Péter Várnai | Semmelweis University (original) (raw)

Papers by Péter Várnai

Research paper thumbnail of A Renox szuperoxid-termelő enzim vizsgálata emlős sejtekben = Characterization of the Renox superoxide-producing enzyme in mammalian cells

1. Kifejlesztettünk egy Nox4 (Renox)-deficiens egérmodellt. Az eddigi vizsgálataink szerint a Nox... more 1. Kifejlesztettünk egy Nox4 (Renox)-deficiens egérmodellt. Az eddigi vizsgálataink szerint a Nox4 jelenléte nem nélkülözhetetlen az élethez és az állatok konstitutív vérképzése is normálisnak tűnik. 2. Kimutattuk, hogy az eddig ismeretlen funkciójú p50RhoGAP fehérje fontos szerepet játszhaz a Rho és a Rab G-fehérjék jelátviteli útvonalainak összekapcsolásában. 3. Dr Thomas Leto laboratóriumával együttműködésben megállapítottuk, hogy a Rac1 kismólsúlyú GTP-kötő fehérje fontos szerepet játszik a Nox1-et és Nox3-at tartalmazó NADPH oxidázok szabályozásában. 4. A Nox4 élettani funkcióját kutatva sikerült azonosítanunk egy olyan új emberi peroxidázt amely fontos szerepet játszhat a reaktív oxigénszármazékok hatásának közvetítésében. | 1. We have developed a Nox4 (Renox)-deficient mouse model. Preliminary analysis of this model indicates that the function of Nox4 is not essential for life and Nox4 has no role in the regulation of constitutive haematopoiesis. 2. We have shown that the p50...

Research paper thumbnail of Calcium entry is regulated by Zn2+ in relation to extracellular ionic environment in human airway epithelial cells

Respiratory Physiology & Neurobiology, 2010

The extracellular pH, sodium and divalent cation concentrations influence the ATP-induced changes... more The extracellular pH, sodium and divalent cation concentrations influence the ATP-induced changes in cytosolic Ca(2+) concentration ([Ca(2+)](i)). This elevation of [Ca(2+)](i) and activation of Ca(2+)-dependent Cl(-) channels represent a possible therapeutic approach in cystic fibrosis (CF). We investigated the changes of [Ca(2+)](i) in different external ionic environment, and P2X purinergic receptors (P2XRs) expression in the control and CF airway epithelial cells. The parallel removal of Na(+) and alkalinization of the extracellular solution increased the amplitude of sustained ATP-induced Ca(2+) signals independent of wild-type or mutant CFTR expression. The ATP-induced Ca(2+) entry was either inhibited or stimulated by Zn(2+) depending on the extracellular Na(+) concentration. In Na(+)-free environment, Zn(2+) and other divalent cations elicited a biphasic Ca(2+) signal. Immunohistochemical data suggest that, multiple subtypes of P2XRs are expressed in these airway epithelial cells. In conclusion, Ca(2+) entry is finely regulated by external ionic environment. Therefore, we speculate that properly compiled aerosols could influence efficacy of zinc-based therapy in CF.

Research paper thumbnail of Chronic repeated restraint stress increases prolactin-releasing peptide/tyrosine-hydroxylase ratio with gender-related differences in the rat brain

Journal of Neurochemistry, 2007

Research paper thumbnail of The MUMO (minimal under-restraining minimal over-restraining) method for the determination of native state ensembles of proteins

Journal of Biomolecular NMR, 2007

While reliable procedures for determining the conformations of proteins are available, methods fo... more While reliable procedures for determining the conformations of proteins are available, methods for generating ensembles of structures that also reflect their flexibility are much less well established. Here we present a systematic assessment of the ability of ensemble-averaged molecular dynamics simulations with ensemble-averaged NMR restraints to simultaneously reproduce the average structure of proteins and their associated dynamics. We discuss the effects that under-restraining (overfitting) and over-restraining (underfitting) have on the structures generated in ensemble-averaged molecular simulations. We then introduce the MUMO (minimal under-restraining minimal over-restraining) method, a procedure in which different observables are averaged over a different number of molecules. As both over-restraining and under-restraining are significantly reduced in the MUMO method, it is possible to generate ensembles of conformations that accurately characterize both the structure and the dynamics of native states of proteins. The application of the MUMO method to the protein ubiquitin yields a high-resolution structural ensemble with an RDC Q-factor of 0.19.

