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Klaus Groschner

Publications and source records attributed to Klaus Groschner.

At least 19 recordsLinked to original sources
PubMed2006

Dynamic but not constitutive association of calmodulin with rat TRPV6 channels enables fine tuning of Ca2+-dependent inactivation.

The Ca(2+)-selective TRPV6 as well as the L-type Ca(2+) channel are regulated by the Ca(2+)-binding protein calmodulin (CaM). Here, we investigated the interaction of CaM with rat (r)TRPV6 in response to alterations of intracellular Ca(2+), employing Ca(2+)-imaging and patch-clamp techniques. Additionally, confocal Förster resonance energy transfer (FRET) microscopy on living cells was utilized as a key method to visualize in vivo protein-protein interactions essential for CaM regulation of rTRPV6 activity. The effects of overexpressed CaM or its Ca(2+)-insensitive mutant (CaM(MUT)) was probed on various rTRPV6 mutants and fragments in an attempt to elucidate the molecular mechanism of Ca(2+)/CaM-dependent regulation and to pinpoint the physiologically relevant rTRPV6-CaM interaction site. A significant reduction of rTRPV6 activity, as well as an increase in current inactivation, were observed when CaM was overexpressed in addition to endogenous CaM. The Ca(2+)-insensitive CaM(MUT), however, failed to affect rTRPV6-derived currents. Accordingly, live cell confocal FRET microscopy revealed a robust interaction for CaM but not CaM(MUT) with rTRPV6, suggesting a strict Ca(2+) dependence for their association. Indeed, interaction of rTRPV6 or its C terminus with CaM increased with rising intracellular Ca(2+) levels, as observed by dynamic FRET measurements. An rTRPV6Delta(695-727) mutant with the very C-terminal end deleted, yielded Ca(2+) currents with a markedly reduced inactivation in accordance with a lack of CaM interaction as substantiated by FRET microscopy. These results, in contrast with those for CaM-dependent L-type Ca(2+) channel inactivation, demonstrate a dynamic association of CaM with the very C-terminal end of rTRPV6 (aa 695-727), and this enables acceleration of the rate of rTRPV6 current inactivation with increasing intracellular CaM concentrations.

Animals↗
PubMed2006

Cellular cholesterol controls TRPC3 function: evidence from a novel dominant-negative knockdown strategy.

TRPC3 (canonical transient receptor potential protein 3) has been suggested to be a component of cation channel complexes that are targeted to cholesterol-rich lipid membrane microdomains. In the present study, we investigated the potential role of membrane cholesterol as a regulator of cellular TRPC3 conductances. Functional experiments demonstrated that cholesterol loading activates a non-selective cation conductance and a Ca2+ entry pathway in TRPC3-overexpressing cells but not in wild-type HEK-293 (human embryonic kidney 293) cells. The cholesterol-induced membrane conductance exhibited a current-to-voltage relationship similar to that observed upon PLC (phospholipase C)-dependent activation of TRPC3 channels. Nonetheless, the cholesterol-activated conductance lacked negative modulation by extracellular Ca2+, a typical feature of agonist-activated TRPC3 currents. Involvement of TRPC3 in the cholesterol-dependent membrane conductance was further corroborated by a novel dominant-negative strategy for selective blockade of TRPC3 channel activity. Expression of a TRPC3 mutant, which contained a haemagglutinin epitope tag in the second extracellular loop, conferred antibody sensitivity to both the classical PLC-activated as well as the cholesterol-activated conductance in TRPC3-expressing cells. Moreover, cholesterol loading as well as PLC stimulation was found to increase surface expression of TRPC3. Promotion of TRPC3 membrane expression by cholesterol was persistent over 30 min, while PLC-mediated enhancement of plasma membrane expression of TRPC3 was transient in nature. We suggest the cholesterol content of the plasma membrane as a determinant of cellular TRPC3 activity and provide evidence for cholesterol dependence of TRPC3 surface expression.

