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Cation Current (cation + current)
Kinds of Cation Current Selected AbstractsDeath and survival of heterozygous Lurcher Purkinje cells In vitroDEVELOPMENTAL NEUROBIOLOGY, Issue 8 2009Hadi S. Zanjani Abstract The differentiation and survival of heterozygous Lurcher (+/Lc) Purkinje cells in vitro was examined as a model system for studying how chronic ionic stress affects neuronal differentiation and survival. The Lurcher mutation in the ,2 glutamate receptor (GluR,2) converts an orphan receptor into a membrane channel that constitutively passes an inward cation current. In the GluR,2+/Lc mutant, Purkinje cell dendritic differentiation is disrupted and the cells degenerate following the first week of postnatal development. To determine if the GluR,2+/Lc Purkinje cell phenotype is recapitulated in vitro, +/+, and +/Lc Purkinje cells from postnatal Day 0 pups were grown in either isolated cell or cerebellar slice cultures. GluR,2+/+ and GluR,2+/Lc Purkinje cells appeared to develop normally through the first 7 days in vitro (DIV), but by 11 DIV GluR,2+/Lc Purkinje cells exhibited a significantly higher cation leak current. By 14 DIV, GluR,2+/Lc Purkinje cell dendrites were stunted and the number of surviving GluR,2+/Lc Purkinje cells was reduced by 75% compared to controls. However, treatment of +/Lc cerebellar cultures with 1-naphthyl acetyl spermine increased +/Lc Purkinje cell survival to wild type levels. These results support the conclusion that the Lurcher mutation in GluR,2 induces cell autonomous defects in differentiation and survival. The establishment of a tissue culture system for studying cell injury and death mechanisms in a relatively simple system like GluR,2+/Lc Purkinje cells will provide a valuable model for studying how the induction of a chronic inward cation current in a single cell type affects neuronal differentiation and survival. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2009 [source] N -methyl- d -aspartate, hyperpolarization-activated cation current (Ih) and ,-aminobutyric acid conductances govern the risk of epileptogenesis following febrile seizures in rat hippocampusEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 7 2010Mohamed Ouardouz Abstract Febrile seizures are the most common types of seizure in children, and are generally considered to be benign. However, febrile seizures in children with dysgenesis have been associated with the development of temporal lobe epilepsy. We have previously shown in a rat model of dysgenesis (cortical freeze lesion) and hyperthermia-induced seizures that 86% of these animals developed recurrent seizures in adulthood. The cellular changes underlying the increased risk of epileptogenesis in this model are not known. Using whole cell patch-clamp recordings from CA1 hippocampal pyramidal cells, we found a more pronounced increase in excitability in rats with both hyperthermic seizures and dysgenesis than in rats with hyperthermic seizures alone or dysgenesis alone. The change was found to be secondary to an increase in N -methyl- d -aspartate (NMDA) receptor-mediated excitatory postsynaptic currents (EPSCs). Inversely, hyperpolarization-activated cation current was more pronounced in naïve rats with hyperthermic seizures than in rats with dysgenesis and hyperthermic seizures or with dysgenesis alone. The increase in GABAA -mediated inhibition observed was comparable in rats with or without dysgenesis after hyperthermic seizures, whereas no changes were observed in rats with dysgenesis alone. Our work indicates that in this two-hit model, changes in NMDA receptor-mediated EPSCs may facilitate epileptogenesis following febrile seizures. Changes in the hyperpolarization-activated cation currents may represent a protective reaction and act by damping the NMDA receptor-mediated hyperexcitability, rather than converting inhibition into excitation. These findings provide a new hypothesis of cellular changes following hyperthermic seizures in predisposed individuals, and may help in the design of therapeutic strategies to prevent epileptogenesis following prolonged febrile seizures. [source] AMPA and metabotropic glutamate receptors cooperatively