Research paper thumbnail of Dependence of STIM1/Orai1-mediated Calcium Entry on Plasma Membrane Phosphoinositides

Journal of Biological Chemistry, 2009

Recent studies identified two main components of store-operated calcium entry (SOCE): the endopla... more Recent studies identified two main components of store-operated calcium entry (SOCE): the endoplasmic reticulum-localized Ca2+ sensor protein, STIM1, and the plasma membrane (PM)-localized Ca2+ channel, Orai1/CRACM1. In the present study, we investigated the phosphoinositide dependence of Orai1 channel activation in the PM and of STIM1 movements from the tubular to PM-adjacent endoplasmic reticulum regions during Ca2+ store depletion. Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) levels were changed either with agonist stimulation or by chemically induced recruitment of a phosphoinositide 5-phosphatase domain to the PM, whereas PtdIns4P levels were decreased by inhibition or down-regulation of phosphatidylinositol 4-kinases (PI4Ks). Agonist-induced phospholipase C activation and PI4K inhibition, but not isolated PtdIns(4,5)P(2) depletion, substantially reduced endogenous or STIM1/Orai1-mediated SOCE without preventing STIM1 movements toward the PM upon Ca2+ store depletion. Patch clamp analysis of cells overexpressing STIM1 and Orai1 proteins confirmed that phospholipase C activation or PI4K inhibition greatly reduced I(CRAC) currents. These results suggest an inositide requirement of Orai1 activation but not STIM1 movements and indicate that PtdIns4P rather than PtdIns(4,5)P2 is a likely determinant of Orai1 channel activity.

Research paper thumbnail of Live cell imaging of phosphoinositides with expressed inositide binding protein domains

Methods, 2008

Inositol lipids and calcium signaling has been inseparable twins during the 1980s when the molecu... more Inositol lipids and calcium signaling has been inseparable twins during the 1980s when the molecular details of phospholipase C-mediated generation of inositol 1,4,5-trisphosphate (InsP3) and its Ca2+ mobilizing action were discovered. Since then, both the Ca2+ and inositol lipid signaling fields have hugely expanded and the tools allowing dissection of the finest details of their molecular organization also followed closely.

Research paper thumbnail of Mutation in the V2 vasopressin receptor gene, AVPR2, causes nephrogenic syndrome of inappropriate diuresis

Kidney international, 2015

Nephrogenic syndrome of inappropriate antidiuresis (NSIAD) is a recently discovered rare disease ... more Nephrogenic syndrome of inappropriate antidiuresis (NSIAD) is a recently discovered rare disease caused by gain-of-function mutations of the V2 vasopressin receptor gene, AVPR2. To date, mutations of Phe229 and Arg137 have been identified as gain-of-function in the V2 vasopressin receptor (V2R). These receptor mutations lead to hyponatremia, which may lead to clinical symptoms in infants. Here we present a newly identified I130N substitution in exon 2 of the V2R gene in a family, causing NSIAD. This I130N mutation resulted in constitutive activity of the V2R with constitutive cyclic adenosine monophosphate (cAMP) generation in HEK293 cells. This basal activity could be blocked by the inverse agonist tolvaptan and arginine-vasopressin stimulation enhanced the cAMP production of I130N-V2R. The mutation causes a biased receptor conformation as the basal cAMP generation activity of I130N does not lead to interaction with β-arrestin. The constitutive activity of the mutant receptor cause...

Research paper thumbnail of Calcium-Induced Redox Microdimains at the ER-Mitochondrial Interface

Biophysical Journal, 2014

Research paper thumbnail of Measurement of Inositol 1,4,5-Trisphosphate in Living Cells Using an Improved Set of Resonance Energy Transfer-Based Biosensors

PLOS ONE, 2015

Improved versions of inositol-1,4,5-trisphosphate (InsP 3 ) sensors were created to follow intrac... more Improved versions of inositol-1,4,5-trisphosphate (InsP 3 ) sensors were created to follow intracellular InsP 3 changes in single living cells and in cell populations. Similar to previous InsP 3 sensors the new sensors are based on the ligand binding domain of the human type-I InsP 3 receptor (InsP 3 R-LBD), but contain a mutation of either R265K or R269K to lower their InsP 3 binding affinity. Tagging the InsP 3 R-LBD with N-terminal Cerulean and C-terminal Venus allowed measurement of InsP 3 in single-cell FRET experiments. Replacing Cerulean with a Luciferase enzyme allowed experiments in multi-cell format by measuring the change in the BRET signal upon stimulation. These sensors faithfully followed the agonistinduced increase in InsP 3 concentration in HEK 293T cells expressing the Gq-coupled AT1 angiotensin receptor detecting a response to agonist concentration as low as 10 pmol/L. Compared to the wild type InsP 3 sensor, the mutant sensors showed an improved off-rate, enabling a more rapid and complete return of the signal to the resting value of InsP 3 after termination of M3 muscarinic receptor stimulation by atropine. For parallel measurements of intracellular InsP 3 and Ca 2+ levels in BRET experiments, the Cameleon D3 Ca 2+ sensor was modified by replacing its CFP with luciferase. In these experiments depletion of plasma membrane PtdIns(4,5)P 2 resulted in the fall of InsP 3 level, followed by the decrease of the Ca 2+ -signal evoked by the stimulation of the AT1 receptor. In contrast, when type-III PI 4-kinases were inhibited with a high concentration of wortmannin or a more specific inhibitor, A1, the decrease of the Ca 2+ -signal preceded the fall of InsP 3 level indicating an InsP 3 -, independent, direct regulation of capacitative Ca 2+ influx by plasma membrane inositol lipids. Taken together, our results indicate that the improved InsP 3 sensor can be used to monitor both the increase and decrease of InsP 3 levels in live cells suitable for high-throughput BRET applications.

