Biomedical subjects

B Nilius

Publications and source records attributed to B Nilius.

At least 19 recordsLinked to original sources

PubMed2007

Modulation of synaptic plasticity and Tau phosphorylation by wild-type and mutant presenilin1.

The function of presenilin1 (PS1) in intra-membrane proteolysis is undisputed, as is its role in neurodegeneration in FAD, in contrast to its exact function in normal conditions. In this study, we analyzed synaptic plasticity and its underlying mechanisms biochemically in brain of mice with a neuron-specific deficiency in PS1 (PS1(n-/-)) and compared them to mice that expressed human mutant PS1[A246E] or wild-type PS1. PS1(n-/-) mice displayed a subtle impairment in Schaffer collateral hippocampal long-term potentiation (LTP) as opposed to normal LTP in wild-type PS1 mice, and a facilitated LTP in mutant PS1[A246E] mice. This finding correlated with, respectively, increased and reduced NMDA receptor responses in PS1[A246E] mice and PS1(n-/-) mice in hippocampal slices. Postsynaptically, levels of NR1/NR2B NMDA-receptor subunits and activated alpha-CaMKII were reduced in PS1(n-/-) mice, while increased in PS1[A246E] mice. In addition, PS1(n-/-) mice, displayed reduced paired pulse facilitation, increased synaptic fatigue and lower number of total and docked synaptic vesicles, implying a presynaptic function for wild-type presenilin1, unaffected by the mutation in PS1[A246E] mice. In contrast to the deficiency in PS1, mutant PS1 activated GSK-3beta by decreasing phosphorylation on Ser-9, which correlated with increased phosphorylation of protein tau at Ser-396-Ser-404 (PHF1/AD2 epitope). The synaptic functions of PS1, exerted on presynaptic vesicles and on postsynaptic NMDA-receptor activity, were concluded to be independent of alterations in GSK-3beta activity and phosphorylation of protein tau.

Animals↗

PubMed2007

Insights into TRPM4 function, regulation and physiological role.

In the current review we will summarise data from the recent literature describing molecular and functional properties of TRPM4. Together with TRPM5, these channels are up till now the only molecular candidates for a class of non-selective, Ca(2+)-impermeable cation channels which are activated by elevated Ca2+ levels in the cytosol. Apart from intracellular Ca2+, TRPM4 activation is also dependent on membrane potential. Additionally, channel activity is modulated by ATP, phosphatidylinositol bisphosphate (PiP2), protein kinase C (PKC) phosphorylation and heat. The molecular determinants for channel activation, permeation and modulation are increasingly being clarified, and will be discussed here in detail. The physiological role of Ca(2+)-activated non-selective cation channels is unclear, especially in the absence of gene-specific knock-out mice, but evidence indicates a role as a regulator of membrane potential, and thus the driving force for Ca2+ entry from the extracellular medium.

Animals↗

PubMed2007

TRPM8.

Originally cloned as a prostate-specific protein, TRPM8 is now best known as a cold- and menthol-activated channel implicated in thermosensation. In this chapter we provide a brief review of current knowledge concerning the biophysical properties, gating mechanisms, pharmacology and (patho)physiology of this TRP channel.

Animals↗

PubMed2007

Influence of temperature on taste perception.

Daily experience tells us that temperature has a strong influence on how we taste. Despite the longstanding interest of many specialists in this aspect of taste, we are only starting to understand the molecular mechanisms underlying the temperature dependence of different taste modalities. Recent research has led to the identification of some strong thermosensitive molecules in the taste transduction pathway. The cold activation of the epithelial Na(+) channel and the heat activation of the taste variant of the vanilloid receptor (TRPV1t) may underlie the temperature dependence of salt responses. Heat activation of the transient receptor potential channel TRPM5 explains the enhancement of sweet taste perception by warm temperatures. Current development of methods to study taste cell physiology will help to determine the contribution of other temperature-sensitive events in the taste transduction pathways. Vice versa, the analysis of the thermodynamic properties of these events may assist to unveil the nature of several taste processes.

