Novel pyrazole compounds for pharmacological discrimination between receptor-operated and store-operated Ca(2+) entry pathways.

BACKGROUND AND PURPOSE: Pyrazole derivatives have recently been suggested as selective blockers of transient receptor potential cation (TRPC) channels but their ability to distinguish between the TRPC and Orai pore complexes is ill-defined. This study was designed to characterize a series of pyrazole derivatives in terms of TRPC/Orai selectivity and to delineate consequences of selective suppression of these pathways for mast cell activation. EXPERIMENTAL APPROACH: Pyrazoles were generated by microwave-assisted synthesis and tested for effects on Ca(2+) entry by Fura-2 imaging and membrane currents by patch-clamp recording. Experiments were performed in HEK293 cells overexpressing TRPC3 and in RBL-2H3 mast cells, which express classical store-operated Ca(2+) entry mediated by Orai channels. The consequences of inhibitory effects on Ca(2+) signalling in RBL-2H3 cells were investigated at the level of both degranulation and nuclear factor of activated T-cells activation. KEY RESULTS: Pyr3, a previously suggested selective inhibitor of TRPC3, inhibited Orai1- and TRPC3-mediated Ca(2+) entry and currents as well as mast cell activation with similar potency. By contrast, Pyr6 exhibited a 37-fold higher potency to inhibit Orai1-mediated Ca(2+) entry as compared with TRPC3-mediated Ca(2+) entry and potently suppressed mast cell activation. The novel pyrazole Pyr10 displayed substantial selectivity for TRPC3-mediated responses (18-fold) and the selective block of TRPC3 channels by Pyr10 barely affected mast cell activation. CONCLUSIONS AND IMPLICATIONS: The pyrazole derivatives Pyr6 and Pyr10 are able to distinguish between TRPC and Orai-mediated Ca(2+) entry and may serve as useful tools for the analysis of cellular functions of the underlying Ca(2+) channels.

TRPC3: a multifunctional, pore-forming signalling molecule.

TRPC3 represents one of the first identified mammalian relatives of the Drosophila trp gene product. Despite intensive biochemical and biophysical characterization as well as numerous attempts to uncover its physiological role in native cell systems, this channel protein still represents one of the most enigmatic members of the transient receptor potential (TRP) superfamily. TRPC3 is significantly expressed in brain and heart and likely to play a role in both non-excitable as well as excitable cells, being potentially involved in a wide spectrum of Ca2+ signalling mechanisms. Its ability to associate with a variety of partner proteins apparently enables TRPC3 to form different cation channels in native cells. TRPC3 cation channels display unique gating and regulatory properties that allow for recognition and integration of multiple input stimuli including lipid mediators and cellular Ca2+ gradients as well as redox signals. The physiological/pathophysiological functions of this highly versatile cation channel protein are as yet barely delineated. Here we summarize current knowledge on properties and possible signalling functions of TRPC3 and discuss the potential biological relevance of this signalling molecule.

Phospholipase C-dependent control of cardiac calcium homeostasis involves a TRPC3-NCX1 signaling complex.

OBJECTIVE: Members of the classical transient receptor potential protein (TRPC) family are considered as key components of phospholipase C (PLC)-dependent Ca2+ signaling. Previous results obtained in the HEK 293 expression system suggested a physical and functional coupling of TRPC3 to the cardiac-type Na+/Ca2+ exchanger, NCX1 (sodium calcium exchanger 1). This study was designed to test for expression of TRPC3 (transient receptor potential channel 3) and for the existence of a native TRPC3/NCX1 signaling complex in rat cardiac myocytes. METHODS: Protein expression and cellular distribution were determined by Western blot and immunocytochemistry. Protein-protein interactions were investigated by reciprocal co-immunoprecipitation and glutathione S-transferase (GST)-pulldown experiments. Recruitment of protein complexes into the plasma membrane was assayed by surface biotinylation. The functional role of TRPC3 was investigated by fluorimetric recording of angiotensin II-induced calcium signals employing a dominant negative knockdown strategy. RESULTS: TRPC3 immunoreactivity was observed in surface plasma membrane regions and in an intracellular membrane system. Co-immunolabeling of TRPC3 and NCX1 indicated significant co-localization of the two proteins. Both co-immunoprecipitation and GST-pulldown experiments demonstrated association of TRPC3 with NCX1. PLC stimulation was found to trigger NCX-mediated Ca2+ entry, which was dependent on TRPC3-mediated Na+ loading of myocytes. This NCX-mediated Ca2+ signaling was significantly suppressed by expression of a dominant negative fragment of TRPC3. PLC stimulation was associated with increased membrane presentation of both TRPC3 and NCX1. CONCLUSION: These results suggest a PLC-dependent recruitment of a TRPC3-NCX1 complex into the plasma membrane as a pivotal mechanism for the control of cardiac Ca2+ homeostasis.