Research paper thumbnail of Regulation and kinetics of angiotensin II-induced gene activation in human and rat adrenocortical cells

Acta Physiologica Hungarica

Research paper thumbnail of Inositol lipid binding and membrane localization of isolated pleckstrin homology (PH) domains. Studies on the PH domains of phospholipase C delta 1 and p130

The Journal of biological chemistry, Jan 26, 2002

The relationship between the ability of isolated pleckstrin homology (PH) domains to bind inosito... more The relationship between the ability of isolated pleckstrin homology (PH) domains to bind inositol lipids or soluble inositol phosphates in vitro and to localize to cellular membranes in live cells was examined by comparing the PH domains of phospholipase Cdelta(1) (PLCdelta(1)) and the recently cloned PLC-like protein p130 fused to the green fluorescent protein (GFP). The prominent membrane localization of PLCdelta(1)PH-GFP was paralleled with high affinity binding to inositol 1,4,5-trisphosphate (InsP(3)) as well as to phosphatidylinositol 4,5-bisphosphate-containing lipid vesicles or nitrocellulose membrane strips. In contrast, no membrane localization was observed with p130PH-GFP despite its InsP(3) and phosphatidylinositol 4,5-bisphosphate-binding properties being comparable with those of PLCdelta(1)PH-GFP. The N-terminal ligand binding domain of the type I InsP(3) receptor also failed to localize to the plasma membrane despite its 5-fold higher affinity to InsP(3) than the PH ...

Research paper thumbnail of Interaction of neuronal calcium sensor-1 (NCS-1) with phosphatidylinositol 4-kinase beta stimulates lipid kinase activity and affects membrane trafficking in COS-7 cells

The Journal of biological chemistry, Jan 26, 2001

Phosphatidylinositol 4-kinases (PI4K) catalyze the first step in the synthesis of phosphatidylino... more Phosphatidylinositol 4-kinases (PI4K) catalyze the first step in the synthesis of phosphatidylinositol 4,5-bisphosphate, an important lipid regulator of several cellular functions. Here we show that the Ca(2+)-binding protein, neuronal calcium sensor-1 (NCS-1), can physically associate with the type III PI4Kbeta with functional consequences affecting the kinase. Recombinant PI4Kbeta, but not its glutathione S-transferase-fused form, showed enhanced PI kinase activity when incubated with recombinant NCS-1, but only if the latter was myristoylated. Similarly, in vitro translated NCS-1, but not its myristoylation-defective mutant, was found associated with recombinant- or in vitro translated PI4Kbeta in PI4Kbeta-immunoprecipitates. When expressed in COS-7 cells, PI4Kbeta and NCS-1 formed a complex that could be immunoprecipitated with antibodies against either proteins, and PI 4-kinase activity was present in anti-NCS-1 immunoprecipitates. Expressed NCS-1-YFP showed co-localization wit...

Research paper thumbnail of How accurately can we image inositol lipids in living cells?

Trends in Pharmacological Sciences, 2000

Search by Subject Search using Medical Subject Headings (< b> MeSH</b>), a controlled... more Search by Subject Search using Medical Subject Headings (< b> MeSH</b>), a controlled vocabulary for indexing life sciences content.< br/> Note that some records do not have MeSH. These include Patents and the latest PubMed and PubMed Central records.

Research paper thumbnail of Visualization of Cellular Phosphoinositide Pools with GFP-Fused Protein-Domains

Current Protocols in Cell Biology, 2001

Research paper thumbnail of Activation of calcium current in voltage-clamped rat glomerulosa cells by potassium ions

The Journal of physiology, Jan 15, 1995

1. We examined Ca2+ influx mechanisms using the whole-cell patch-clamp technique in primary cultu... more 1. We examined Ca2+ influx mechanisms using the whole-cell patch-clamp technique in primary cultures of rat glomerulosa cells. 2. Depolarization of the plasma membrane, as studied by a stepwise or ramp depolarization technique, activated low-threshold, transient (T-type) and high-threshold, long-lasting (L-type) voltage-dependent calcium channels (VDCCs). 3. Extracellular K+ activated an inward current (Ig1), even in voltage-clamped cells. This phenomenon was observed within the physiological concentration range, beginning at 4.6 mM K+o (as opposed to the control level of 3.6 mM K+o). Increased cell conductance and increased background noise indicated that Ig1 is evoked by enhanced channel activity. Potassium induced no outward current in the voltage range examined (-120 to +60 mV). 4. When non-permeable anions were present only in the pipette and Na+ and Mg2+ were omitted from the bath, K+ still activated the current. Ig1 was blocked by 100 microM cadmium but was insensitive to 2 m...