Calcium Signaling↗
PubMed2006

TRPC3 and TRPC4 associate to form a redox-sensitive cation channel. Evidence for expression of native TRPC3-TRPC4 heteromeric channels in endothelial cells.

Canonical transient receptor potential proteins (TRPC) have been proposed to form homo- or heteromeric cation channels in a variety of tissues, including the vascular endothelium. Assembly of TRPC multimers is incompletely understood. In particular, heteromeric assembly of distantly related TRPC isoforms is still a controversial issue. Because we have previously suggested TRPC proteins as the basis of the redox-activated cation conductance of porcine aortic endothelial cells (PAECs), we set out to analyze the TRPC subunit composition of endogenous endothelial TRPC channels and report here on a redox-sensitive TRPC3-TRPC4 channel complex. The ability of TRPC3 and TRPC4 proteins to associate and to form a cation-conducting pore complex was supported by four lines of evidence as follows: 1) Co-immunoprecipitation experiments in PAECs and in HEK293 cells demonstrated the association of TRPC3 and TRPC4 in the same complex. 2) Fluorescence resonance energy transfer analysis demonstrated TRPC3-TRPC4 association, involving close proximity between the N terminus of TRPC4 and the C terminus of TRPC3 subunits. 3) Co-expression of TRPC3 and TRPC4 in HEK293 cells generated a channel that displayed distinct biophysical and regulatory properties. 4) Expression of dominant-negative TRPC4 proteins suppressed TRPC3-related channel activity in the HEK293 expression system and in native endothelial cells. Specifically, an extracellularly hemagglutinin (HA)-tagged TRPC4 mutant, which is sensitive to blockage by anti-HA-antibody, was found to transfer anti-HA sensitivity to both TRPC3-related currents in the HEK293 expression system and the redox-sensitive cation conductance of PAECs. We propose TRPC3 and TRPC4 as subunits of native endothelial cation channels that are governed by the cellular redox state.

Animals↗
PubMed2006

Intracellular pH as a determinant of vascular smooth muscle function.

Intracellular pH (pHi) is a physiological parameter that is intimately linked to contractility, growth and proliferation of vascular smooth muscle (VSM). Regarding contractility, no general unifying concept of pHi regulation but a rather complex relation between pHi signals and vascular tone has been revealed so far. The modulation of vasotone by pHi depends on the type of blood vessel as well as on the pattern of regulatory input signals. In addition, changes in pHi have been recognized as an important cellular signal to determine the fate of cells in terms of proliferation or apoptosis. Cellular sensors for pHi include a variety of ion transport systems which control intracellular Ca2+ gradients and are likely to serve as a link between pHi and cell functions. Here we provide an overview on the potential targets and mechanisms that transduce pHi signals in VSM. The role of pHi-sensing signaling complexes and localized pHi signaling as the basis of diversity of pHi regulation of VSM function is discussed.

Animals↗
PubMed2006

TRPC4 expression determines sensitivity of the platelet-type capacitative Ca2+ entry channel to intracellular alkalosis.

The present study was designed to analyze the molecular basis of the intracellular pH-dependent capacitative Ca2+ entry (CCE) of human platelets and megakaryocytic cells, specifically to test the hypothesis that members of the classical transient receptor potential (TRPC) protein family are involved in the CCE pathway that is promoted by intracellular alkalosis. Human platelets as well as the tested megakaryocytic cell lines (CMK cells, MEG-01 cells) and HEK293 cells displayed thapsigargin-induced CCE and responded to monensin with comparable elevation in intracellular pH. Promotion of CCE by monensin-induced intracellular alkalosis, however, was profound in mature platelets, moderate in CMK cells and lacking in MEG-01 cells as well as in HEK293 cells. Analysis of the TRPC expression pattern by immunoblotting revealed that mature platelets and CMK cells express TRPC4 along with TRPC1 and TRPC3, while TRPC4 is lacking in MEG-01 cells. HEK293 cells displayed CCE characteristics as well as lack of TRPC4 expression similar to MEG-01 cells. Over-expression of TRPC4 in HEK293 cells was found to result in a gain of pH-sensitivity of CCE with clearly detectable promotion of CCE in response to monensin. These results suggest that platelet CCE channel complexes contain TRPC4 as a molecular component that determines sensitivity of CCE to intracellular alkalosis.