Distinct Ca(2+)-permeable cation currents are activated by internal Ca(2+)-store depletion in RBL-2H3 cells and human salivary gland cells, HSG and HSY.

Store-operated Ca(2+) influx, suggested to be mediated via store-operated cation channel (SOC), is present in all cells. The molecular basis of SOC, and possible heterogeneity of these channels, are still a matter of controversy. Here we have compared the properties of SOC currents ( I(SOC)) in human submandibular glands cells (HSG) and human parotid gland cells (HSY) with I(CRAC) (Ca(2+) release-activated Ca(2+) current) in RBL cells. Internal Ca(2+) store-depletion with IP(3) or thapsigargin activated cation channels in all three cell types. 1 muM Gd(3+) blocked channel activity in all cells. Washout of Gd(3+) induced partial recovery in HSY and HSG but not RBL cells. 2-APB reversibly inhibited the channels in all cells. I(CRAC )in RBL cells displayed strong inward rectification with E(rev)(Ca) = >+90 mV and E(rev) (Na) = +60 mV. I(SOC) in HSG cells showed weaker rectification with E(rev)(Ca) = +25 mV and E(rev)(Na) = +10 mV. HSY cells displayed a linear current with E(rev) = +5 mV, which was similar in Ca(2+)- or Na(+)-containing medium. pCa/ pNa was >500, 40, and 4.6 while pCs / pNa was 0.1,1, and 1.3, for RBL, HSG, and HSY cells, respectively. Evidence for anomalous mole fraction behavior of Ca(2+)/Na(+) permeation was obtained with RBL and HSG cells but not HSY cells. Additionally, channel inactivation with Ca(2+) + Na(+) or Na(+) in the bath was different in the three cell types. In aggregate, these data demonstrate that distinct store-dependent cation currents are stimulated in RBL, HSG, and HSY cells. Importantly, these data suggest a molecular heterogeneity, and possibly cell-specific differences in the function, of these channels.

Extracellular pH affects platelet aggregation associated with modulation of store-operated Ca(2+) entry.

The pH dependence of store-operated Ca(2+) influx (SOCI) into human platelets, as well as its physiological consequence, aggregation, was studied. In Ca(2+)-free medium, thapsigargin (1 microM) induced a small increase in intracellular free-Ca(2+) ([Ca(2+)](i)), which was not affected by changes in extracellular pH. The addition of Ca(2+) (0.5-3 mM) after Ca(2+) store depletion caused by thapsigargin resulted in concentration-dependent increases in [Ca(2+)](i) (SOCI), which were strongly inhibited by SKF-96365 (100 microM), an inhibitor of receptor-mediated Ca(2+) entry. SOCI was inhibited by acidosis (pH 6.9) and augmented by alkalosis (pH 7.9). The addition of Ca(2+) (0.5-3 mM) to platelets, which were kept in Ca(2+)-free medium, slightly but significantly increased [Ca(2+)](i). This Ca(2+) leak entry was also decreased and increased by extracellular acidosis (pH 6.9) and alkalosis (pH 7.9), respectively, but not affected by SKF-96365. Neither thapsigargin (1 microM) stimulation in Ca(2+)-free solution nor elevation of extracellular Ca(2+) alone was sufficient to induce platelet aggregation. In contrast, the addition of Ca(2+) (1 mM) to platelets activated by thapsigargin resulted in aggregation, which was markedly inhibited by SKF-96365 (100 microM). Platelet aggregation associated with SOCI was also inhibited by extracellular acidosis (pH 6.9) and augmented by extracellular alkalosis (pH 7.9). These results suggest that acidosis-induced inhibition, as well as alkalosis-induced promotion of platelet aggregation, involve pH effects on SOCI.