generate inspiratory-like depolarization in mouse respiratory neurons in vitroEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 12 2008Ryland W. Pace Abstract Excitatory transmission mediated by AMPA receptors is critical for respiratory rhythm generation. However, the role of AMPA receptors has not been fully explored. Here we tested the functional role of AMPA receptors in inspiratory neurons of the neonatal mouse preBötzinger complex (preBötC) using an in vitro slice model that retains active respiratory function. Immediately before and during inspiration, preBötC neurons displayed envelopes of depolarization, dubbed inspiratory drive potentials, that required AMPA receptors but largely depended on the Ca2+ -activated non-specific cation current (ICAN). We showed that AMPA receptor-mediated depolarization opened voltage-gated Ca2+ channels to directly evoke ICAN. Inositol 1,4,5-trisphosphate receptor-mediated intracellular Ca2+ release also evoked ICAN. Inositol 1,4,5-trisphosphate receptors acted downstream of group I metabotropic glutamate receptor activity but, here too, AMPA receptor-mediated Ca2+ influx was essential to trigger the metabotropic glutamate receptor contribution to inspiratory drive potential generation. This study helps to elucidate the role of excitatory transmission in respiratory rhythm generation in vitro. AMPA receptors in preBötC neurons initiate convergent signaling pathways that evoke post-synaptic ICAN, which underlies inspiratory drive potentials. The coupling of AMPA receptors with ICAN suggests that latent burst-generating intrinsic conductances are recruited by excitatory synaptic interactions among preBötC neurons in the context of respiratory network activity in vitro, exemplifying a rhythmogenic mechanism based on emergent properties of the network. [source] Muscarinic control of graded persistent activity in lateral amygdala neuronsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 11 2006Alexei V. Egorov Abstract The cholinergic system is crucially involved in several cognitive processes including attention, learning and memory. Muscarinic actions have profound effects on the intrinsic firing pattern of neurons. In principal neurons of the entorhinal cortex (EC), muscarinic receptors activate an intrinsic cation current that causes multiple self-sustained spiking activity, which represents a potential mechanism for transiently sustaining information about novel items. The amygdala appears to be important for experience-dependent learning by emotional arousal, and cholinergic muscarinic influences are essential for the amygdala-mediated modulation of memory. Here we show that principal neurons from the lateral nucleus of the amygdala (LA) can generate intrinsic graded persistent activity that is similar to EC layer V cells. This firing behavior is linked to muscarinic activation of a calcium-sensitive non-specific cation current and can be mimicked by stimulation of cholinergic afferents that originate from the nucleus basalis of Meynert (n. M). Moreover, we demonstrate that the projections from the n. M. are essential and sufficient for the control and modulation of graded firing activity in LA neurons. We found that activation of these cholinergic afferents (i) is required to maintain and to increase firing rates in a graded manner, and (ii) is sufficient for the graded increases of stable discharge rates even without an associated up-regulation of Ca2+. The induction of persistent activity was blocked by flufenamic acid or 2-APB and remained intact after Ca2+ -store depletion with thapsigargin. The internal ability of LA neurons to generate graded persistent activity could be essential for amygdala-mediated memory operations. [source] Environmental manipulations early in development alter seizure activity, Ih and HCN1 protein expression later in lifeEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 12 2006Ulrich Schridde Abstract Although absence epilepsy has a genetic origin, evidence from an animal model (Wistar Albino Glaxo/Rijswijk; WAG/Rij) suggests that seizures are sensitive to environmental manipulations. Here, we show that manipulations of the early rearing environment (neonatal handling, maternal deprivation) of