Alkalosis↗
PubMed2005

Na(+) entry and modulation of Na(+)/Ca(2+) exchange as a key mechanism of TRPC signaling.

Ion channels formed by canonical transient receptor potential (TRPC) proteins are considered to be key players in cellular Ca(2+) homeostasis. As permeation of Ca(2+) through TRPC homo- and/or heteromeric channels has been repeatedly demonstrated, analysis of the physiological role of TRPC proteins was so far based on the concept that these proteins form regulated Ca(2+) entry channels. The well-recognized lack of cation selectivity of TRPC channels and the ability to generate substantial monovalent conductances that govern membrane potential and cation gradients were barely appreciated as a physiologically relevant issue. Nonetheless, recent studies suggest monovalent, specifically Na(+) permeation through TRPC cation channels as an important event in TRPC signaling. TRPC-mediated Na(+) entry may be converted into a distinct pattern of cellular Ca(2+) signals by interaction with Na(+)/Ca(2+) exchanger proteins. This review discusses current concepts regarding the link between Na(+) entry through TRPC channels and cellular Ca(2+) signaling.

Animals↗
PubMed2005

Molecular analysis of a store-operated and 2-acetyl-sn-glycerol-sensitive non-selective cation channel. Heteromeric assembly of TRPC1-TRPC3.

We have reported that internal Ca2+ store depletion in HSY cells stimulates a nonselective cation current which is distinct from I(CRAC) in RBL cells and TRPC1-dependent I(SOC) in HSG cells (Liu, X., Groschner, K., and Ambudkar, I. S. (2004) J. Membr. Biol. 200, 93-104). Here we have analyzed the molecular composition of this channel. Both thapsigargin (Tg) and 2-acetyl-sn-glycerol (OAG) stimulated similar non-selective cation currents and Ca2+ entry in HSY cells. The effects of Tg and OAG were not additive. HSY cells endogenously expressed TRPC1, TRPC3, and TRPC4 but not TRPC5 or TRPC6. Immunoprecipitation of TRPC1 pulled down TRPC3 but not TRPC4. Conversely, TRPC1 co-immunoprecipitated with TRPC3. Expression of antisense TRPC1 decreased (i) Tg- and OAG-stimulated currents and Ca2+ entry and (ii) the level of endogenous TRPC1 but not TRPC4. Antisense TRPC3 similarly reduced Ca2+ entry and endogenous TRPC3. Yeast two-hybrid analysis revealed an interaction between NTRPC1 and NTRPC3 (CTRPC1-CTRPC3, CTRPC3-CTRPC1, or CTRPC1-NTRPC3 did not interact), which was confirmed by glutathione S-transferase (GST) pull-down assays (GST-NTRPC3 pulled down TRPC1 and vice versa). Expression of NTRPC1 or NTRPC3 induced similar dominant suppression of Tg- and OAG-stimulated Ca2+ entry. NTRPC3 did not alter surface expression of TRPC1 or TRPC3 but disrupted TRPC1-TRPC3 association. In aggregate, our data demonstrate that TRPC1 and TRPC3 co-assemble, via N-terminal interactions, to form a heteromeric store-operated non-selective cation channel in HSY cells. Thus selective association between TRPCs generate distinct store-operated channels. Diversity of store-operated channels might be related to the physiology of the different cell types.

Biotinylation↗
PubMed2005

TRPC3: a versatile transducer molecule that serves integration and diversification of cellular signals.