Expression of Trp3 determines sensitivity of capacitative Ca2+ entry to nitric oxide and mitochondrial Ca2+ handling: evidence for a role of Trp3 as a subunit of capacitative Ca2+ entry channels.

The role of Trp3 in cellular regulation of Ca(2+) entry by NO was studied in human embryonic kidney (HEK) 293 cells. In vector-transfected HEK293 cells (controls), thapsigargin (TG)-induced (capacitative Ca(2+) entry (CCE)-mediated) intracellular Ca(2+) signals and Mn(2+) entry were markedly suppressed by the NO donor 2-(N,N-diethylamino)diazenolate-2-oxide sodium salt (3 microm) or by authentic NO (100 microm). In cells overexpressing Trp3 (T3-9), TG-induced intracellular Ca(2+) signals exhibited an amplitude similar to that of controls but lacked sensitivity to inhibition by NO. Consistently, NO inhibited TG-induced Mn(2+) entry in controls but not in T3-9 cells. Moreover, CCE-mediated Mn(2+) entry into T3-9 cells exhibited a striking sensitivity to inhibition by extracellular Ca(2+), which was not detectable in controls. Suppression of mitochondrial Ca(2+) handling with the uncouplers carbonyl cyanide m-chlorophenyl hydrazone (300 nm) or antimycin A(1) (-AA(1)) mimicked the inhibitory effect of NO on CCE in controls but barely affected CCE in T3-9 cells. T3-9 cells exhibited enhanced carbachol-stimulated Ca(2+) entry and clearly detectable cation currents through Trp3 cation channels. NO as well as carbonyl cyanide m-chlorophenyl hydrazone slightly promoted carbachol-induced Ca(2+) entry into T3-9 cells. Simultaneous measurement of cytoplasmic Ca(2+) and membrane currents revealed that Trp3 cation currents are inhibited during Ca(2+) entry-induced elevation of cytoplasmic Ca(2+), and that this negative feedback regulation is blunted by NO. Our results demonstrate that overexpression of Trp3 generates phospholipase C-regulated cation channels, which exhibit regulatory properties different from those of endogenous CCE channels. Moreover, we show for the first time that Trp3 expression determines biophysical properties as well as regulation of CCE channels by NO and mitochondrial Ca(2+) handling. Thus, we propose Trp3 as a subunit of CCE channels.

Intracellular alkalinization augments alpha(1)-adrenoceptor-mediated vasoconstriction by promotion of Ca(2+) entry through the non-L-type Ca(2+) channels.

Modulation by intracellular pH of the vasoconstriction induced by alpha-adrenoceptor agonists was investigated in isolated guinea pig aorta. NH(4)Cl (15 mM) increased intracellular pH of aortic smooth muscle cells by about 0.2 pH unit and significantly augmented KCl-induced contraction of aortic strips, whereas simultaneous administration of NH(4)Cl (15 mM) plus Na(+) propionate (30 mM) failed to affect intracellular pH or contractility. NH(4)Cl (15 mM) potentiated contractions induced by alpha-adrenoceptor agonists, norepinephrine, phenylephrine and clonidine. Contraction induced by alpha(1)-selective adrenoceptor agonist, phenylephrine, but not that induced by norepinephrine or clonidine, was insensitive to inhibition by verapamil (1 microM). Phenylephrine-induced contraction was not affected by NH(4)Cl in Ca(2+)-free medium whereas extracellular Ca(2+)-induced contraction of phenylephrine-stimulated aorta was significantly augmented by NH(4)Cl. Consistently, 45Ca(2+)uptake into phenylephrine 1 microM)-stimulated aortic strips was increased by incubation with NH(4)Cl. The potentiating effects of NH(4)Cl on both phenylephrine-induced Ca(2+) entry and contraction were antagonized by Na(+) propionate. These results suggest that intracellular alkalinization facilitates alpha(1)-adrenoceptor-mediated vasoconstriction by facilitation of an agonist-induced Ca(2+) entry pathway that is independent of L-type Ca(2+) channels.