WAG/Rij rats leads to a pronounced decrease in seizure activity later in life. Recent observations link seizure activity in WAG/Rij rats to the hyperpolarization-activated cation current (Ih) in the somatosensory cortex, the site of seizure generation. Therefore, we investigated whether the alterations in seizure activity between rats reared differently might be correlated with changes in Ih and its channel subunits hyperpolarization-activated cation channel HCN1, 2 and 4. Whole-cell recordings from layer 5 pyramidal neurons, in situ hybridization and Western blot of the somatosensory cortex revealed an increase in Ih and HCN1 in neonatal handled and maternal deprived, compared to control rats. The increase was specific to HCN1 protein expression and did not involve HCN2/4 protein expression, or mRNA expression of any of the subunits (HCN1, 2, 4). Our findings provide the first evidence that relatively mild changes in the neonatal environment have a long-term impact of absence seizures, Ih and HCN1, and suggest that an increase of Ih and HCN1 is associated with absence seizure reduction. Our findings shed new light on the role of Ih and HCN in brain functioning and development and demonstrate that genetically determined absence seizures are quite sensitive for early interventions. [source] Calcium-dependent K current in plasma membranes of dermal cells of developing bean cotyledonsPLANT CELL & ENVIRONMENT, Issue 2 2004W.-H. ZHANG ABSTRACT In developing seeds of bean (Phaseolus vulgaris L.), phloem-imported assimilates (largely sucrose and potassium) are released from coats to seed apoplasm and subsequently retrieved by the dermal cell complexes of cotyledons. To investigate the mechanisms of K+ uptake by the cotyledons, protoplasts of dermal cell complexes were isolated and whole-cell currents across their plasma membranes were measured with the patch-clamp technique. A weakly rectified cation current displaying a voltage-dependent blockade by external Ca2+ and acidic pH, dominated the conductance of the protoplasts. The P haseolus v ulgaris Cotyledon Dermal-cell pH and Calcium-dependent Cation Conductance (Pv-CD-pHCaCC) was highly selective for K+ over Ca2+ and Cl,. For K+ current through Pv-CD-pHCaCC a sigmoid shaped current,voltage (I,V) curve was observed with negative conductance at voltages between ,200 and ,140 mV. This negative K+ conductance was Ca2+ dependent. With other univalent cations (Na+, Rb+, NH4+) the currents were smaller and were not Ca2+ dependent. Reversal potentials remained constant when external K+ was substituted with these cations, suggesting that Pv-CD-pHCaCC channels were non-selective. The Pv-CD-pHCaCC would provide a pathway for K+ and other univalent cation influx into developing cotyledons. These cation influxes could be co-ordinated with sucrose influx via pH and Ca2+dependence. [source] Functional expression of the hyperpolarization-activated, non-selective cation current If in immortalized HL-1 cardiomyocytesTHE JOURNAL OF PHYSIOLOGY, Issue 1 2002Laura Sartiani HL-1 cells are adult mouse atrial myocytes induced to proliferate indefinitely by SV40 large T antigen. These cells beat spontaneously when confluent and express several adult cardiac cell markers including the outward delayed rectifier K+ channel. Here, we examined the presence of a hyperpolarization-activated If current in HL-1 cells using the whole-cell patch-clamp technique on isolated cells enzymatically dissociated from the culture at confluence. Cell membrane capacitance (Cm) ranged from 5 to 53 pF. If was detected in about 30 % of the cells and its occurrence was independent of the stage of the culture. If maximal slope conductance was 89.7 ± 0.4 pS pF,1 (n= 10). If current in HL-1 cells showed typical characteristics of native cardiac If current: activation threshold between ,50 and ,60 mV, half-maximal activation potential of ,83.1 ± 0.7 mV (n= 50), reversal potential at ,20.8 ± 1.5 mV (n= 10), time-dependent activation by hyperpolarization and blockade by 4 mm Cs+. In half of the cells tested, activation of adenylyl cyclase by the forskolin analogue L858051 (20 ,m) induced both a ,6 mV positive shift of the half-activation potential and a ,37 % increase in