Transient receptor potential (TRP) proteins have been recognized as sensors for a wide variety of external and internal signals involved in maintenance of cellular homeostasis and control of physiological functions. Evidence of a striking versatility in terms of signal integration and transduction has been reported for members of the canonical (or classical) TRP subfamily (TRPCs). TRPC species are cation channel subunits and emerge as multifunctional signal transduction molecules that are able to function as components of divergent signalplexes. Results obtained in heterologous expression systems suggest TRPC3 as a paradigm of multifunctional signal transduction by a cation channel protein. TRPC3 serves cellular Ca(2+) signaling by multiple mechanisms and may control a variety of distinct physiological functions. In this review, we summarize current knowledge on the properties and possible signaling partners of TRPC3, and discuss the role of TRPC3 channel proteins in cellular signaling networks.

Animals↗
PubMed2004

Cholesterol- and caveolin-rich membrane domains are essential for phospholipase A2-dependent EDHF formation.

OBJECTIVE: Cholesterol-rich membrane domains, which contain the scaffold protein caveolin-1 (Cav-1) (caveolae), represent an important structural element involved in endothelial signal transduction. The present study was designed to investigate the role of these signaling platforms in the generation of endothelial-derived hyperpolarizing factor (EDHF). METHODS: Caveolae were disrupted by cholesterol depletion with methyl-beta-cyclodextrin (MbetaCD 10 mM). MbetaCD-induced modulation of non-nitric oxide-/non-prostanoid-dependent (EDHF)-mediated vasorelaxation was studied in pig coronary arteries. Effects of MbetaCD on endothelial Ca(2+) signaling and phospholipase A(2) (cPLA(2)) activity were determined using fura-2 imaging and measurement of [(3)H]-arachidonate mobilization in cultured pig aortic endothelial cells (PAEC). Cellular localization of caveolin-1 and phospholipase A(2) was investigated by cell fractionation, and interaction of cPLA(2) with caveolin-1 was tested by immunoprecipitation experiments. RESULTS: MbetaCD inhibited EDHF-mediated relaxations of pig coronary arteries induced by bradykinin (100 nM) or ionomycin (300 nM) but not relaxations induced by the NO donor DEA/NO (1 microM). Exposure of arteries to cholesterol-saturated MbetaCD failed to affect EDHF-mediated relaxations. Cholesterol depletion with MbetaCD did not affect bradykinin or ionomycin-induced Ca(2+) signaling in pig aortic endothelial cells, but was associated with enhanced basal and reduced Ca(2+)-dependent release of arachidonic acid (AA). Cell fractionation experiments indicated targeting of cPLA(2) to low density, caveolin-1 rich membranes and immunoprecipitation experiments demonstrated association of phospholipase A(2) with the scaffold protein of caveolae, caveolin-1. Cholesterol depletion with MbetaCD did not disrupt the interaction between cPLA(2) and caveolin-1 but prevented targeting of cPLA(2) to low density membranes. Exogenous supplementation of arachidonic acid after cholesterol depletion partially restored EHDF responses in pig coronary arteries. CONCLUSION: The integrity of caveolin-1-containing membrane microdomains is prerequisite for arachidonic acid recruitment and EDHF signaling in porcine arteries.

Animals↗
PubMed2004

CaT1 knock-down strategies fail to affect CRAC channels in mucosal-type mast cells.

CaT1, the calcium transport protein 1 encoded by TRPV6, is able to generate a Ca(2+) conductance similar but not identical to the classical CRAC current in mucosal-type mast cells. Here we show that CaT1-derived Ca(2+) entry into HEK293 cells is effectively inhibited either by expression of various dominant negative N-terminal fragments of CaT1 (N(334)-CaT1, N(198)-CaT1 and N(154)-CaT1) or by antisense suppression. By contrast, the endogenous CRAC current of the mast cells was unaffected by CaT1 antisense and siRNA knockdown but markedly suppressed by two (N(334)-CaT1, N(198)-CaT1) of the dominant negative N-CaT1 fragments. Inhibition of CRAC current was not an unspecific, toxic effect, as inward rectifier K(+) and MagNuM currents of the mast cells were not significantly affected by these N-CaT1 fragments. The shortest N(154)-CaT1 fragment inhibited CaT1-derived currents in mast cells, but failed to inhibit CRAC currents. Thus, the structural requirements of rCaT N-terminal fragments for inhibition of rCaT1 and CRAC channels are different. These results together with the lack of CaT1 antisense and siRNA effects on currents render it unlikely that CaT1 is a component of native CRAC channels in mast cells. The data further demonstrate a novel strategy for CRAC current inhibition by an N-terminal structure of CaT1.