Animals↗

PubMed2007

Regulation of TRP channels: a voltage-lipid connection.

TRP (transient receptor potential) channels respond to a plethora of stimuli in a fine-tuned manner. We show here that both membrane potential and the level of PI (phosphatidylinositol) phosphates are efficient regulators of TRP channel gating. Recent work has shown that this regulation applies to several members of the TRPV (TRP vanilloid) subfamily (TRPV1 and TRPV5) and the TRPM (TRP melastatin) subfamily (TRPM4/TRPM5/TRPM7/TRPM8), whereas regulation of members of the TRPC subfamily is still disputed. The mechanism whereby PIP(2) (PI 4,5-bisphosphate) acts on TRPM4, a Ca(2+)- and voltage-activated channel, is shown in detail in this paper: (i) PIP(2) may bind directly to the channel, (ii) PIP(2) induces sensitization to activation by Ca(2+), and (iii) PIP(2) shifts the voltage dependence towards negative and physiologically more meaningful potentials. A PIP(2)-binding pocket seems to comprise a part of the TRP domain and especially pleckstrin homology domains in the C-terminus.

Amino Acid Sequence↗

PubMed2006

A novel function of capsaicin-sensitive TRPV1 channels: involvement in cell migration.

Cell migration relies on a tight temporal and spatial regulation of the intracellular Ca2+ concentration ([Ca2+]i). [Ca2+]i in turn depends on Ca2+ influx via channels in the plasma membrane whose molecular nature is still largely unknown for migrating cells. A mechanosensitive component of the Ca2+ influx pathway was suggested. We show here that the capsaicin-sensitive transient receptor potential channel TRPV1, that plays an important role in pain transduction, is one of the Ca2+ influx channels involved in cell migration. Activating TRPV1 channels with capsaicin leads to an acceleration of human hepatoblastoma (HepG2) cells pretreated with hepatocyte growth factor (HGF). The speed rises by up to 50% and the displacement is doubled. Patch clamp experiments revealed the presence of capsaicin and resiniferatoxin (RTX)-sensitive currents. In contrast, HepG2 cells kept in the absence of HGF are not accelerated by capsaicin and express no capsaicin- or RTX-sensitive current. The TRPV1 antagonist capsazepine prevents the stimulation of migration and inhibits capsaicin-sensitive currents. Finally, we compared the contribution of capsaicin-sensitive TRPV1 channels to cell migration with that of mechanosensitive TRPV4 channels that are also expressed in HepG2 cells. A specific TRPV4 agonist, 4alpha-phorbol 12,13-didecanoate, does not increase the displacement. In summary, we assigned a novel role to capsaicin-sensitive TRPV1 channels. They are important Ca2+ influx channels required for cell migration.

Calcium↗

PubMed2006

The effect of imatinib mesylate on the contractility of isolated rabbit myometrial strips.

BACKGROUND: C-kit receptor expressing interstitial cells generate and coordinate the electrical signals that control peristalsis in the gut. However, the function of interstitial cells in the myometrium is not known. METHODS: (1) Sections of rabbit myometrium were subjected to immunohistochemical staining for the c-kit receptor. (2) Spontaneously contracting myometrial strips from New Zealand White rabbits near term were mounted in an organ bath and attached to a tension-recording device. The effect of increased concentrations of the c-kit receptor antagonist imatinib mesylate on these contractions was observed. The main outcome measures were the change in frequency, amplitude and duration of contraction. RESULTS: (1) Multipolar cells expressing c-kit were identified in the fibromuscular septum confirming the presence of interstitial cells in rabbit myometrium. (2) Imatinib decreased the amplitude of contractions by approximately 20% at 100 microM. No effect was seen at lower concentrations. No effect of imatinib on frequency or duration of contractions was observed at any of the concentrations studied. CONCLUSIONS: In isolated rabbit myometrium, acute inhibition of the c-kit receptor by imatinib mesylate affects only the amplitude of spontaneous contractions at concentrations, the equivalent of x10-100 the normal therapeutic concentration.