Comparison of neuronal and endothelial isoforms of nitric oxide synthase in stably transfected HEK 293 cells.

The neuronal and endothelial isoforms of nitric oxide (NO) synthase (nNOS and eNOS, respectively) both catalyze the production of NO but are regulated differently. Stably transfected HEK 293 cell lines containing nNOS, eNOS, and a soluble mutant of eNOS were therefore established to compare their activity in a common cellular environment. NOS activity was determined by measuring L-[3H]citrulline production in homogenates and intact cells, the conversion of oxyhemoglobin to methemoglobin, and the production of cGMP. The results indicate that nNOS is more active than eNOS, both in unstimulated as well as calcium-stimulated cells. Under basal conditions, the soluble mutant of eNOS appeared to be slightly more active than wild-type eNOS in terms of NO and cGMP formation, suggesting that membrane association may be crucial for inhibition of basal NO release but is not required for stimulation by Ca2+-mobilizing agents. The maximal activity of soluble guanylate cyclase was significantly reduced by transfection with wild-type eNOS due to downregulation of mRNA expression. These results demonstrate that nNOS and eNOS behave differently even in an identical cellular environment.

S-nitrosation controls gating and conductance of the alpha 1 subunit of class C L-type Ca(2+) channels.

Modulation of smooth muscle, L-type Ca(2+) channels (class C, Ca(V)1.2b) by thionitrite S-nitrosoglutathione (GSNO) was investigated in the human embryonic kidney 293 expression system at the level of whole-cell and single-channel currents. Extracellular administration of GSNO (2 mM) rapidly reduced whole-cell Ba(2+) currents through channels derived either by expression of alpha1C-b or by coexpression of alpha1C-b plus beta2a and alpha2-delta. The non-thiol nitric oxide (NO) donors 2,2-diethyl-1-nitroso-oxhydrazin (2 mM) and 3-morpholinosydnonimine-hydrochloride (2 mM), which elevated cellular cGMP levels to a similar extent as GSNO, failed to affect Ba(2+) currents significantly. Intracellular administration of copper ions, which promote decomposition of the thionitrite, antagonized its inhibitory effect, and loading of cells with high concentrations of dithiothreitol (2 mM) prevented the effect of GSNO on alpha1C-b channels. Intracellular loading of cells with oxidized glutathione (2 mM) affected neither alpha1C-b channel function nor their modulation by GSNO. Analysis of single-channel behavior revealed that GSNO inhibited Ca(2+) channels mainly by reducing open probability. The development of GSNO-induced inhibition was associated with the transient occurrence of a reduced conductance state of the channel. Our results demonstrate that GSNO modulates the alpha1 subunit of smooth muscle L-type Ca(2+) channels by an intracellular mechanism that is independent of NO release and stimulation of guanylyl cyclase. We suggest S-nitrosation of intracellularly located sulfhydryl groups as an important determinant of Ca(2+) channel gating and conductance.

Molecular determinant for run-down of L-type Ca2+ channels localized in the carboxyl terminus of the 1C subunit.

