrial
    The Saccharomyces cerevisiae strain ,1278b possesses two putative aquaporins, Aqy1-1p and Aqy2-1p. Previous work demonstrated that Aqy1-1p functions as a water channel in Xenopus oocyte. However, no function could be attributed to Aqy2-1p in this system. Specific antibodies were used to follow the expression of Aqy1-1p and Aqy2-1p in the yeast. Aqy1-1p was never detected whatever the growth phase and culture conditions tested. In contrast, Aqy2-1p was detected only during the exponential growth phase in rich medium containing glucose. Aqy2-1p expression was repressed by hyper-osmotic culture conditions. Both immunocytochemistry and biochemical subcellular fractionation demonstrated that Aqy2-1p is located on the endoplasmic reticulum (ER) as well as on the plasma membrane. In microsomal vesicles enriched in ER, a water channel activity due to Aqy2-1p was detected by stopped-flow analysis. Our results show that the expression of aquaporins is tightly controlled. The physiological relevance of aquaporin-mediated water transport in yeast is discussed. [source]

    RESEARCH ARTICLE: Fungicidal activity of amiodarone is tightly coupled to calcium influx

    FEMS YEAST RESEARCH, Issue 3 2008
    Sabina Muend
    Abstract The antiarrhythmic drug amiodarone has microbicidal activity against fungi, bacteria and protozoa. In Saccharomyces cerevisiae, amiodarone triggers an immediate burst of cytosolic Ca2+, followed by cell death markers. Ca2+ transients are a common response to many forms of environmental insults and toxic compounds, including osmotic and pH shock, endoplasmic reticulum stress, and high levels of mating pheromone. Downstream signaling events involving calmodulin, calcineurin and the transcription factor Crz1 are critical in mediating cell survival in response to stress. In this study we asked whether amiodarone induced Ca2+ influx was beneficial, toxic or a bystander effect unrelated to the fungicidal effect of the drug. We show that downregulation of Ca2+ channel activity in stationary phase cells correlates with increased resistance to amiodarone. In actively growing cells, extracellular Ca2+ modulated the size and shape of the Ca2+ transient and directly influenced amiodarone toxicity. Paradoxically, protection was achieved both by removal of external Ca2+ or by adding high levels of CaCl2 (10 mM) to block the drug induced Ca2+ burst. Our results support a model in which the fungicidal activity of amiodarone is mediated by Ca2+ stress, and highlight the pathway of Ca2+ mediated cell death as a promising target for antifungal drug development. [source]

    Antiepileptic Drugs: How They Work in Headache

    HEADACHE, Issue 2001
    F. Michael Cutrer MD
    Antiepileptic drugs (AEDs) are promising agents for the prevention of migraine and other head pain. Migraine and epilepsy share several clinical features and respond to many of the same pharmacologic agents, suggesting that similar mechanisms may be involved in their pathophysiology. The mechanisms of action of AEDs are not fully understood, and a single drug may have more than one mechanism, both in epilepsy and in migraine. Valproate, topiramate, and gabapentin are likely to affect nociception by modulating gamma-aminobutyric acid- (GABA-) and/or glutamate-mediated neurotransmission. All three AEDs enhance GABA-mediated inhibition. Valproate and gabapentin interfere with GABA metabolism to prevent its ultimate conversion to succinate, and topiramate potentiates GABA-mediated inhibition by facilitating the action of GABA receptors. In addition, topiramate acts directly on non- N -methyl-D-aspartate, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid/kainate glutamate receptors. Valproate, topiramate, and possibly gabapentin inhibit sodium ion channels. All three drugs modulate calcium ion channel activity. Valproate blocks T-type calcium ion channels; topiramate inhibits high-voltage-activated L-type calcium ion channels; and gabapentin binds to the ,2, subunit of L-type calcium ion channels. AEDs may be useful in migraine prevention through such mechanisms as modulating the biochemical phenomena of aura or acting directly on the nociceptive system. Further evaluations of AEDs in migraine models will provide a better understanding of the pathophysiology and prevention of migraine. [source]

    Genotype-dependent sensitivity of hepatitis C virus to inhibitors of the p7 ion channel,

    HEPATOLOGY, Issue 6 2008
    Stephen Griffin
    The hepatitis C virus (HCV) p7 protein plays a critical role during particle formation in cell culture and is required for virus replication in chimpanzees. The discovery that it displayed cation channel activity in vitro led to its classification within the "viroporin" family of virus-coded ion channel proteins, which includes the influenza A virus (IAV) M2 protein. Like M2, p7 was proposed as a potential target for much needed new HCV therapies, and this was supported by our finding that the M2 inhibitor, amantadine, blocked its activity in vitro. Since then, further compounds have been shown to inhibit p7 function but the relationship between inhibitory effects in vitro and efficacy against infectious virus is controversial. Here, we have sought to validate multiple p7 inhibitor compounds using a parallel approach combining the HCV infectious culture system and a rapid throughput in vitro assay for p7 function. We identify a genotype-dependent and subtype-dependent sensitivity of HCV to p7 inhibitors, in which results in cell culture largely mirror the sensitivity of recombinant protein in vitro; thus building separate sensitivity profiles for different p7 sequences. Inhibition of virus entry also occurred, suggesting that p7 may be a virion component. Second site effects on both cellular and viral processes were identified for several compounds in addition to their efficacy against p7 in vitro. Nevertheless, for some compounds antiviral effects were specific to a block of ion channel function. Conclusion: These data validate p7 inhibitors as prototype therapies for chronic HCV disease. (HEPATOLOGY 2008;48:1779-1790.) [source]

    KCa2 channels transiently downregulated during spatial learning and memory in rats