Research paper thumbnail of Investigation of the Fate of Type I Angiotensin Receptor after Biased Activation

Molecular Pharmacology, 2015

Biased agonism on the type I angiotensin receptor (AT1-R) can achieve different outcomes, via act... more Biased agonism on the type I angiotensin receptor (AT1-R) can achieve different outcomes, via activation of G protein-dependent and -independent cellular responses. In this study, we investigated whether the biased activation of the AT1-R can lead to different regulation and intracellular processing of the receptor. We analyzed β-arrestin binding, endocytosis and subsequent trafficking steps such as early and late phases of recycling of the AT1-R in HEK293 cells expressing wild type or biased mutant receptors in response to different ligands. We used Renilla luciferase tagged receptors and yellow fluorescent protein (YFP) tagged β-arrestin2, Rab5, Rab7 and Rab11 proteins in bioluminescence resonance energy transfer (BRET) measurements to follow the fate of the receptor after stimulation. We found that not only is the signaling of the receptor different upon using selective ligands, but the fate within the cells is also determined by the type of the stimulation. β-arrestin binding and the internalization kinetics of the angiotensin II (AngII)-stimulated AT1-R differed from those stimulated by the biased agonists. Similarly, AngII-stimulated wild type AT1-R showed differences compared to a biased mutant AT1-R (DRY/AAY AT1-R) regarding β-arrestin binding and endocytosis. We suggest that the differences in the internalization kinetics of the receptor in response to biased agonist stimulation are due to the differences in plasma membrane PtdIns(4,5)P2 depletion. Moreover, the stability of the β-arrestin binding is a major determinant of the later fate of the internalized AT1-R receptor.

Research paper thumbnail of Active Arf6 recruits ARNO/cytohesin GEFs to the PM by binding their PH domains

Molecular biology of the cell, 2007

ARNO is a soluble guanine nucleotide exchange factor (GEF) for the Arf family of GTPases. Althoug... more ARNO is a soluble guanine nucleotide exchange factor (GEF) for the Arf family of GTPases. Although in biochemical assays ARNO prefers Arf1 over Arf6 as a substrate, its localization in cells at the plasma membrane (PM) suggests an interaction with Arf6. In this study, we found that ARNO activated Arf1 in HeLa and COS-7 cells resulting in the recruitment of Arf1 on to dynamic PM ruffles. By contrast, Arf6 was activated less by ARNO than EFA6, a canonical Arf6 GEF. Remarkably, Arf6 in its GTP-bound form recruited ARNO to the PM and the two proteins could be immunoprecipitated. ARNO binding to Arf6 was not mediated through the catalytic Sec7 domain, but via the pleckstrin homology (PH) domain. Active Arf6 also bound the PH domain of Grp1, another ARNO family member. This interaction was direct and required both inositol phospholipids and GTP. We propose a model of sequential Arf activation at the PM whereby Arf6-GTP recruits ARNO family GEFs for further activation of other Arf isoforms.

Research paper thumbnail of Trans-mitochondrial coordination of cristae at regulated membrane junctions

Nature Communications, 2015

Reminiscent of bacterial quorum sensing, mammalian mitochondria participate in interorganelle com... more Reminiscent of bacterial quorum sensing, mammalian mitochondria participate in interorganelle communication. However, physical structures that enhance or enable interactions between mitochondria have not been defined. Here we report that adjacent mitochondria exhibit coordination of inner mitochondrial membrane cristae at inter-mitochondrial junctions (IMJs). These electron-dense structures are conserved across species, resistant to genetic disruption of cristae organization, dynamically modulated by mitochondrial bioenergetics, independent of known inter-mitochondrial tethering proteins mitofusins and rapidly induced by the stable rapprochement of organelles via inducible synthetic linker technology. At the associated junctions, the cristae of adjacent mitochondria form parallel arrays perpendicular to the IMJ, consistent with a role in electrochemical coupling. These IMJs and associated cristae arrays may provide the structural basis to enhance the propagation of intracellular bioenergetic and apoptotic waves through mitochondrial networks within cells.

Research paper thumbnail of STIM and Orai: the long-awaited constituents of store-operated calcium entry

Trends in Pharmacological Sciences, 2009

Rapid changes in cytosolic Ca 2+ concentrations [Ca 2+ ] i are the most commonly used signals in ... more Rapid changes in cytosolic Ca 2+ concentrations [Ca 2+ ] i are the most commonly used signals in biology to regulate a whole host of cellular functions including contraction, secretion and gene activation. A widely utilized form of Ca 2+ influx is termed store-operated Ca 2+ entry (SOCE) due to its control by the Ca 2+ content of the endoplasmic reticulum (ER). The underlying molecular mechanism of SOCE has eluded identification until recently when two groups of proteins, the ER Ca 2+ sensors, STIM1 and -2, and the plasma membrane channels, Orai1, -2 and -3 have been identified. These landmark discoveries have allowed impressive progress in clarifying how these proteins work in concert and what developmental and cellular processes require their participation most. As we begin to better understand the biology of the STIM and Orai proteins, the attention to the pharmacological tools to influence their functions quickly follow suit. This review will briefly summarize recent developments in this exciting area of Ca 2+ signaling.