1. The role of the sequence 1572-1651 in the C-terminal tail of the alpha1C subunit in run-down of Ca2+ channels was studied by comparing functional properties of the conventional alpha1C,77 channel with those of three isoforms carrying alterations in this motif. 2. The pore-forming alpha1C subunits were co-expressed with alpha2delta and beta2a subunits in HEK-tsA201 cells, a subclone of the human embryonic kidney cell line, and studied by whole-cell and single-channel patch-clamp techniques. 3. Replacement of amino acids 1572-1651 in alpha1C,77 with 81 different amino acids leading to alpha1C,86 significantly altered run-down behaviour. Run-down of Ba2+ currents was rapid with alpha1C,77 channels, but was slow with alpha1C,86. 4. Transfer of the alpha1C,86 segments L (amino acids 1572-1598) or K (amino acids 1595-1652) into the alpha1C,77 channel yielded alpha1C,77L and alpha1C,77K channels, respectively, the run-down of which resembled more that of alpha1C,77. These results demonstrate that a large stretch of sequence between residues 1572 and 1652 of alpha1C,86 renders Ca2+ channels markedly resistant to run-down. 5. The protease inhibitor calpastatin added together with ATP was able to reverse the run-down of alpha1C,77 channels. Calpastatin expression was demonstrated in the HEK-tsA cells by Western blot analysis. 6. These results indicate a significant role of the C-terminal sequence 1572-1651 of the alpha1C subunit in run-down of L-type Ca2+ channels and suggest this sequence as a target site for a modulatory effect by endogenous calpastatin.

Coassembly of Trp1 and Trp3 proteins generates diacylglycerol- and Ca2+-sensitive cation channels.

To analyze the functional consequences of coassembly of transient receptor potential 1 (Trp1) and Trp3 channel proteins, we characterized membrane conductances and divalent cation entry derived by separate overexpression and by coexpression of both Trp isoforms. Trp1 expression generated a 1-oleoyl-2-acetyl-sn-glycerol (OAG)-activated conductance that was detectable only in Ca(2+)-free extracellular solution. Trp3 expression gave rise to an OAG-activated conductance that was suppressed but clearly detectable at physiological Ca(2+) concentrations. Coexpression of both species resulted in a constitutively active, OAG-sensitive conductance, which exhibited distinctive cation selectivity and high sensitivity to inhibition by intracellular Ca(2+). Trp1-expressing cells displayed only modest carbachol-induced Ca(2+) entry and lacked OAG-induced Sr(2+) entry, whereas Trp3-expressing cells responded to both agents with a substantial divalent cation entry. Coexpression of Trp1 plus Trp3 suppressed carbachol-induced Ca(2+) entry compared with Trp3 expression and abolished OAG-induced Sr(2+) entry signals. We concluded that coassembly of Trp1 and Trp3 resulted in the formation of oligomeric Trp channels that are subject to regulation by phospholipase C and Ca(2+). The distinguished Ca(2+) sensitivity of these Trp1/Trp3 hetero-oligomers appeared to limit Trp-mediated Ca(2+) signals and may be of importance for negative feedback control of Trp function in mammalian cells.

A sequence in the carboxy-terminus of the alpha(1C) subunit important for targeting, conductance and open probability of L-type Ca(2+) channels.

The role of the 80-amino acid motif 1572-1651 in the C-terminal tail of alpha(1C) Ca(2+) channel subunits was studied by comparing properties of the conventional alpha(1C,77) channel expressed in HEK-tsA201 cells to three isoforms carrying alterations in this motif. Replacement of amino acids 1572-1651 in alpha(1C,77) with 81 non-identical residues leading to alpha(1C,86) impaired membrane targeting and cluster formation of the channel. Similar to alpha(1C, 86), substitution of its 1572-1598 (alpha(1C,77L)) or 1595-1652 (alpha(1C,77K)) segments into the alpha(1C,77) channel yielded single-channel Ba(2+) currents with increased inactivation, reduced open probability and unitary conductance, when compared to the alpha(1C,77) channel. Thus, the C-terminal sequence 1572-1651 of the alpha(1C) subunit is important for membrane targeting, permeation and open probability of L-type Ca(2+) channels.

Modulation of the smooth-muscle L-type Ca2+ channel alpha1 subunit (alpha1C-b) by the beta2a subunit: a peptide which inhibits binding of beta to the I-II linker of alpha1 induces functional uncoupling.

