Biomedical subjects

Christian Rosker

Publications and source records attributed to Christian Rosker.

6 recordsLinked to original sources

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↗

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

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↗