the fully activated If current. RT-PCR analysis of the hyperpolarization-activated, cyclic nucleotide-gated channels (HCN) expressed in HL-1 cells demonstrated major contributions of HCN1 and HCN2 channel isoforms to If current. Cytosolic Ca2+ oscillations in spontaneously beating HL-1 cells were measured in Fluo-3 AM-loaded cells using a fast-scanning confocal microscope. The oscillation frequency ranged from 1.3 to 5 Hz and the spontaneous activity was stopped in the presence of 4 mm Cs+. Action potentials from HL-1 cells had a triangular shape, with an overshoot at +15 mV and a maximal diastolic potential of ,69 mV, i.e. more negative than the threshold potential for If activation. In conclusion, HL-1 cells display a hyperpolarization-activated If current which might contribute to the spontaneous contractile activity of these cells. [source] A modelling study of locomotion-induced hyperpolarization of voltage threshold in cat lumbar motoneuronesTHE JOURNAL OF PHYSIOLOGY, Issue 2 2002Yue Dai During fictive locomotion the excitability of adult cat lumbar motoneurones is increased by a reduction (a mean hyperpolarization of ,6.0 mV) of voltage threshold (Vth) for action potential (AP) initiation that is accompanied by only small changes in AP height and width. Further examination of the experimental data in the present study confirms that Vth lowering is present to a similar degree in both the hyperpolarized and depolarized portions of the locomotor step cycle. This indicates that Vth reduction is a modulation of motoneurone membrane currents throughout the locomotor state rather than being related to the phasic synaptic input within the locomotor cycle. Potential ionic mechanisms of this locomotor-state-dependent increase in excitability were examined using three five-compartment models of the motoneurone innervating slow, fast fatigue resistant and fast fatigable muscle fibres. Passive and active membrane conductances were set to produce input resistance, rheobase, afterhyperpolarization (AHP) and membrane time constant values similar to those measured in adult cat motoneurones in non-locomoting conditions. The parameters of 10 membrane conductances were then individually altered in an attempt to replicate the hyperpolarization of Vth that occurs in decerebrate cats during fictive locomotion. The goal was to find conductance changes that could produce a greater than 3 mV hyperpolarization of Vth with only small changes in AP height (< 3 mV) and width (< 1.2 ms). Vth reduction without large changes in AP shape could be produced either by increasing fast sodium current or by reducing delayed rectifier potassium current. The most effective Vth reductions were achieved by either increasing the conductance of fast sodium channels or by hyperpolarizing the voltage dependency of their activation. These changes were particularly effective when localized to the initial segment. Reducing the conductance of delayed rectifier channels or depolarizing their activation produced similar but smaller changes in Vth. Changes in current underlying the AHP, the persistent Na+ current, three Ca2+ currents, the ,h' mixed cation current, the ,A' potassium current and the leak current were either ineffective in reducing Vth or also produced gross changes in the AP. It is suggested that the increased excitability of motoneurones during locomotion could be readily accomplished by hyperpolarizing the voltage dependency of fast sodium channels in the axon hillock by a hitherto unknown neuromodulatory action. [source] Genistein potentiates activity of the cation channel TRPC5 independently of tyrosine kinasesBRITISH JOURNAL OF PHARMACOLOGY, Issue 7 2010Ching-On Wong Background and purpose:, TRPC5 is a Ca2+ -permeable channel with multiple modes of activation. We have explored the effects of genistein, a plant-derived isoflavone, on TRPC5 activity, and the mechanism(s) involved. Experimental approach:, Effects of genistein on TRPC5 channels were investigated in TRPC5-over-expressing human embryonic kidney 293 (HEK) cells and bovine aortic endothelial cells (BAECs) using fluorescent Ca2+ imaging and electrophysiological techniques. Key results:, In TRPC5-over-expressing HEK cells, genistein stimulated