Modulation of the smooth-muscle Ca(2+) channel alpha1C-b subunit by the auxiliary beta2a subunit was studied in the HEK 293 (cell line from human embryonic kidney cells) expression system. In addition, we tested whether the alpha1-beta interaction in functional channels is sensitive to an 18-amino-acid synthetic peptide that corresponds to the sequence of the defined major interaction domain in the cytoplasmic I-II linker of alpha1C (AID-peptide). Ca(2+) channels derived by co-expression of alpha1C-b and beta2a subunits exhibited an about 3-fold higher open probability (P(o)) than alpha1C-b channels. High-P(o) gating of alpha1C-b.beta2a channels was associated with the occurrence of long-lasting channel openings [mean open time (tau)>10 ms] which were rarely observed in alpha1C-b channels. Modulation of fast gating by the beta2a subunit persisted in the cell-free, inside-out recording configuration. Biochemical experiments showed that the AID-peptide binds with appreciable affinity to beta2 subunits of native Ca(2+) channels. Binding of the beta2 protein to immobilized AID-peptide was specifically inhibited (K(i) of 100 nM) by preincubation with free (uncoupled) AID-peptide, but not by a corresponding scrambled peptide. Administration of the AID-peptide (10 microM) to the cytoplasmic side of inside-out patches induced a substantial reduction of P(o) of alpha1C-b.beta2a channels. The scrambled control peptide failed to affect alpha1C-b. beta2a channels, and the AID-peptide (10 microM) did not modify alpha1C-b channel function in the absence of expressed beta2a subunit. Our results demonstrate that the beta2a subunit controls fast gating of alpha1C-b channels, and suggest the alpha1-beta interaction domain in the cytoplasmic I-II linker of alpha1C (AID) as a possible target of modulation of the channel. Moreover, our data are consistent with a model of alpha1-beta interaction that is based on multiple interaction sites, including AID as a determinant of the affinity of the alpha1-beta interaction.

Inhibitory effects of aclarubicin on nitric oxide production in aortic smooth muscle cells and macrophages.

The effects of aclaruhicin (ACR), an anthracycline antibiotic, on inducible nitric oxide (NO) synthesis was investigated in rat aortic smooth muscle cells (RASMCs) and RAW macrophages. ACR at concentrations as low as 0.1 microM significantly inhibited NO production induced by interleukin-1beta in RASMCs. About 5- to 10-fold higher concentrations of ACR were required for inhibition of interferon-gamma and lipopolipopolysaccharide-induced NO production in RAW cells. When ACR was subsequently administered to inducible NO synthase (iNOS) induction, the NO production was barely suppressed in RASMCs. Moreover, ACR (up to 10 microM) lacked direct inhibitory effects on iNOS activity in homogenates of these cells. ACR (0.1 microM) inhibited the expression of iNOS protein and mRNA in RASMCs without concomitant cytotoxic effects. ACR (>0.5 microM)-induced inhibition of NO production in RAW cells was associated with substantial cytotoxic effects as shown by measurement of lactate dehydrogenase release. These results suggest that ACR is a potent inhibitor of iNOS induction in vascular smooth muscle, but inhibits iNOS induction in macrophage only at high cytotoxic

IK.ACh activation by arachidonic acid occurs via a G-protein-independent pathway mediated by the GIRK1 subunit.

The molecular target of arachidonic-acid-derived metabolites, serving as second messengers that activate atrial acetylcholine-activated potassium current (IK.ACh) in addition to G-protein beta/gamma subunits (Gbeta/gamma), is unknown. Co-expression of two isoforms of G-protein-activated, inwardly rectifying potassium channels (GIRKs) in oocytes of Xenopus laevis revealed that these heterologous co-expressed GIRKs, which are responsible for the formation of IK.ACh in the atrium, are activated by arachidonic acid metabolites, like their counterparts in atrial cells. The expression of homooligomeric GIRK1(F137S) and GIRK4wt channels revealed that this activatory mechanism is specific to the GIRKI subunit. Sequestrating available Gbeta/gamma by overexpression of C-betaARK (a Gbeta/gamma binding protein) failed to abolish the activation of GIRK currents by arachidonic acid. From our experiments we conclude that the GIRKI subunit itself is the molecular target for regulation of GIRK channels by arachidonic acid metabolites.