Animals↗

PubMed2006

Structure-function relationship of the TRP channel superfamily.

Transient receptor potential (TRP) channels are involved in the perception of a wide range of physical and chemical stimuli, including temperature and osmolarity changes, light, pain, touch, taste and pheromones, and in the initiation of cellular responses thereupon. Since the last decade, rapid progress has been made in the identification and characterization of new members of the TRP superfamily. They constitute a large superfamily of cation channels that are expressed in almost all cell types in both invertebrates and vertebrates. This review summarizes and discusses the current knowledge on the TRP protein structure and its impact on the regulation of the channel function.

Animals↗

PubMed2005

Modulation of the Ca2 permeable cation channel TRPV4 by cytochrome P450 epoxygenases in vascular endothelium.

TRPV4 is a broadly expressed Ca2+-permeable cation channel in the vanilloid subfamily of transient receptor potential channels. TRPV4 gates in response to a large variety of stimuli, including cell swelling, warm temperatures, the synthetic phorbol ester 4alpha-phorbol 12,13-didecanoate (4alpha-PDD), and the endogenous lipid arachidonic acid (AA). Activation by cell swelling and AA requires cytochrome P450 (CYP) epoxygenase activity to convert AA to epoxyeicosatrienoic acids (EETs) such as 5,6-EET, 8,9-EET, which both act as direct TRPV4 agonists. To evaluate the role of TRPV4 and its modulation by the CYP pathway in vascular endothelial cells, we performed Ca2+ imaging and patch-clamp measurements on mouse aortic endothelial cells (MAECs) isolated from wild-type and TRPV4(-/-) mice. All TRPV4-activating stimuli induced robust Ca2+ responses in wild-type MAECs but not in MAECs isolated from TRPV4(-/-) mice. Upregulation of CYP2C expression by preincubation with nifedipine enhanced the responses to AA and cell swelling in wild-type MAECs, whereas responses to other stimuli remained unaffected. Conversely, inhibition of CYP2C9 activity with sulfaphenazole abolished the responses to AA and hypotonic solution (HTS). Moreover, suppression of EET hydrolysis using 1-adamantyl-3-cyclo-hexylurea or indomethacin, inhibitors of soluble epoxide hydrolases (sEHs), and cyclooxygenases, respectively, enhanced the TRPV4-dependent responses to AA, HTS, and EETs but not those to 4alpha-PDD or heat. Together, our data establish that CYP-derived EETs modulate the activity of TRPV4 channels in endothelial cells and shows the unraveling of novel modulatory pathways via CYP2C modulation and sEH inhibition.

8,11,14-Eicosatrienoic Acid↗

PubMed2004

Cell volume response to hyposmotic shock and elevated cAMP in bovine trabecular meshwork cells.