TRPC5-mediated Ca2+ influx, concentration dependently (EC50= 93 µM). Genistein and lanthanum activated TRPC5 channels synergistically. Effects of genistein on TRPC5 channels were mimicked by daidzein (100 µM), a genistein analogue inactive as a tyrosine kinase inhibitor, but not by known tyrosine kinase inhibitors herbimycin (2 µM), PP2 (20 µM) and lavendustin A (10 µM). Action of genistein on TRPC5 channels was not affected by an oestrogen receptor inhibitor ICI-182780 (50 µM) or a phospholipase C inhibitor U73122 (10 µM), suggesting genistein did not act through oestrogen receptors or phospholipase C. In BAECs, genistein (100 µM) stimulated TRPC5-mediated Ca2+ influx. In patch clamp studies, both genistein (50 µM) and daidzein (50 µM) augmented TRPC5-mediated whole-cell cation current in TRPC5 over-expressing HEK cells. Genistein stimulated TRPC5 channel activity in excised inside-out membrane patch, suggesting that its action was relatively direct and did not require cytosolic factors. Conclusions and implications:, The present study is the first to demonstrate stimulation of a TRP channel by isoflavones. Genistein is a lipophilic compound able to stimulate TRPC5 activity in TRPC5-over-expressing HEK cells and in native vascular endothelial cells. [source] N -methyl- d -aspartate, hyperpolarization-activated cation current (Ih) and ,-aminobutyric acid conductances govern the risk of epileptogenesis following febrile seizures in rat hippocampusEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 7 2010Mohamed Ouardouz Abstract Febrile seizures are the most common types of seizure in children, and are generally considered to be benign. However, febrile seizures in children with dysgenesis have been associated with the development of temporal lobe epilepsy. We have previously shown in a rat model of dysgenesis (cortical freeze lesion) and hyperthermia-induced seizures that 86% of these animals developed recurrent seizures in adulthood. The cellular changes underlying the increased risk of epileptogenesis in this model are not known. Using whole cell patch-clamp recordings from CA1 hippocampal pyramidal cells, we found a more pronounced increase in excitability in rats with both hyperthermic seizures and dysgenesis than in rats with hyperthermic seizures alone or dysgenesis alone. The change was found to be secondary to an increase in N -methyl- d -aspartate (NMDA) receptor-mediated excitatory postsynaptic currents (EPSCs). Inversely, hyperpolarization-activated cation current was more pronounced in naïve rats with hyperthermic seizures than in rats with dysgenesis and hyperthermic seizures or with dysgenesis alone. The increase in GABAA -mediated inhibition observed was comparable in rats with or without dysgenesis after hyperthermic seizures, whereas no changes were observed in rats with dysgenesis alone. Our work indicates that in this two-hit model, changes in NMDA receptor-mediated EPSCs may facilitate epileptogenesis following febrile seizures. Changes in the hyperpolarization-activated cation currents may represent a protective reaction and act by damping the NMDA receptor-mediated hyperexcitability, rather than converting inhibition into excitation. These findings provide a new hypothesis of cellular changes following hyperthermic seizures in predisposed individuals, and may help in the design of therapeutic strategies to prevent epileptogenesis following prolonged febrile seizures. [source] Cellular expression and functional characterization of four hyperpolarization-activated pacemaker channels in cardiac and neuronal tissuesFEBS JOURNAL, Issue 6 2001Sven Moosmang Hyperpolarization-activated cation currents (Ih) have been identified in cardiac pacemaker cells and a variety of central and peripheral neurons. Four members of a gene family encoding hyperpolarization-activated, cyclic nucleotide-gated cation channels (HCN1,4) have been cloned recently. Native Ih currents recorded from different cell types exhibit distinct activation kinetics. To determine if this diversity of Ih currents may be caused by differential expression of HCN channel isoforms, we investigated the cellular distribution of the transcripts of HCN1,4 in the murine sinoatrial node, retina