Calcium↗
PubMed2004

Ca(2+) signaling by TRPC3 involves Na(+) entry and local coupling to the Na(+)/Ca(2+) exchanger.

TRPC3 has been suggested as a key component of phospholipase C-dependent Ca(2+) signaling. Here we investigated the role of TRPC3-mediated Na(+) entry as a determinant of plasmalemmal Na(+)/Ca(2+) exchange. Ca(2+) signals generated by TRPC3 overexpression in HEK293 cells were found to be dependent on extracellular Na(+), in that carbachol-stimulated Ca(2+) entry into TRPC3 expressing cells was significantly suppressed when extracellular Na(+) was reduced to 5 mm. Moreover, KB-R9743 (5 microm) an inhibitor of the Na(+)/Ca(2+) exchanger (NCX) strongly suppressed TRPC3-mediated Ca(2+) entry but not TRPC3-mediated Na(+) currents. NCX1 immunoreactivity was detectable in HEK293 as well as in TRPC3-overexpressing HEK293 cells, and reduction of extracellular Na(+) after Na(+) loading with monensin resulted in significant rises in intracellular free Ca(2+) (Ca(2+)(i)) of HEK293 cells. Similar rises in Ca(2+)(i) were recorded in TRPC3-overexpressing cells upon the reduction of extracellular Na(+) subsequent to stimulation with carbachol. These increases in Ca(2+)(i) were associated with outward membrane currents at positive potentials and inhibited by KB-R7943 (5 microm), chelation of extracellular Ca(2+), or dominant negative suppression of TRPC3 channel function. This suggests that Ca(2+) entry into TRPC3-expressing cells involves reversed mode Na(+)/Ca(2+) exchange. Cell fractionation experiments demonstrated co-localization of TRPC3 and NCX1 in low density membrane fractions, and co-immunoprecipitation experiments provided evidence for association of TRPC3 and NCX1. Glutathione S-transferase pull-down experiments revealed that NCX1 interacts with the cytosolic C terminus of TRPC3. We suggest functional and physical interaction of nonselective TRPC cation channels with NCX proteins as a novel principle of TRPC-mediated Ca(2+) signaling.

Anti-Arrhythmia Agents↗
PubMed2004

Role of TRP channels in oxidative stress.

Increasing evidence suggests a pivotal role of reactive oxygen species (ROS) as well as reactive nitrogen species (RNS) in human pathophysiology. A typical target of ROS/RNS signalling is Ca2+ channels which mediate both long-term as well as acute cellular responses to oxidative stress. We have previously reported that cation channels related to the Drosophila transient receptor potential gene product (TRPC proteins) are likely to serve as redox sensors in the vascular endothelium, and demonstrated that TRPC3 expression is a determinant of the nitric oxide sensitivity of store-operated Ca2+ signalling. Experiments with TRPC species overexpressed in HEK293 cells confirmed that TRPC3 and TRPC4 are able to form redox sensitive cation channels. A key mechanism involved in redox activation of TRPC3 appears to be ROS-induced promotion of protein tyrosine phosphorylation and stimulation of phospholipase C activity. In addition, oxidative stress-induced disruption of caveolin 1-rich lipid raft domains, which interfere with functional TRPC channels, is likely to contribute to redox modulation of TRP proteins and to oxidative stress-induced changes in cellular Ca2+ signalling. Taken together, our data suggest TRPC species serve as a link between cellular redox state and Ca2+ homeostasis. Thus, modulation of these cellular redox sensors may offer unique opportunities for therapeutic interventions.