PURPOSE: Hyposmolar perfusion of intact trabecular meshwork (TM) induces a decrease in its hydraulic conductivity (Lp). However, exposure to agents that elevate intracellular cAMP in TM cells increases Lp. Since volume of TM cells could directly influence porosity of the TM and hence Lp, this study has investigated changes in volume in response to acute hyposmotic shock (i.e. regulatory volume decrease or RVD) and elevated cAMP in cultured TM cells. METHODS: Bovine trabecular meshwork cells (BTMC), grown on glass coverslips and loaded with the fluorescent dye MQAE, were used to measure rapid changes in cell volume using the principle of dynamic fluorescence quenching. Activation of volume-regulated anion channels (VRAC) was assessed by measuring volume-sensitive Cl(-) currents (I(Cl,swell)) in the whole cell configuration of the patch clamp technique and by determining the swelling-induced enhancement in I(-) permeability using the halide-sensitivity of MQAE. Expressions of ClC (chloride channels of the ClC gene family), P-glycoprotein (Pgp), and cystic fibrosis transmembrane regulator (CFTR) Cl(-) channels were examined by RT-PCR. Elevation of cAMP in response to forskolin was confirmed by determining the phosphorylation of cAMP response element-binding protein and activating transcription factor-1 (CREB, ATF-1), which form the downstream targets of protein kinase A. RESULTS: As a response to hyposmotic shock, there was an acute increase in cell volume but there was no robust RVD. Patch clamp experiments showed activation of a characteristic Cl(-) current in response to cell swelling. This Cl(-) current was inhibited by NPPB (100microM) and fluoxetine (50microM), both of which are known blockers of VRAC. Experiments, which used the halide-sensitivity of MQAE, also indicated a 9-fold increase in I(-) influx upon cell swelling (8.9+/-4.6; n=9), consistent with activation of a VRAC-like Cl(-) current. To examine whether RVD is limited by K(+) conductance, the swollen cells were exposed to gramicidin, which is known to induce cation channel activity. Such a maneuver led to secondary swelling with [Na(+)](o)=140mM but a rapid shrinkage [Na(+)](o)=8mM indicating that the RVD is limited by cationic conductance necessary for K(+) efflux. Exposure to forskolin, which resulted in CREB and ATF-1 phosphorylation, caused a reversible decrease in cell volume (14.5+/-5%; n=20) under isosmotic and hyposmotic conditions. RT-PCR analysis confirmed expression of ClC-2, ClC-5, and Pgp Cl(-) channels in bovine TM cells. However, ClC-3 and CFTR were not expressed. CONCLUSIONS: TM cells respond to acute hyposmotic shock in an osmometric manner, but their RVD is limited by K(+) conductance. The lack of CFTR expression and decrease in cell volume in response to forskolin concomitant with hyposmolarity suggest that elevated cAMP activates a K(+) conductance. Thus, the altered resistance to aqueous outflow in response to hyposmotic perfusion of the TM and elevated cAMP may be attributed to persistent cell swelling and cell shrinkage, respectively.

Animals↗

PubMed2003

Cell swelling, heat, and chemical agonists use distinct pathways for the activation of the cation channel TRPV4.

TRPV4 is a Ca(2+)- and Mg(2+)-permeable cation channel within the vanilloid receptor subgroup of the transient receptor potential (TRP) family, and it has been implicated in Ca(2+)-dependent signal transduction in several tissues, including brain and vascular endothelium. TRPV4-activating stimuli include osmotic cell swelling, heat, phorbol ester compounds, and 5',6'-epoxyeicosatrienoic acid, a cytochrome p450 epoxygenase metabolite of arachidonic acid (AA). It is presently unknown how these distinct activators converge on opening of the channel. Here, we demonstrate that blockers of phospholipase A(2) (PLA(2)) and cytochrome p450 epoxygenase inhibit activation of TRPV4 by osmotic cell swelling but not by heat and 4alpha-phorbol 12,13-didecanoate. Mutating a tyrosine residue (Tyr-555) in the N-terminal part of the third transmembrane domain to an alanine strongly impairs activation of TRPV4 by 4alpha-phorbol 12,13-didecanoate and heat but has no effect on activation by cell swelling or AA. We conclude that TRPV4-activating stimuli promote channel opening by means of distinct pathways. Cell swelling activates TRPV4 by means of the PLA(2)-dependent formation of AA, and its subsequent metabolization to 5',6'-epoxyeicosatrienoic acid by means of a cytochrome p450 epoxygenase-dependent pathway. Phorbol esters and heat operate by means of a distinct, PLA(2)- and cytochrome p450 epoxygenase-independent pathway, which critically depends on an aromatic residue at the N terminus of the third transmembrane domain.

Animals↗

PubMed2003

TRPs make sense.

Drosophila flies with the trp mutation exhibit impaired vision due to the lack of a specific Ca2+ influx pathway in the photoreceptors. The identification of the trp gene product as a Ca2+-permeable ion channel and the search for TRP homologues in flies, worms and mammals has opened the way to the discovery of a whole superfamily of cation channels, baptized TRP channels. In contrast to voltage-gated K+, Na+, or Ca2+ channels, with whom they share their transmembrane architecture, TRP channels are not activated by voltage but by a variety of signals including intra- and extracellular ligands, Ca2+-store depletion and mechanical or thermal stress. Due to the promiscuity of these gating mechanisms, TRP channels are privileged candidates as primary sensing molecules for the recognition and integration of physical and chemical signals from the environment. In this review we discuss recent evidence that implicates members of the TRP superfamily in sensory signal transduction.