and dorsal root ganglion (DRG) by in situ hybridization. In the sinoatrial node, the most prominently expressed HCN channel is HCN4, whereas HCN2 and HCN1 are detected there at moderate and low levels, respectively. Retinal photoreceptors express high levels of HCN1, whereas HCN2, 3 and 4 were not found in these cells. In DRG neurons, the dominant HCN transcript is HCN1, followed by HCN2. We next determined the functional properties of recombinant HCN1,4 channels expressed in HEK293 cells. All four channel types gave rise to Ih currents but displayed marked differences in their activation kinetics. Our results suggest that the heterogeneity of native Ih currents is generated, at least in part, by the tissue-specific expression of HCN channel genes. [source] Interleukin-1, Release in the Supraoptic Nucleus Area During Osmotic Stimulation Requires Neural FunctionJOURNAL OF NEUROENDOCRINOLOGY, Issue 11 2008J. Y. Summy-Long Interleukin (IL)-1, is present throughout the magnocellular neuroendocrine system and co-depletes with oxytocin and vasopressin from the neural lobe during salt-loading. To examine whether IL-1, is released from the dendrites/soma of magnocellular neurones during osmotic stimulation, microdialysis adjacent to the supraoptic nucleus (SON) in conscious rats was combined with immunocapillary electrophoresis and laser-induced fluorescence detection to quantify cytokine in 5-min dialysates collected before (0,180 min; basal), and after (180,240 min), hypertonic saline injected s.c. (1.5 m NaCl). Osmotic release of IL-1, was compared after inhibiting local voltage-gated channels for Na+ (tetrodotoxin) and Ca2+ (cadmium and nickel) or by reducing intracellular Ca2+ stores (thapsigargin). Immunohistochemistry combined with microdialysis was used to localise cytokine sources (IL-1,+) and microglia (OX-42+). Under conditions of microdialysis, the basal release of IL-1,+ in the SON area was measurable and stable (pg/ml; mean ± SEM) from 0,60 min (2.2 ± 0.06), 60,120 min (2.32 ± 0.05) and 120,180 min (2.33 ± 0.06), likely originating locally from activated microglia (OX42+; IL-1,+; ameboid, hypertrophied) and magnocellular neurones expressing IL-1,. In response to osmotic stimulation, IL-1, increased progressively in dialysates of the SON area by a mechanism dependent on intracellular Ca2+ stores sensitive to thapsigargin and, similar to dendritic secretion of oxytocin and vasopressin, required local voltage-gated Na+ and Ca2+ channels for activation by osmoregulatory pathways from the forebrain. During osmotic stimulation, neurally dependent release of IL-1, in the SON area likely upregulates osmosensitive cation currents on magnocellular neurones (observed in vitro by others), to facilitate dendritic release of neurohypophysial hormones. [source] Electrophysiologically "complex" glial cells freshly isolated from the hippocampus are immunopositive for the chondroitin sulfate proteoglycan NG2JOURNAL OF NEUROSCIENCE RESEARCH, Issue 6 2003Gary P. Schools Abstract We have recently described a subgroup of isolated glial fibrillary acidic protein-positive (GFAP+) hippocampal astrocytes that predominantly express outwardly rectifying currents (which we term "ORAs" for outwardly rectifying astrocytes), which are similar to the currents already described for hippocampal GFAP, "complex glia." We now report that post-recording staining of cells that were first selected as "complex" by morphology and then confirmed by their electrophysiological characteristics were NG2+ ,90% of the time. Also, the morphology of freshly isolated NG2+ cells differs from that of isolated GFAP+ ORAs in having a smaller and round cell body with thinner processes, which usually are collapsed back onto the soma. Upon detailed examination, NG2+ cells were found to differ quantitatively in some electrophysiological characteristics from GFAP+ ORAs. The outward, transient K+ currents (IKa) in the NG2+ cells showed a slower decay than the IKa in ORAs, and their density decreased in NG2+ cells from older animals. The other two major cation currents, the voltage-activated Na+ and outwardly delayed rectifier K+ currents, were similar in NG2+ cells and ORAs. To further distinguish isolated complex cells