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Author(s): Karel Talavera (corresponding author) [1]; Keiko Yasumatsu [2]; Thomas Voets [1]; Guy Droogmans [1]; Noriatsu Shigemura [2]; Yuzo Ninomiya [2]; Robert F. Margolskee [3]; Bernd Nilius [1] TRPM5, a [...]

Arachidonic acid (AA) modulates T-type [Ca.sup.2+] channels and is therefore a potential regulator of diverse cell functions, including neuronal and cardiac excitability. The underlying mechanism of modulation is unknown. Here we analyze the effects of AA on the T-type [Ca.sup.2+] channel [[alpha].sub.1G]; heterologously expressed in HEK-293 cells. AA inhibited [[alpha].sub.1G] currents within a few minutes, regardless of preceding exposure to inhibitors of AA metabolism (ETYA and 17-ODYA). Current inhibition was also observed in cell-free inside-out patches, indicating a membrane-delimited interaction of AA with the channel. AA action was consistent with a decrease of the open probability without changes in the size of unitary currents. AA shifted the inactivation curve to more negative potentials, increased the speed of macroscopic inactivation, and decreased the extent of recovery, from inactivation at--80 mV but not at--110 mV. AA induced a slight increase of activation near the threshold and did not significantly change the deactivation kinetics or the rectification pattern. We observed a tonic current inhibition, regardless of whether the channels were held in resting or inactivated states during AA perfusion, suggesting a state-independent interaction with the channel. Model simulations indicate that AA inhibits T-type currents by switching the channels into a nonavailable conformation and by affecting transitions between inactivated states, which results in the negative shift of the inactivation curve. Slow-inactivating [[alpha].sub.1G]; mutants showed an increased 'affinity, for AA with respect to the wild type. indicating that the structural determinants of fast inactivation are involved in the AA-channel interaction. KEY WORDS: Low voltage-activated calcium channel * fatty acid * kinetic model * inactivation * mutant

Author(s): Thomas Voets (corresponding author) [1]; Guy Droogmans [1]; Ulrich Wissenbach [2]; Annelies Janssens [1]; Veit Flockerzi [2]; Bernd Nilius [1] Mammals sense ambient temperature through primary afferent sensory neurons [...]

The vanilloid receptor-1 (VR1, now TRPV1) was the founding member of a subgroup of cation channels within the TRP family. The TRPV subgroup contains six mammalian members, which all function as [Ca.sup.2+] entry channels gated by a variety of physical and chemical stimuli. TRPV4, which displays 45% sequence identity with TRPV1, is characterized by a surprising gating promiscuity: it is activated by hypotonic cell swelling, heat, synthetic 4[alpha]-phorbols, and several endogenous substances including arachidonic acid (AA), the endocannabinoids anandamide and 2-AG, and cytochrome P-450 metabolites of AA, such as epoxyeicosatrienoic acids. This review summarizes data on TRPV4 as a paradigm of gating diversity in this subfamily of [Ca.sup.2+] entry channels. transient receptor potential; calcium channels; vanilloid receptor

Author(s): Hiroyuki Watanabe [1, 2]; Joris Vriens [1, 2]; Jean Prenen [1]; Guy Droogmans [1]; Thomas Voets [1]; Bernd Nilius (corresponding author) [1] TRPV4 is a widely expressed cation channel [...]

TRPV6 (CaT1/ECaC2), a highly [Ca.sup.2+]-selective member of the TRP superfamily of cation channels, becomes permeable to monovalent cations in the absence of extracellular divalent cations. The monovalent currents display characteristic voltage-dependent gating and almost absolute inward rectification. Here, we show that these two features are dependent on the voltage-dependent block/unblock of the channel by intracellular [Mg.sup.2+]. [Mg.sup.2+] blocks the channel by binding to a site within the transmembrane electrical field where it interacts with permeant cations. The block is relieved at positive potentials, indicating that under these conditions [Mg.sup.2+] is able to permeate the selectivity filter of the channel. Although sizeable outward monovalent currents were recorded in the absence of intracellular [Mg.sup.2+], outward conductance is still ~10 times lower than inward conductance under symmetric, divalent-free ionic conditions. This [Mg.sup.2+]-independent rectification was preserved in inside-out patches and not altered by high intracellular concentrations of spermine, indicating that TRPV6 displays intrinsic rectification. Neutralization of a single aspartate residue within the putative pore loop abolished the [Mg.sup.2+] sensitivity of the channel, yielding voltage-independent, moderately inwardly rectifying monovalent currents in the presence of intracellular [Mg.sup.2+]. The effects of intracellular [Mg.sup.2+] on TRPV6 are partially reminiscent of the gating mechanism of inwardly rectifying [K.sup.+] channels and may represent a novel regulatory mechanism for TRPV6 function in vivo. KEY WORD: voltage-dependent gating * pore block * calcium channel * TRP channel * CRAC channel