Research paper thumbnail of The effect of hormone-induced PtdIns (4, 5) P2 depletion on endocytosis suggests the importance of local regulation of inositol lipid signalling

Endocrine Abstracts, 2014

Research paper thumbnail of A Renox szuperoxid-termelő enzim vizsgálata emlős sejtekben = Characterization of the Renox superoxide-producing enzyme in mammalian cells

1. Kifejlesztettünk egy Nox4 (Renox)-deficiens egérmodellt. Az eddigi vizsgálataink szerint a Nox... more 1. Kifejlesztettünk egy Nox4 (Renox)-deficiens egérmodellt. Az eddigi vizsgálataink szerint a Nox4 jelenléte nem nélkülözhetetlen az élethez és az állatok konstitutív vérképzése is normálisnak tűnik. 2. Kimutattuk, hogy az eddig ismeretlen funkciójú p50RhoGAP fehérje fontos szerepet játszhaz a Rho és a Rab G-fehérjék jelátviteli útvonalainak összekapcsolásában. 3. Dr Thomas Leto laboratóriumával együttműködésben megállapítottuk, hogy a Rac1 kismólsúlyú GTP-kötő fehérje fontos szerepet játszik a Nox1-et és Nox3-at tartalmazó NADPH oxidázok szabályozásában. 4. A Nox4 élettani funkcióját kutatva sikerült azonosítanunk egy olyan új emberi peroxidázt amely fontos szerepet játszhat a reaktív oxigénszármazékok hatásának közvetítésében. | 1. We have developed a Nox4 (Renox)-deficient mouse model. Preliminary analysis of this model indicates that the function of Nox4 is not essential for life and Nox4 has no role in the regulation of constitutive haematopoiesis. 2. We have shown that the p50...

Research paper thumbnail of Calcium entry is regulated by Zn2+ in relation to extracellular ionic environment in human airway epithelial cells

Respiratory Physiology & Neurobiology, 2010

The extracellular pH, sodium and divalent cation concentrations influence the ATP-induced changes... more The extracellular pH, sodium and divalent cation concentrations influence the ATP-induced changes in cytosolic Ca(2+) concentration ([Ca(2+)](i)). This elevation of [Ca(2+)](i) and activation of Ca(2+)-dependent Cl(-) channels represent a possible therapeutic approach in cystic fibrosis (CF). We investigated the changes of [Ca(2+)](i) in different external ionic environment, and P2X purinergic receptors (P2XRs) expression in the control and CF airway epithelial cells. The parallel removal of Na(+) and alkalinization of the extracellular solution increased the amplitude of sustained ATP-induced Ca(2+) signals independent of wild-type or mutant CFTR expression. The ATP-induced Ca(2+) entry was either inhibited or stimulated by Zn(2+) depending on the extracellular Na(+) concentration. In Na(+)-free environment, Zn(2+) and other divalent cations elicited a biphasic Ca(2+) signal. Immunohistochemical data suggest that, multiple subtypes of P2XRs are expressed in these airway epithelial cells. In conclusion, Ca(2+) entry is finely regulated by external ionic environment. Therefore, we speculate that properly compiled aerosols could influence efficacy of zinc-based therapy in CF.

Research paper thumbnail of Chronic repeated restraint stress increases prolactin-releasing peptide/tyrosine-hydroxylase ratio with gender-related differences in the rat brain

Journal of Neurochemistry, 2007

Research paper thumbnail of The MUMO (minimal under-restraining minimal over-restraining) method for the determination of native state ensembles of proteins

Journal of Biomolecular NMR, 2007

While reliable procedures for determining the conformations of proteins are available, methods fo... more While reliable procedures for determining the conformations of proteins are available, methods for generating ensembles of structures that also reflect their flexibility are much less well established. Here we present a systematic assessment of the ability of ensemble-averaged molecular dynamics simulations with ensemble-averaged NMR restraints to simultaneously reproduce the average structure of proteins and their associated dynamics. We discuss the effects that under-restraining (overfitting) and over-restraining (underfitting) have on the structures generated in ensemble-averaged molecular simulations. We then introduce the MUMO (minimal under-restraining minimal over-restraining) method, a procedure in which different observables are averaged over a different number of molecules. As both over-restraining and under-restraining are significantly reduced in the MUMO method, it is possible to generate ensembles of conformations that accurately characterize both the structure and the dynamics of native states of proteins. The application of the MUMO method to the protein ubiquitin yields a high-resolution structural ensemble with an RDC Q-factor of 0.19.