from outwardly rectifying GFAP+ astrocytes, we performed immunocytochemistry for glial markers in fixed, freshly isolated rat hippocampal glia. NG2+ cells were negative for GFAP and also for the astrocytic glutamate transporters GLT-1 and GLAST. Thus the isolated hippocampal NG2+ glial cells, though having an electrophysiological phenotype similar to that of ORAs, are an immunologically and morphologically distinct glial cell population and most likely represent NG2+ cells in situ. © 2003 Wiley-Liss, Inc. [source] Immunohistochemical localization of Ih channel subunits, HCN1,4, in the rat brainTHE JOURNAL OF COMPARATIVE NEUROLOGY, Issue 3 2004Takuya Notomi Abstract Hyperpolarization-activated cation currents (Ih) contribute to various physiological properties and functions in the brain, including neuronal pacemaker activity, setting of resting membrane potential, and dendritic integration of synaptic input. Four subunits of the Hyperpolarization-activated and Cyclic-Nucleotide-gated nonselective cation channels (HCN1,4), which generate Ih, have been cloned recently. To better understand the functional diversity of Ih in the brain, we examined precise immunohistochemical localization of four HCNs in the rat brain. Immunoreactivity for HCN1 showed predominantly cortical distribution, being intense in the neocortex, hippocampus, superior colliculus, and cerebellum, whereas those for HCN3 and HCN4 exhibited subcortical distribution mainly concentrated in the hypothalamus and thalamus, respectively. Immunoreactivity for HCN2 had a widespread distribution throughout the brain. Double immunofluorescence revealed colocalization of immunoreactivity for HCN1 and HCN2 in distal dendrites of pyramidal cells in the hippocampus and neocortex. At the electron microscopic level, immunogold particles for HCN1 and HCN2 had similar distribution patterns along plasma membrane of dendritic shafts in layer I of the neocortex and stratum lacunosum moleculare of the hippocampal CA1 area, suggesting that these subunits could form heteromeric channels. Our results further indicate that HCNs are localized not only in somato-dendritic compartments but also in axonal compartments of neurons. Immunoreactivity for HCNs often occurred in preterminal rather than terminal portions of axons and in specific populations of myelinated axons. We also found HCN2-immunopositive oligodendrocytes including perineuronal oligodendrocytes throughout the brain. These results support previous electrophysiological findings and further suggest unexpected roles of Ih channels in the brain. J. Comp. Neurol. 471:241,276, 2004. © 2004 Wiley-Liss, Inc. [source] TRPC channels function independently of STIM1 and Orai1THE JOURNAL OF PHYSIOLOGY, Issue 10 2009Wayne I. DeHaven Recent studies have defined roles for STIM1 and Orai1 as calcium sensor and calcium channel, respectively, for Ca2+ -release activated Ca2+ (CRAC) channels, channels underlying store-operated Ca2+ entry (SOCE). In addition, these proteins have been suggested to function in signalling and constructing other channels with biophysical properties distinct from the CRAC channels. Using the human kidney cell line, HEK293, we examined the hypothesis that STIM1 can interact with and regulate members of a family of non-selective cation channels (TRPC) which have been suggested to also function in SOCE pathways under certain conditions. Our data reveal no role for either STIM1 or Orai1 in signalling of TRPC channels. Specifically, Ca2+ entry seen after carbachol treatment in cells transiently expressing TRPC1, TRPC3, TRPC5 or TRPC6 was not enhanced by the co-expression of STIM1. Further, knockdown of STIM1 in cells expressing TRPC5 did not reduce TRPC5 activity, in contrast to one published report. We previously reported in stable TRPC7 cells a Ca2+ entry which was dependent on TRPC7 and appeared store-operated. However, we show here that this TRPC7-mediated entry was also not dependent on either STIM1 or Orai1, as determined by RNA interference (RNAi) and expression of a constitutively active mutant of STIM1. Further, we determined that this entry was not actually store-operated, but instead TRPC7 activity which appears to be regulated by SERCA. Importantly, endogenous