Animals↗

PubMed2003

Homo- and heterotetrameric architecture of the epithelial Ca2+ channels TRPV5 and TRPV6.

The molecular assembly of the epithelial Ca(2+) channels (TRPV5 and TRPV6) was investigated to determine the subunit stoichiometry and composition. Immunoblot analysis of Xenopus laevis oocytes expressing TRPV5 and TRPV6 revealed two specific bands of 75 and 85-100 kDa, corresponding to the core and glycosylated proteins, respectively, for each channel. Subsequently, membranes of these oocytes were sedimented on sucrose gradients. Immuno blotting revealed that TRPV5 and TRPV6 complexes migrate with a mol. wt of 400 kDa, in line with a tetrameric structure. The tetrameric stoichiometry was confirmed in an electrophysiological analysis of HEK293 cells co-expressing concatemeric channels together with a TRPV5 pore mutant that reduced Cd(2+) sensitivity and voltage-dependent gating. Immuno precipitations using membrane fractions from oocytes co-expressing TRPV5 and TRPV6 demonstrated that both channels can form heteromeric complexes. Expression of all possible heterotetrameric TRPV5/6 complexes in HEK293 cells resulted in Ca(2+) channels that varied with respect to Ca(2+)-dependent inactivation, Ba(2+) selectivity and pharmacological block. Thus, Ca(2+)-transporting epithelia co-expressing TRPV5 and TRPV6 can generate a pleiotropic set of functional heterotetrameric channels with different Ca(2+) transport kinetics.

Animals↗

PubMed2003

Amazing chloride channels: an overview.

AIM: This review describes molecular and functional properties of the following Cl- channels: the ClC family of voltage-dependent Cl- channels, the cAMP-activated transmembrane conductance regulator (CFTR), Ca2+ activated Cl- channels (CaCC) and volume-regulated anion channels (VRAC). If structural data are available, their relationship with the function of Cl- channels will be discussed. We also describe shortly some recently discovered channels, including high conductance Cl- channels and the family of bestrophins. We illustrate the growing physiological importance of these channels in the plasma membrane and in intracellular membranes, including their involvement in transepithelial transport, pH regulation of intracellular organelles, regulation of excitability and volume regulation. Finally, we discuss the role of Cl- channels in various diseases and describe the pathological phenotypes observed in knockout mice models.

Animals↗

PubMed2002

Block of volume-regulated anion channels by selective serotonin reuptake inhibitors.

We have used the whole-cell patch clamp technique to study the effects of the commonly used antidepressants sertraline, paroxetine, citalopram and fluvoxamine on the volume-regulated anion channel (VRAC) in endothelial cells. It was the purpose of the present experiments to investigate whether VRAC block is a general property of this group of selective serotonin reuptake inhibitors (SSRIs). At pH 7.4, all SSRIs induced a fast and reversible block of the volume-sensitive chloride current ( I(Cl,swell)), with an IC(50) value of 2.1+/-0.5 microM for sertraline, 2.7+/-0.2 microM for paroxetine, 12.3+/-1.4 microM for fluvoxamine and 27.7+/-2.8 microM for citalopram. The block was enhanced at more alkaline pH, indicating that it is mediated by the uncharged form. This study describes the effects of a variety of SSRIs on an anion channel. Our data reveal a potent block and suggest a hydrophobic interaction of high affinity between the uncharged SSRI and volume-regulated anion channels. We conclude that VRAC block is a general property of this pharmacological class of selective serotonin reuptake inhibitors.

Antidepressive Agents↗

PubMed2002

Current understanding of mammalian TRP homologues.