    HIPPOCAMPUS, Issue 3 2010
    Bedel Mpari
    Abstract Small-conductance calcium-activated potassium channels (KCa2) are essential components involved in the modulation of neuronal excitability, underlying learning and memory. Recent evidence suggests that KCa2 channel activity reduces synaptic transmission in a postsynaptic NMDA receptor-dependent manner and is modulated by long-term potentiation. We used radioactive in situ hybridization and apamin binding to investigate the amount of KCa2 subunit mRNA and KCa2 proteins in brain structures involved in learning and memory at different stages of a radial-arm maze task in naive, pseudoconditioned, and conditioned rats. We observed significant differences in KCa2.2 and KCa2.3, but not KCa2.1 mRNA levels, between conditioned and pseudoconditioned rats. KCa2.2 levels were transiently reduced in the dorsal CA fields of the hippocampus, whereas KCa2.3 mRNA levels were reduced in the dorsal and ventral CA fields of the hippocampus, entorhinal cortex, and basolateral amygdaloid nucleus in conditioned rats, during early stages of learning. Levels of apamin-binding sites displayed a similar pattern to KCa2 mRNA levels during learning. Spatial learning performance was positively correlated with levels of apamin-binding sites and KCa2.3 mRNA in the dorsal CA1 field and negatively correlated in the dorsal CA3 field. These findings suggest that KCa2 channels are transiently downregulated in the early stages of learning and that regulation of KCa2 channel levels is involved in the modification of neuronal substrates underlying new information acquisition. 2009 Wiley-Liss, Inc. [source]

    N-terminal CFTR missense variants severely affect the behavior of the CFTR chloride channel,

    HUMAN MUTATION, Issue 5 2008
    G.G. Gen
    Abstract Over 1,500 cystic fibrosis transmembrane conductance regulator (CFTR) gene sequence variations have been identified in patients with cystic fibrosis (CF) and related disorders involving an impaired function of the CFTR chloride channel. However, detailed structure,function analyses have only been established for a few of them. This study aimed evaluating the impact of eight N-terminus CFTR natural missense changes on channel behavior. By site-directed mutagenesis, we generated four CFTR variants in the N-terminal cytoplasmic tail (p.P5L, p.S50P, p.E60K, and p.R75Q) and four in the first transmembrane segment of membrane-spanning domain 1 (p.G85E/V, p.Y89C, and p.E92K). Immunoblot analysis revealed that p.S50P, p.E60K, p.G85E/V, and p.E92K produced only core-glycosylated proteins. Immunofluorescence and whole cell patch-clamp confirmed intracellular retention, thus reflecting a defect of CFTR folding and/or trafficking. In contrast, both p.R75Q and p.Y89C had a glycosylation pattern and a subcellular distribution comparable to the wild-type CFTR, while the percentage of mature p.P5L was considerably reduced, suggesting a major biogenesis flaw on this channel. Nevertheless, whole-cell chloride currents were recorded for all three variants. Single-channel patch-clamp analyses revealed that the channel activity of p.R75Q appeared similar to that of the wild-type CFTR, while both p.P5L and p.Y89C channels displayed abnormal gating. Overall, our results predict a major impact of the CFTR missense variants analyzed, except p.R75Q, on the CF phenotype and highlight the importance of the CFTR N-terminus on channel physiology. Hum Mutat 29(5), 738,749, 2008. 2008 Wiley-Liss, Inc. [source]

    Cholesterol and Kir channels

    IUBMB LIFE, Issue 8 2009
    Irena Levitan
    Abstract To date, most of the major types of Kir channels, Kir2s, Kir3s, Kir4s, and Kir6s, have been found to partition into cholesterol-rich membrane domains and/or to be regulated by changes in the level of membrane cholesterol. Surprisingly, however, in spite of the structural similarities between different Kirs, effects of cholesterol on different types of Kir channels vary from cholesterol-induced decrease in the current density (Kir2 channels) to the loss of channel activity by cholesterol depletion (Kir4 channels) and loss of channel coupling by different mediators (Kir3 and Kir6 channels). Recently, we have gained initial insights into the mechanisms responsible for cholesterol-induced suppression Kir2 channels, but mechanisms underlying cholesterol sensitivity of other Kir channels are mostly unknown. The goal of this review is to present a summary of the current knowledge of the distinct effects of cholesterol on different types of Kir channels in vitro and in vivo. 2009 IUBMB IUBMB Life 61(8): 781,790, 2009 [source]

    Behavior of Nonselective Cation Channels and Large-Conductance Ca2+ -Activated K+ Channels Induced by Dynamic Changes in Membrane Stretch in Cultured Smooth Muscle Cells of Human Coronary Artery

    Stretch-Activated Ion Channels. Introduction: The effects of membrane stretch on ion channels were investigated in cultured smooth muscle cells of human coronary artery. Methods and Results: In the cell-attached configuration, membrane stretch with negative pressure induced two types of stretch-activated (SA) ion channels: a nonselective cation channel and a large-conductance Ca2+ -activated K+ (BKCa) channel. The single-channel conductances of SA cation and BKCa channels were 26 and 203 pS, respectively. To elucidate the mechanism of activation of these SA channels and to minimize mechanical disruption, a sinusoidal change in pipette pressure was applied to the on-cell membrane patch. During dynamic changes in pipette pressure, increases in SA cation channel activity was found to coincide with increases in BKCa channel activity. In the continued presence of cyclic stretch, the activity of SA cation channels gradually diminished. However, after termination of cyclic stretch, BKCa channel activity was greatly enhanced, but the activity of SA cation channels disappeared. Conclusion: This study is the first to demonstrate that the behavior of SA cation and BKCa channels in coronary smooth muscle cells is differentially susceptible to dynamic changes in membrane tension. [source]