TRPC activity was also not regulated by STIM1. In vascular smooth muscle cells, arginine-vasopressin (AVP) activated non-selective cation currents associated with TRPC6 activity were not affected by RNAi knockdown of STIM1, while SOCE was largely inhibited. Finally, disruption of lipid rafts significantly attenuated TRPC3 activity, while having no effect on STIM1 localization or the development of ICRAC. Also, STIM1 punctae were found to localize in regions distinct from lipid rafts. This suggests that TRPC signalling and STIM1/Orai1 signalling occur in distinct plasma membrane domains. Thus, TRPC channels appear to be activated by mechanisms dependent on phospholipase C which do not involve the Ca2+ sensor, STIM1. [source] Characteristics and physiological role of hyperpolarization activated currents in mouse cold thermoreceptorsTHE JOURNAL OF PHYSIOLOGY, Issue 9 2009Patricio Orio Hyperpolarization-activated currents (Ih) are mediated by the expression of combinations of hyperpolarization-activated, cyclic nucleotide-gated (HCN) channel subunits (HCN1,4). These cation currents are key regulators of cellular excitability in the heart and many neurons in the nervous system. Subunit composition determines the gating properties and cAMP sensitivity of native Ih currents. We investigated the functional properties of Ih in adult mouse cold thermoreceptor neurons from the trigeminal ganglion, identified by their high sensitivity to moderate cooling and responsiveness to menthol. All cultured cold-sensitive (CS) neurons expressed a fast activating Ih, which was fully blocked by extracellular Cs+ or ZD7288 and had biophysical properties consistent with those of heteromeric HCN1,HCN2 channels. In CS neurons from HCN1(,/,) animals, Ih was greatly reduced but not abolished. We find that Ih activity is not essential for the transduction of cold stimuli in CS neurons. Nevertheless, Ih has the potential to shape the excitability of CS neurons. First, Ih blockade caused a membrane hyperpolarization in CS neurons of about 5 mV. Furthermore, impedance power analysis showed that all CS neurons had a prominent subthreshold membrane resonance in the 5,7 Hz range, completely abolished upon blockade of Ih and absent in HCN1 null mice. This frequency range matches the spontaneous firing frequency of cold thermoreceptor terminals in vivo. Behavioural responses to cooling were reduced in HCN1 null mice and after peripheral pharmacological blockade of Ih with ZD7288, suggesting that Ih plays an important role in peripheral sensitivity to cold. [source] Antagonist effect of flufenamic acid on TRPM2 cation channels activated by hydrogen peroxideCELL BIOCHEMISTRY AND FUNCTION, Issue 4 2007Mustafa Naz Abstract The melastatin-related transient receptor potential channel TRPM2 is a plasma membrane Ca2+ -permeable cation channel that is activated by hydrogen peroxide (H2O2) as a consequence of oxidative stress although the channel activation by H2O2 appears to represent a cell-specific process in cells with endogenous expression of TRPM2. Flufenamic acid (FA) is a non-steroidal anti-inflammatory compound. Whether H2O2 activates or FA inhibits TRPM2 channels in Chinese hamster ovary (CHO) cell is currently unknown. Due to lack of known antogonists of this channel, we demonstrate in CHO cells that FA inhibits TRPM2 activated by extracellular H2O2. CHO cells were transfected with cDNA coding for TRPM2. Cells were studied with the conventional whole-cell patch clamp technique. The intracellular solution used EDTA (10,mM) as chelator for Ca2+ and heavy metal ions. H2O2 (10,mM) and FA (0.1,mM) were applied extracellularly. Non-selective cation currents were consistently induced by H2O2. The time cause of H2O2 effects was characterized by a delay of 2,5,min and a slow current induction to reach a plateau. The H2O2 - induced inward current was effectively inhibited by 0.1,mM FA applied extracellularly. In conclusion, we have demonstrated that FA is an effective antogonist of TRPM2 channels and H2O2activated currents in CHO cells. FA in CHO cells may be considered, at best, a starting point for the development of TRPM2 channel blockers. Copyright © 2006 John Wiley & Sons, Ltd. [source] | |||||||||||||