Calcium↗
PubMed2003

Crosstalk between voltage-independent Ca2+ channels and L-type Ca2+ channels in A7r5 vascular smooth muscle cells at elevated intracellular pH: evidence for functional coupling between L-type Ca2+ channels and a 2-APB-sensitive cation channel.

This study was designed to investigate the role of voltage-independent and voltage-dependent Ca2+ channels in the Ca2+ signaling associated with intracellular alkalinization in A7r5 vascular smooth muscle cells. Extracellular administration of ammonium chloride (20 mmol/L) resulted in elevation of intracellular pH and activation of a sustained Ca2+ entry that was inhibited by 2-amino-ethoxydiphenyl borate (2-APB, 200 micromol/L) but not by verapamil (10 micro;mol/L). Alkalosis-induced Ca2+ entry was mediated by a voltage-independent cation conductance that allowed permeation of Ca2+ (PCa/PNa approximately 6), and was associated with inhibition of L-type Ca2+ currents. Alkalosis-induced inhibition of L-type Ca2+ currents was dependent on the presence of extracellular Ca2+ and was prevented by expression of a dominant-negative mutant of calmodulin. In the absence of extracellular Ca2+, with Ba2+ or Na+ as charge carrier, intracellular alkalosis failed to inhibit but potentiated L-type Ca2+ channel currents. Inhibition of Ca2+ currents through voltage-independent cation channels by 2-APB prevented alkalosis-induced inhibition of L-type Ca2+ currents. Similarly, 2-APB prevented vasopressin-induced activation of nonselective cation channels and inhibition of L-type Ca2+ currents. We suggest the existence of a pH-controlled Ca2+ entry pathway that governs the activity of smooth muscle L-type Ca2+ channels due to control of Ca2+/calmodulin-dependent negative feedback regulation. This Ca2+ entry pathway exhibits striking similarity with the pathway activated by stimulation of phospholipase-C-coupled receptors, and may involve a similar type of cation channel. We demonstrate for the first time the tight functional coupling between these voltage-independent Ca2+ channels and classical voltage-gated L-type Ca2+ channels.

Ammonium Chloride↗
PubMed2003

Vascular actions of anthracycline antibiotics.

Anthracycline antibiotics are of particular value in the therapy of malignant diseases and exert profound effects not only on tumor cells but also on cells in the cardiovascular system. These quinone drugs affect vascular tone by a multitude of mechanisms, including acute modulation of Ca(2+) homeostasis, altered expression of membrane proteins and enzymes that are involved in the control of smooth muscle contraction, and generation of autoregulatory mediators, such as nitric oxide and endothelin. Anthracyclines interfere with blood coagulation-fibrinolysis balance due to its effects on the production of prostacyclin, plasminogen activator and plasminogen activator inhibitor in the endothelium. Moreover, anthracyclines are thought to be the modulators of angiogenesis. The intensity and quality of anthracycline actions on blood vessel function are highly variable and may depend not only on the chemical structure of anthracycline but also on the type of blood vessel as well as the metabolic and redox status of the vascular tissue. Vascular actions of anthracyclines are possibly involved in both beneficial as well as toxic and undesirable side-effects such as tumor progress. Further investigations are required to clarify the relation between specific modifications of vascular cell function and clinical events observed during antineoplastic therapy with anthracyclines.

Animals↗
PubMed2002

Desensitization of endothelial nitric oxide synthase by receptor agonists.