Calcium influx into the cell from the extracellular medium is crucial for important processes including muscle contraction, secretion and gene expression. This calcium influx is mainly mediated through calcium influx channels, which on the basis of their activation mechanism can be subdivided in voltage-gated calcium channels, which have already been thoroughly characterized and non-voltage-gated calcium permeable channels. This latter group includes ion channels activated by binding of extra and intracellular messengers, mechanical stress or depletion of intracellular calcium stores. Currently little molecular data is available concerning this class of calcium influx channels. However, recent studies have indicated that members of the transient receptor potential (TRP) family of ion channels can function as calcium influx channels both in excitable and non-excitable tissues. On the basis of structural information the TRP family is subdivided in three main subfamilies: the TRPC (canonical) group, the TRPV (vanilloid) group and the TRPM (melastatin) group. The cloning and characterization of members of this cation channel family has exploded during recent years, leading to a plethora of data concerning TRPs in a variety of tissues and species, including mammals, insects and yeast. This review summarizes the currently available information concerning members of the TRP family expressed in mammalian tissues.

Animals↗

PubMed2001

Function and expression of the epithelial Ca(2+) channel family: comparison of mammalian ECaC1 and 2.

1. The epithelial Ca(2+) channel (ECaC) family represents a unique group of Ca(2+)-selective channels that share limited homology to the ligand-gated capsaicin receptors, the osmolarity-sensitive channel OTRPC4, as well as the transient receptor potential family. Southern blot analysis demonstrated that this family is restricted to two members, ECaC1 and ECaC2 (also named CaT1). 2. RT-PCR analysis demonstrated that the two channels are co-expressed in calbindin-D-containing epithelia, including small intestine, pancreas and placenta, whereas kidney and brain only express ECaC1 and stomach solely ECaC2. 3. From an electrophysiological point of view, ECaC1 and ECaC2 are highly similar channels. Differences concern divalent cation permeability, the kinetics of Ca(2+)-dependent inactivation and recovery from inactivation. 4. Ruthenium red is a potent blocker of ECaC activity. Interestingly, ECaC2 has a 100-fold lower affinity for ruthenium red (IC(50) 9 +/- 1 microM) than ECaC1 (IC(50) 121 +/- 13 nM). 5. ECaCs are modulated by intracellular Mg(2+) and ATP. ECaC1 and ECaC2 activity rapidly decay in the absence of intracellular ATP. This effect is further accelerated at higher intracellular Mg(2+) concentrations. 6. In conclusion, ECaC1 and ECaC2 are homologous channels, with an almost identical pore region. They can be discriminated by their sensitivity for ruthenium red and show differences in Ca(2+)-dependent regulation.

Adenosine Triphosphate↗

PubMed2001

CaT1 and the calcium release-activated calcium channel manifest distinct pore properties.

The calcium release-activated calcium channel (CRAC) is a highly Ca(2+)-selective ion channel that is activated on depletion of inositol triphosphate (IP(3))-sensitive intracellular Ca(2+) stores. It was recently reported that CaT1, a member of the TRP family of cation channels, exhibits the unique biophysical properties of CRAC, which led to the conclusion that CaT1 comprises all or part of the CRAC pore (Yue, L., Peng, J. B., Hediger, M. A., and Clapham, D. E. (2001) Nature 410, 705-709). Here, we directly compare endogenous CRAC with heterologously expressed CaT1 and show that they manifest several clearly distinct properties. CaT1 can be distinguished from CRAC in the following features: sensitivity to store-depleting agents; inward rectification in the absence of divalent cations; relative permeability to Na(+) and Cs(+); effect of 2-aminoethoxydiphenyl borate (2-APB). Moreover, CaT1 displays a mode of voltage-dependent gating that is fully absent in CRAC and originates from the voltage-dependent binding/unbinding of Mg(2+) inside the channel pore. Our results imply that the pores of CaT1 and CRAC are not identical and indicate that CaT1 is a Mg(2+)-gated channel not directly related to CRAC.

Calcium Channels↗