Research paper thumbnail of Dependence of STIM1/Orai1-mediated Calcium Entry on Plasma Membrane Phosphoinositides

Journal of Biological Chemistry, 2009

Recent studies identified two main components of store-operated calcium entry (SOCE): the endopla... more Recent studies identified two main components of store-operated calcium entry (SOCE): the endoplasmic reticulum-localized Ca2+ sensor protein, STIM1, and the plasma membrane (PM)-localized Ca2+ channel, Orai1/CRACM1. In the present study, we investigated the phosphoinositide dependence of Orai1 channel activation in the PM and of STIM1 movements from the tubular to PM-adjacent endoplasmic reticulum regions during Ca2+ store depletion. Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) levels were changed either with agonist stimulation or by chemically induced recruitment of a phosphoinositide 5-phosphatase domain to the PM, whereas PtdIns4P levels were decreased by inhibition or down-regulation of phosphatidylinositol 4-kinases (PI4Ks). Agonist-induced phospholipase C activation and PI4K inhibition, but not isolated PtdIns(4,5)P(2) depletion, substantially reduced endogenous or STIM1/Orai1-mediated SOCE without preventing STIM1 movements toward the PM upon Ca2+ store depletion. Patch clamp analysis of cells overexpressing STIM1 and Orai1 proteins confirmed that phospholipase C activation or PI4K inhibition greatly reduced I(CRAC) currents. These results suggest an inositide requirement of Orai1 activation but not STIM1 movements and indicate that PtdIns4P rather than PtdIns(4,5)P2 is a likely determinant of Orai1 channel activity.

Research paper thumbnail of Live cell imaging of phosphoinositides with expressed inositide binding protein domains

Methods, 2008

Inositol lipids and calcium signaling has been inseparable twins during the 1980s when the molecu... more Inositol lipids and calcium signaling has been inseparable twins during the 1980s when the molecular details of phospholipase C-mediated generation of inositol 1,4,5-trisphosphate (InsP3) and its Ca2+ mobilizing action were discovered. Since then, both the Ca2+ and inositol lipid signaling fields have hugely expanded and the tools allowing dissection of the finest details of their molecular organization also followed closely.

Research paper thumbnail of Mutation in the V2 vasopressin receptor gene, AVPR2, causes nephrogenic syndrome of inappropriate diuresis

Kidney international, 2015

Nephrogenic syndrome of inappropriate antidiuresis (NSIAD) is a recently discovered rare disease ... more Nephrogenic syndrome of inappropriate antidiuresis (NSIAD) is a recently discovered rare disease caused by gain-of-function mutations of the V2 vasopressin receptor gene, AVPR2. To date, mutations of Phe229 and Arg137 have been identified as gain-of-function in the V2 vasopressin receptor (V2R). These receptor mutations lead to hyponatremia, which may lead to clinical symptoms in infants. Here we present a newly identified I130N substitution in exon 2 of the V2R gene in a family, causing NSIAD. This I130N mutation resulted in constitutive activity of the V2R with constitutive cyclic adenosine monophosphate (cAMP) generation in HEK293 cells. This basal activity could be blocked by the inverse agonist tolvaptan and arginine-vasopressin stimulation enhanced the cAMP production of I130N-V2R. The mutation causes a biased receptor conformation as the basal cAMP generation activity of I130N does not lead to interaction with β-arrestin. The constitutive activity of the mutant receptor cause...

Research paper thumbnail of Calcium-Induced Redox Microdimains at the ER-Mitochondrial Interface

Biophysical Journal, 2014

Research paper thumbnail of Measurement of Inositol 1,4,5-Trisphosphate in Living Cells Using an Improved Set of Resonance Energy Transfer-Based Biosensors

PLOS ONE, 2015

Improved versions of inositol-1,4,5-trisphosphate (InsP 3 ) sensors were created to follow intrac... more Improved versions of inositol-1,4,5-trisphosphate (InsP 3 ) sensors were created to follow intracellular InsP 3 changes in single living cells and in cell populations. Similar to previous InsP 3 sensors the new sensors are based on the ligand binding domain of the human type-I InsP 3 receptor (InsP 3 R-LBD), but contain a mutation of either R265K or R269K to lower their InsP 3 binding affinity. Tagging the InsP 3 R-LBD with N-terminal Cerulean and C-terminal Venus allowed measurement of InsP 3 in single-cell FRET experiments. Replacing Cerulean with a Luciferase enzyme allowed experiments in multi-cell format by measuring the change in the BRET signal upon stimulation. These sensors faithfully followed the agonistinduced increase in InsP 3 concentration in HEK 293T cells expressing the Gq-coupled AT1 angiotensin receptor detecting a response to agonist concentration as low as 10 pmol/L. Compared to the wild type InsP 3 sensor, the mutant sensors showed an improved off-rate, enabling a more rapid and complete return of the signal to the resting value of InsP 3 after termination of M3 muscarinic receptor stimulation by atropine. For parallel measurements of intracellular InsP 3 and Ca 2+ levels in BRET experiments, the Cameleon D3 Ca 2+ sensor was modified by replacing its CFP with luciferase. In these experiments depletion of plasma membrane PtdIns(4,5)P 2 resulted in the fall of InsP 3 level, followed by the decrease of the Ca 2+ -signal evoked by the stimulation of the AT1 receptor. In contrast, when type-III PI 4-kinases were inhibited with a high concentration of wortmannin or a more specific inhibitor, A1, the decrease of the Ca 2+ -signal preceded the fall of InsP 3 level indicating an InsP 3 -, independent, direct regulation of capacitative Ca 2+ influx by plasma membrane inositol lipids. Taken together, our results indicate that the improved InsP 3 sensor can be used to monitor both the increase and decrease of InsP 3 levels in live cells suitable for high-throughput BRET applications.