Evidence for a role of Trp proteins in the oxidative stress-induced membrane conductances of porcine aortic endothelial cells.

OBJECTIVE: Expression of homologues of the Drosophila transient receptor potential (Trp) protein has recently been demonstrated for vascular endothelium. Some Trp isoforms such as Trp3, are known to constitute cation conductances with biophysical properties similar to those of the endothelial oxidant-activated cation conductance. Therefore we tested whether Trp proteins provide the molecular basis of the oxidant-induced membrane currents in porcine aortic endothelial cells (ECAP). METHODS: Expression of the Trp3 isoform in ECAP was tested by RT-PCR and subsequent southern blot analysis. In order to knock-out the function of endogenous Trp channels, ECAP were transiently transfected to express NTRP3, a dominant negative fragment of Trp3. Oxidative-stress was introduced by exposure of cells to tert-butylhydroperoxide (tBHP; 400 microM), and membrane current as well as membrane potential were recorded using the conventional whole cell patch-clamp technique. RESULTS: RT-PCR experiments demonstrated the expression of a Trp3 isoform in ECAP. The oxidant tert.-butylhydroperoxide (tBHP) completely depolarized endothelial cells by activation of a cation conductance which allowed significant Na+ inward currents at negative potentials (mean inward current 462 pA at -80 mV). The tBHP-induced currents resembled Trp-related currents in terms of cation selectivity, La3+ sensitivity and lack of voltage dependence. Expression of the N-terminal fragment of hTrp3 (NTRP3), but not of a C-terminal fragment of hTrp3 (CTRP3), abolished the oxidant-induced cation current and reduced membrane depolarization. CONCLUSION: Our results strongly suggest Trp proteins as the molecular basis of endothelial oxidant-activated cation channels. It is concluded that Trp proteins play an important role in the redox sensitivity of the vascular endothelium.

Na(+)/Ca(2+) exchange facilitates Ca(2+)-dependent activation of endothelial nitric-oxide synthase.

Recent evidence suggests the expression of a Na(+)/Ca(2+) exchanger (NCX) in vascular endothelial cells. To elucidate the functional role of endothelial NCX, we studied Ca(2+) signaling and Ca(2+)-dependent activation of endothelial nitric-oxide synthase (eNOS) at normal, physiological Na(+) gradients and after loading of endothelial cells with Na(+) ions using the ionophore monensin. Monensin-induced Na(+) loading markedly reduced Ca(2+) entry and, thus, steady-state levels of intracellular free Ca(2+) ([Ca(2+)](i)) in thapsigargin-stimulated endothelial cells due to membrane depolarization. Despite this reduction of overall [Ca(2+)](i), Ca(2+)-dependent activation of eNOS was facilitated as indicated by a pronounced leftward shift of the Ca(2+) concentration response curve in monensin-treated cells. This facilitation of Ca(2+)-dependent activation of eNOS was strictly dependent on the presence of Na(+) ions during treatment of the cells with monensin. Na(+)-induced facilitation of eNOS activation was not due to a direct effect of Na(+) ions on the Ca(2+) sensitivity of the enzyme. Moreover, the effect of Na(+) was not related to Na(+) entry-induced membrane depolarization or suppression of Ca(2+) entry, since neither elevation of extracellular K(+) nor the Ca(2+) entry blocker 1-(beta-[3-(4-methoxyphenyl)-propoxy]-4-methoxyphenethyl)-1H-imidazol e hydrochloride (SK&F 96365) mimicked the effects of Na(+) loading. The effects of monensin were completely blocked by 3', 4'-dichlorobenzamil, a potent and selective inhibitor of NCX, whereas the structural analog amiloride, which barely affects Na(+)/Ca(2+) exchange, was ineffective. Consistent with a pivotal role of Na(+)/Ca(2+) exchange in Ca(2+)-dependent activation of eNOS, an NCX protein was detected in caveolin-rich membrane fractions containing both eNOS and caveolin-1. These results demonstrate for the first time a crucial role of cellular Na(+) gradients in regulation of eNOS activity and suggest that a tight functional interaction between endothelial NCX and eNOS may take place in caveolae.