RhoA exerts a permissive effect on volumeregulated anion channels in vascular endothelial cells. Am J Physiol Cell Physiol 283: Cl15-C125, 2002. First published February 20, 2002; 10.1152/ajpcell.00038.2001.--Cell swelling triggers in most cell types an outwardly rectifying anion current, [I.sub.Cl.swell], via volume-regulated anion channels (VRACs). We have previously demonstrated in calf pulmonary artery endothelial (CPAE) cells that inhibition of the Rho/Rho kinase/myosin light chain phosphorylation pathway reduces the swelling-dependent activation of [I.sub.Cl.swell]. However, these experiments did not allow us to discriminate between a direct activator role or a permissive effect. We now show that the Rho pathway did not affect VRAC activity if this pathway was activated by transfecting CPAE cells with constitutively active isoforms of G[alpha] (a Rho activating heterotrimeric protein subunit), Rho, or Rho kinase. Furthermore, biochemical and morphological analysis failed to demonstrate activation of the Rho pathway during hypotonic cell swelling. Finally, manipulating the Rho pathway with either guanosine 5'-O-(3-thiotriphosphate) or C3 exoenzyme had no effect on VRACs in caveolin-l-expressing Caco-2 cells. We conclude that the Rho pathway exerts a permissive effect on VRACs in CPAE cells, i.e., swelling-induced opening of VRACs requires a functional Rho pathway, but not an activation of the Rho pathway. cytoskeleton; actin; myosin phosphorylation; RhoA pathway

Mechanical stress induces auto/paracrine ATP release from various cell types, but the mechanisms underlying this release are not well understood. Here we show that the release of ATP induced by hypotonic stress (HTS) in bovine aortic endothelial cells (BAECs) occurs through volume-regulated anion channels (VRAC). Various VRAC inhibitors, such as glibenclamide, verapamil, tamoxifen, and fluoxetine, suppressed the HTS-induced release of ATP, as well as the concomitant [Ca.sup.2+] oscillations and NO production. They did not, however, affect [Ca.sup.2+] oscillations and NO production induced by exogenously applied ATP. Extracellular ATP inhibited VRAC currents in a voltage-dependent manner: block was absent at negative potentials and was manifest at positive potentials, but decreased at highly depolarized potentials. This phenomenon could be described with a 'permeating blocker model,' in which ATP binds with an affinity of 1.0 [+ or -] 0.5 mM at 0 mV to a site at an electrical distance of 0.41 inside the channel. Bound ATP occludes the channel at moderate positive potentials, but permeates into the cytosol at more depolarized potentials. The triphosphate nucleotides UTP, GTP, and CTP, and the adenine nucleotide ADP, exerted a similar voltage-dependent inhibition of VRAC currents at submillimolar concentrations, which could also be described with this model. However, inhibition by ADP was less voltage sensitive, whereas adenosine did not affect VRAC currents, suggesting that the negative charges of the nucleotides are essential for their inhibitory action. The observation that high concentrations of extracellular ADP enhanced the outward component of the VRAC current in low [Cl.sup.-] hypotonic solution and shifted its reversal potential to negative potentials provides more direct evidence for the nucleotide permeability of VRAC. We conclude from these observations that VRAC is a nucleotide-permeable channel, which may serve as a pathway for HTS-induced ATP release in BAEC. KEY WORDS: ATP release * nitric oxide * hypotonic stress * volume-regulated anion channel * calcium

The effects of calix[4]arenes on the volume-regulated anion channel currents in cultured calf pulmonary artery endothelial cells were investigated. Results indicated that calix[4] arenes induce a voltage-dependent inhibition of the swelling-activated Cl- current in endothelial cells. This may probably be due to an occlusion of the pore region at moderately positive potentials which is relieved at more positive potentials.

The chloride current activated by cell swelling (IC1,swell), observed in many mammalian and non-mammalian cell types, is involved in several cellular processes.The voltage-dependent properties of IC1,swell in BCsub3H1 myoblasts is described. Extracellular pH, Mgsuper2+, the permeant anion and channel blockers were found to have a profound impact on the voltage-dependent properties.

Research shows that organic osmolytes such as taurine in endothelial cells (EC) are stimulated by hyposmotic swelling. The outgoing movement of organic osmolytes may be facilitated via anion channels. [3H]taurine-loaded monolayers of calf pulmonary artery EC were used to measure taurine efflux. Anion channel blockers such as tamoxifen and 5-nitro-2-(3-phenylpropylamino)benzoic acid inhibits the outgoing movement of taurine and subsequent swelling process.

The BC3H1 and C2C12 cell lines were analyzed by patch-clamp techniques to determine the activation properties of the volume-activated Cl- current and the pharmacological characteristics of the cell lines. The BC3H1 and C2C12 cells display volume-activated Cl- currents with properties that are similar to volume-activated Cl- currents in other cell types. Patch-clamp analysis of BC3H1 and C2C12 cell lines also indicated the presence of a close correlation between volume-activated Cl- currents and cell proliferation.

Analysis of ionic currents induced by hypotonic extracellular solutions using whole-cell and perforated patches indicates that the currents are mainly carried by Cl- and are Ca2+ independent. The cell-swelling induced by extracellular hypotonic solutions (HTS) activates a Cl- current. This current is inhibited by P-glycoproteins encoded by multi-drug resistance gene (MDR), implying a functional connection between HTS-induced current and activation of P-glycoprotein in human endothelial cells.

Analysis of human endothelial cells provides insights into the shifts in tetracellular calcium concentration, (Ca2+)i, caused by mechanical stretch owing to osmotic cell swelling. Arachidonic acid may modulate the mechanosensitivity in endothelial cells.

Experiments performed on human melanoma cells using the nystatin-perforated patch-clamp technique and calcium ion measurements showed increase in Ca ion concentration with reducing voltage. Ca ion concentration varied with voltage with the maximum around the resting potential. The increase in Ca ion concentration leads to a K ion current that causes hyperpolarization of nonclamped cells, enhancing the effect of membrane potential changes on calcium ion concentrations. Increased K ion pathways contribute to enhanced growth of melanoma cells by promoting the G1-S transition.