Research paper thumbnail of Regulation and kinetics of angiotensin II-induced gene activation in human and rat adrenocortical cells

Acta Physiologica Hungarica

Research paper thumbnail of Inositol lipid binding and membrane localization of isolated pleckstrin homology (PH) domains. Studies on the PH domains of phospholipase C delta 1 and p130

The Journal of biological chemistry, Jan 26, 2002

The relationship between the ability of isolated pleckstrin homology (PH) domains to bind inosito... more The relationship between the ability of isolated pleckstrin homology (PH) domains to bind inositol lipids or soluble inositol phosphates in vitro and to localize to cellular membranes in live cells was examined by comparing the PH domains of phospholipase Cdelta(1) (PLCdelta(1)) and the recently cloned PLC-like protein p130 fused to the green fluorescent protein (GFP). The prominent membrane localization of PLCdelta(1)PH-GFP was paralleled with high affinity binding to inositol 1,4,5-trisphosphate (InsP(3)) as well as to phosphatidylinositol 4,5-bisphosphate-containing lipid vesicles or nitrocellulose membrane strips. In contrast, no membrane localization was observed with p130PH-GFP despite its InsP(3) and phosphatidylinositol 4,5-bisphosphate-binding properties being comparable with those of PLCdelta(1)PH-GFP. The N-terminal ligand binding domain of the type I InsP(3) receptor also failed to localize to the plasma membrane despite its 5-fold higher affinity to InsP(3) than the PH ...

Research paper thumbnail of Interaction of neuronal calcium sensor-1 (NCS-1) with phosphatidylinositol 4-kinase beta stimulates lipid kinase activity and affects membrane trafficking in COS-7 cells

The Journal of biological chemistry, Jan 26, 2001

Phosphatidylinositol 4-kinases (PI4K) catalyze the first step in the synthesis of phosphatidylino... more Phosphatidylinositol 4-kinases (PI4K) catalyze the first step in the synthesis of phosphatidylinositol 4,5-bisphosphate, an important lipid regulator of several cellular functions. Here we show that the Ca(2+)-binding protein, neuronal calcium sensor-1 (NCS-1), can physically associate with the type III PI4Kbeta with functional consequences affecting the kinase. Recombinant PI4Kbeta, but not its glutathione S-transferase-fused form, showed enhanced PI kinase activity when incubated with recombinant NCS-1, but only if the latter was myristoylated. Similarly, in vitro translated NCS-1, but not its myristoylation-defective mutant, was found associated with recombinant- or in vitro translated PI4Kbeta in PI4Kbeta-immunoprecipitates. When expressed in COS-7 cells, PI4Kbeta and NCS-1 formed a complex that could be immunoprecipitated with antibodies against either proteins, and PI 4-kinase activity was present in anti-NCS-1 immunoprecipitates. Expressed NCS-1-YFP showed co-localization wit...

Research paper thumbnail of How accurately can we image inositol lipids in living cells?

Trends in Pharmacological Sciences, 2000

Search by Subject Search using Medical Subject Headings (< b> MeSH</b>), a controlled... more Search by Subject Search using Medical Subject Headings (< b> MeSH</b>), a controlled vocabulary for indexing life sciences content.< br/> Note that some records do not have MeSH. These include Patents and the latest PubMed and PubMed Central records.

Research paper thumbnail of Visualization of Cellular Phosphoinositide Pools with GFP-Fused Protein-Domains

Current Protocols in Cell Biology, 2001

Research paper thumbnail of Activation of calcium current in voltage-clamped rat glomerulosa cells by potassium ions

The Journal of physiology, Jan 15, 1995

1. We examined Ca2+ influx mechanisms using the whole-cell patch-clamp technique in primary cultu... more 1. We examined Ca2+ influx mechanisms using the whole-cell patch-clamp technique in primary cultures of rat glomerulosa cells. 2. Depolarization of the plasma membrane, as studied by a stepwise or ramp depolarization technique, activated low-threshold, transient (T-type) and high-threshold, long-lasting (L-type) voltage-dependent calcium channels (VDCCs). 3. Extracellular K+ activated an inward current (Ig1), even in voltage-clamped cells. This phenomenon was observed within the physiological concentration range, beginning at 4.6 mM K+o (as opposed to the control level of 3.6 mM K+o). Increased cell conductance and increased background noise indicated that Ig1 is evoked by enhanced channel activity. Potassium induced no outward current in the voltage range examined (-120 to +60 mV). 4. When non-permeable anions were present only in the pipette and Na+ and Mg2+ were omitted from the bath, K+ still activated the current. Ig1 was blocked by 100 microM cadmium but was insensitive to 2 m...

