PubMed Health⌕ Search
Home/Archive/PubMed

PubMed · 16448384

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

Annarita Graziani·Christian Rosker·Sepp D Kohlwein·Michael X Zhu·Christoph Romanin·Wolfgang Sattler·Klaus Groschner·Michael Poteser
View original source ↗Export metadata ↓

Abstract

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.

Explore related subjects

Calcium SignalingCarbacholCationsCell LineCholesterolEpitopesGene TargetingGenes, DominantGenes, ReporterHumansIon TransportKidneyMembrane LipidsMutagenesis, InsertionalPatch-Clamp TechniquesRecombinant Fusion ProteinsTRPC Cation ChannelsTransfectionbeta-CyclodextrinsJournal ArticleResearch Support, Non-U.S. Gov't

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Annarita Graziani, Christian Rosker, Sepp D Kohlwein, Michael X Zhu, Christoph Romanin, Wolfgang Sattler, Klaus Groschner, Michael Poteser. 2006-05-15. Cellular cholesterol controls TRPC3 function: evidence from a novel dominant-negative knockdown strategy.. https://doi.org/10.1042/bj20051246

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

PubMed2006

Mutation and divergence of the phospholipase C gene in Neurospora crassa.

In the fungus Neurospora crassa we have used RIP to obtain a presumptive null mutation of the phospholipase C-1 gene, thought to be important in intracellular calcium signaling, notably maintenance of the tip-high calcium gradient. The mutant is viable but has slow, aberrant growth and branching. Hence plc-1 is not required for polar growth at the tip, but is necessary to modulate growth to give normal form. The mutant has residual PLC activity suggesting that this enzyme function can be provided from other parts of the genome. Sequencing natural isolates has shown that the plc-1 gene is highly variable in nature. A large proportion of the variable sites are in a region that is unique to Neurospora. A phylogeny for this gene shows that New and Old World strains have diverged the most. Within the Americas, morphs are found throughout the continent suggesting extensive strain dispersal.

Calcium Signaling↗
PubMed2006

The inositol 1,4,5-trisphosphate receptor (IP3R) and its regulators: sometimes good and sometimes bad teamwork.

In both nonexcitable and excitable cells, the inositol 1,4,5-trisphosphate receptor (IP(3)R) is the primary cytosolic target responsible for the initiation of intracellular calcium (Ca(2+)) signaling. To fulfill this function, the IP(3)R depends on interaction with accessory subunits and regulatory proteins. These include proteins that reside in the lumen of the endoplasmic reticulum (ER), such as chromogranin A and B and ERp44, and cytosolic proteins, such as neuronal Ca(2+) sensor 1, huntingtin, cytochrome c, IP(3)R-binding protein released with inositol 1,4,5-trisphosphate, Homer, and 4.1N. Specific interactions between these modulatory proteins and the IP(3)R have been described, making it clear that the controlled modulation of the IP(3)R by its binding partners is necessary for physiological cell regulation. The functional coupling of these modulators with the IP(3)R can control apoptosis, intracellular pH, the initiation and regulation of neuronal Ca(2+) signaling, exocytosis, and gene expression. The pathophysiological relevance of IP(3)R modulation is apparent when the functional interaction of these proteins is enhanced or abolished by mutation or overexpression. The subsequent deregulation of the IP(3)R leads to pathological changes in Ca(2+) signaling, signal initiation, the amplitude and frequency of Ca(2+) signals, and the duration of the Ca(2+) elevation. Consequences of this deregulation include abnormal growth and apoptosis. Complex regulation of Ca(2+) signaling is required for the cell to live and function, and this difficult task can only be managed when the IP(3)R teams up and acts properly with its numerous binding partners.

Calcium Signaling↗
PubMed2006

Effect of lysophosphatidylglycerol on several signaling molecules in OVCAR-3 human ovarian cancer cells: involvement of pertussis toxin-sensitive G-protein coupled receptor.

In this study, we observed that lysophosphatidylglycerol (LPG) stimulated intracellular calcium ([Ca(2+)](i)) increase in OVCAR-3 human ovarian cancer cells. LPG-stimulated [Ca(2+)](i) increase was inhibited by U-73122 but not by U-73343, suggesting that LPG stimulates calcium signaling via phospholipase C activation. Moreover, pertussis toxin (PTX) almost completely inhibited [Ca(2+)](i) increase by LPG, indicating the activation of PTX-sensitive G-proteins. LPG-induced [Ca(2+)](i) increase was only observed in OVCAR-3 ovarian cancer cells and SK-OV3 ovarian cancer cells among tested several cell types. LPG also induced extracellular signal-regulated kinase (ERK) and Akt phosphorylation in OVCAR-3 ovarian cancer cells. Pertussis toxin did not affect the LPG-induced activation of ERK and Akt phosphorylation. We also found that LPG failed to stimulate NF-kappaB-driven luciferase activity in exogenously LPA(1), LPA(2), or LPA(3)-transfected HepG2 cells. Taken together we suggest that LPG stimulates a membrane bound receptor which is different from well-known LPA receptors (LPA(1), LPA(2), and LPA(3)), resulting in at least two different signaling cascades; one involves a pertussis toxin-sensitive and phospholipase C-dependent [Ca(2+)](i) increase, and the other involves a pertussis toxin-insensitive activation of ERK and Akt in ovarian cancer cells.

Calcium Signaling↗