Stimulation of endothelial cells with receptor agonists leads to an activation of endothelial nitric oxide synthase (eNOS) that only lasts for a short duration. A more prolonged effect, however, is observed in response to non-receptor agonists, such as Ca2+ ionophores or thapsigargin (TG). To investigate the molecular mechanisms underlying the rapid deactivation of eNOS after stimulation with receptor agonists, we measured the time courses of eNOS activation and intracellular free Ca2+ concentration ([Ca2+]i) in response to bradykinin (BK) and ATP. Incubation of porcine aortic endothelial cells with BK (1 microM) in the presence of 3 mM extracellular Ca2+ increased [Ca2+]i from 110 to 350 nM and enhanced the rate of l-[3H]citrulline formation from 0.1 to 5 fmol/min. In the absence of extracellular Ca2+, the BK-induced increase in [Ca2+]i was only marginal (from 30 to 110 nM) and not sufficient to activate eNOS. When Ca(2+) (final concentration 3 mM) was added 10 min after BK, [Ca2+]i increased to 330 nM within 3 min, but interestingly, formation of l-[3H]citrulline was not detectable. A similar phenomenon was observed with ATP, but not with Ca2+ ionophores or TG. This indicates that stimulation of endothelial cells with receptor agonists leads to desensitization of eNOS, which renders the enzyme insensitive to activation by subsequent increases in [Ca2+]i. However, when ATP was added to BK-pretreated cells or, conversely, BK to ATP-pretreated cells, activation of eNOS was comparable with that of untreated cells, suggesting that BK and ATP affect different pools of eNOS. The desensitization of eNOS was reversible, since removal of ATP or BK from the incubation buffer restored the response to the respective agonist within 20 min. In addition to the transient Ca2+ signal, desensitization of eNOS may represent a further mechanism by which endothelial cells rapidly terminate receptor-dependent NO formation.

Animals↗
PubMed2002

Store depletion-activated CaT1 currents in rat basophilic leukemia mast cells are inhibited by 2-aminoethoxydiphenyl borate. Evidence for a regulatory component that controls activation of both CaT1 and CRAC (Ca(2+) release-activated Ca(2+) channel) channels.

The intestinal Ca(2+) transport protein CaT1 encoded by TRPV6 has been reported (Yue, L., Peng, J. B., Hediger, M. A., and Clapham, D. E. (2001) Nature 410, 705-709) to be all or a part of the Ca(2+) release-activated Ca(2+) channel (CRAC). The major characteristic of CRAC is its activation following store depletion. We expressed CaT1 in HEK293 cells and rat basophilic leukemia (RBL) mast cells and measured whole-cell currents by the patch clamp technique. In HEK293 cells, the expression of CaT1 consistently yielded a constitutively active current, the size of which was strongly dependent on the holding potential and duration of voltage ramps. In CaT1-expressing RBL cells, the current was either activated by store depletion or was constitutively active at a higher current density. CaT1 currents could be clearly distinguished from endogenous CRAC by their typical current-voltage relationship in divalent free solution. 2-aminoethoxydiphenyl borate (2-APB), which is considered a blocker of CRAC, was tested for its inhibitory effect on both cell types expressing CaT1. Endogenous CRAC as well as store-dependent CaT1-derived currents of RBL cells were largely blocked by 75 microm 2-APB, whereas constitutively active CaT1 currents in both RBL and HEK293 cells were slightly potentiated. These results indicate that despite the difference in the permeation properties of CRAC and CaT1 channels, the latter are similarly able to form store depletion-activated conductances in RBL mast cells that are inhibited by 2-APB.

Animals↗
PubMed2002

Two ways to feel the pressure: an endothelial Ca(2+) entry channel with dual mechanosensitivity.

One impressive function of the vascular endothelium is its ability to adjust the release of vasoactive mediators such as NO and PGI(2) almost instantaneously to changes in blood flow or blood pressure. Besides this fast feedback response to hemodynamic alterations, the endothelium is subject to long-term adaptations that are crucial for prevention of pathological processes such as atherogenesis. Among the various signals that are sensed by endothelial cells, mechanical forces which arise from pulsatile blood flow are probably most important for fast as well as long-term control of blood vessel function by the endothelium.

Calcium Channels↗