Research paper thumbnail of Investigation of the Fate of Type I Angiotensin Receptor after Biased Activation

Molecular Pharmacology, 2015

Biased agonism on the type I angiotensin receptor (AT1-R) can achieve different outcomes, via act... more Biased agonism on the type I angiotensin receptor (AT1-R) can achieve different outcomes, via activation of G protein-dependent and -independent cellular responses. In this study, we investigated whether the biased activation of the AT1-R can lead to different regulation and intracellular processing of the receptor. We analyzed β-arrestin binding, endocytosis and subsequent trafficking steps such as early and late phases of recycling of the AT1-R in HEK293 cells expressing wild type or biased mutant receptors in response to different ligands. We used Renilla luciferase tagged receptors and yellow fluorescent protein (YFP) tagged β-arrestin2, Rab5, Rab7 and Rab11 proteins in bioluminescence resonance energy transfer (BRET) measurements to follow the fate of the receptor after stimulation. We found that not only is the signaling of the receptor different upon using selective ligands, but the fate within the cells is also determined by the type of the stimulation. β-arrestin binding and the internalization kinetics of the angiotensin II (AngII)-stimulated AT1-R differed from those stimulated by the biased agonists. Similarly, AngII-stimulated wild type AT1-R showed differences compared to a biased mutant AT1-R (DRY/AAY AT1-R) regarding β-arrestin binding and endocytosis. We suggest that the differences in the internalization kinetics of the receptor in response to biased agonist stimulation are due to the differences in plasma membrane PtdIns(4,5)P2 depletion. Moreover, the stability of the β-arrestin binding is a major determinant of the later fate of the internalized AT1-R receptor.

Research paper thumbnail of Active Arf6 recruits ARNO/cytohesin GEFs to the PM by binding their PH domains

Molecular biology of the cell, 2007

ARNO is a soluble guanine nucleotide exchange factor (GEF) for the Arf family of GTPases. Althoug... more ARNO is a soluble guanine nucleotide exchange factor (GEF) for the Arf family of GTPases. Although in biochemical assays ARNO prefers Arf1 over Arf6 as a substrate, its localization in cells at the plasma membrane (PM) suggests an interaction with Arf6. In this study, we found that ARNO activated Arf1 in HeLa and COS-7 cells resulting in the recruitment of Arf1 on to dynamic PM ruffles. By contrast, Arf6 was activated less by ARNO than EFA6, a canonical Arf6 GEF. Remarkably, Arf6 in its GTP-bound form recruited ARNO to the PM and the two proteins could be immunoprecipitated. ARNO binding to Arf6 was not mediated through the catalytic Sec7 domain, but via the pleckstrin homology (PH) domain. Active Arf6 also bound the PH domain of Grp1, another ARNO family member. This interaction was direct and required both inositol phospholipids and GTP. We propose a model of sequential Arf activation at the PM whereby Arf6-GTP recruits ARNO family GEFs for further activation of other Arf isoforms.

Research paper thumbnail of Trans-mitochondrial coordination of cristae at regulated membrane junctions

Nature Communications, 2015

Reminiscent of bacterial quorum sensing, mammalian mitochondria participate in interorganelle com... more Reminiscent of bacterial quorum sensing, mammalian mitochondria participate in interorganelle communication. However, physical structures that enhance or enable interactions between mitochondria have not been defined. Here we report that adjacent mitochondria exhibit coordination of inner mitochondrial membrane cristae at inter-mitochondrial junctions (IMJs). These electron-dense structures are conserved across species, resistant to genetic disruption of cristae organization, dynamically modulated by mitochondrial bioenergetics, independent of known inter-mitochondrial tethering proteins mitofusins and rapidly induced by the stable rapprochement of organelles via inducible synthetic linker technology. At the associated junctions, the cristae of adjacent mitochondria form parallel arrays perpendicular to the IMJ, consistent with a role in electrochemical coupling. These IMJs and associated cristae arrays may provide the structural basis to enhance the propagation of intracellular bioenergetic and apoptotic waves through mitochondrial networks within cells.

Research paper thumbnail of STIM and Orai: the long-awaited constituents of store-operated calcium entry

Trends in Pharmacological Sciences, 2009

Rapid changes in cytosolic Ca 2+ concentrations [Ca 2+ ] i are the most commonly used signals in ... more Rapid changes in cytosolic Ca 2+ concentrations [Ca 2+ ] i are the most commonly used signals in biology to regulate a whole host of cellular functions including contraction, secretion and gene activation. A widely utilized form of Ca 2+ influx is termed store-operated Ca 2+ entry (SOCE) due to its control by the Ca 2+ content of the endoplasmic reticulum (ER). The underlying molecular mechanism of SOCE has eluded identification until recently when two groups of proteins, the ER Ca 2+ sensors, STIM1 and -2, and the plasma membrane channels, Orai1, -2 and -3 have been identified. These landmark discoveries have allowed impressive progress in clarifying how these proteins work in concert and what developmental and cellular processes require their participation most. As we begin to better understand the biology of the STIM and Orai proteins, the attention to the pharmacological tools to influence their functions quickly follow suit. This review will briefly summarize recent developments in this exciting area of Ca 2+ signaling.

Research paper thumbnail of The effect of hormone-induced PtdIns (4, 5) P2 depletion on endocytosis suggests the importance of local regulation of inositol lipid signalling

Endocrine Abstracts, 2014