Current modulation and membrane targeting of the calcium channel alpha1C subunit are independent functions of the beta subunit.

1. The beta subunits of voltage-sensitive calcium channels facilitate the incorporation of channels into the plasma membrane and modulate calcium currents. In order to determine whether these two effects of the beta subunit are interdependent or independent of each other we studied plasma membrane incorporation of the channel subunits with green fluorescent protein and immunofluorescence labelling, and current modulation with whole-cell and single-channel patch-clamp recordings in transiently transfected human embryonic kidney tsA201 cells. 2. Coexpression of rabbit cardiac muscle alpha1C with rabbit skeletal muscle beta1a, rabbit heart/brain beta2a or rat brain beta3 subunits resulted in the colocalization of alpha1C with beta and in a marked translocation of the channel complexes into the plasma membrane. In parallel, the whole-cell current density and single-channel open probability were increased. Furthermore, the beta2a isoform specifically altered the voltage dependence of current activation and the inactivation kinetics. 3. A single amino acid substitution in the beta subunit interaction domain of alpha1C (alpha1CY467S) disrupted the colocalization and plasma membrane targeting of both subunits without affecting the beta subunit-induced modulation of whole-cell currents and single-channel properties. 4. These results show that the modulation of calcium currents by beta subunits can be explained by beta subunit-induced changes of single-channel properties, but the formation of stable alpha1C-beta complexes and their increased incorporation into the plasma membrane appear not to be necessary for functional modulation.

The cardiac acetylcholine-activated, inwardly rectifying K+-channel subunit GIRK1 gives rise to an inward current induced by free oxygen radicals.

Reactive oxygen species (ROS) play a crucial role in pathophysiology of the cardiovascular system. The present study was designed to analyze the redox sensitivity of G-protein-activated inward rectifier K+ (GIRK) channels, which control cardiac contractility and excitability. GIRK1 subunits were heterologously expressed in Xenopus laevis oocytes and the resulting K+ currents were measured with the two-electrode voltage clamp technique. Oxygen free radicals generated by the hypoxanthine/xanthine oxidase system led to a marked increase in the current through GIRK channels, termed superoxide-induced current (I(SO)). Furthermore, I(SO) did not depend on G-protein-dependent activation of GIRK currents by coexpressed muscarinic m2-receptors, but could also be observed when no agonist was present in the bathing solution. Niflumic acid at a concentration of 0.5 mmol/l did not abolish I(SO), whereas 100 micromol/l Ba2+ attenuated I(SO) completely. Catalase (10(6) i.u./l) failed to suppress I(SO), whereas H2O2 concentration was kept close to zero, as measured by chemiluminescence. Hence, we conclude that O2*- or a closely related species is responsible for I(SO) induction. Our results demonstrate a significant redox sensitivity of GIRK1 channels and suggest redox-activation of G-protein-activated inward rectifier K+ channels as a key mechanism in oxidative stress-associated cardiac dysfunction.

Trp proteins form store-operated cation channels in human vascular endothelial cells.

Members of the Trp protein family have been suggested as the structural basis of store-operated cation conductances. With this study, we provide evidence for the expression of three isoforms of Trp (hTrp1, 3 and 4) in human umbilical vein endothelial cells (HUVEC). The role of Trp proteins in store regulation of endothelial membrane conductances was tested by expression of an N-terminal fragment of hTrp3 (N-TRP) which exerts a dominant negative effect on Trp channel function presumably due to suppression of channel assembly. Depletion of intracellular Ca2+ stores with IP3 (100 microM) or thapsigargin (100 nM) induced a substantial cation conductance in sham-transfected HUVEC as well as in HUVEC transfected with hTrp3. In contrast, HUVEC transfected with N-TRP failed to exhibit store-operated currents. Our results suggest the involvement of Trp related proteins in the store-operated cation conductance of human vascular endothelial cells.