Biomedical subjects

C Romanin

Publications and source records attributed to C Romanin.

At least 19 recordsLinked to original sources

PubMed2001

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.

Anthranilate Synthase↗

PubMed2001

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.

Calcium Channel Blockers↗

PubMed2001

Single-molecule imaging of l-type Ca(2+) channels in live cells.

L-type Ca(2+) channels are an important means by which a cell regulates the Ca(2+) influx into the cytosol on electrical stimulation. Their structure and dynamics in the plasma membrane, including their molecular mobility and aggregation, is of key interest for the in-depth understanding of their function. Construction of a fluorescent variant by fusion of the yellow-fluorescent protein to the ion channel and expression in a human cell line allowed us to address its dynamic embedding in the membrane at the level of individual channels in vivo. We report on the observation of individual fluorescence-labeled human cardiac L-type Ca(2+) channels using wide-field fluorescence microscopy in living cells. Our fluorescence and electrophysiological data indicate that L-type Ca(2+) channels tend to form larger aggregates which are mobile in the plasma membrane.

Bacterial Proteins↗

PubMed2001

The elderly cancer patient: a nursing perspective.

Since cancer incidence tends to increase with age, health professionals will encounter ever-greater numbers of older people with cancer. Elderly cancer patients present complex problems that need comprehensive physical and psychosocial support. In order to give specialised care to this segment of the population, a multidisciplinary approach must be used; only in this way can an individualised treatment program be provided. Oncology nurses are an important component of this team and can contribute significantly to the panorama of needs of this segment of the population, which include the prevention and early detection of cancer, the use of state-of-the-art treatments, patient education, care during and after hospitalisation and quality of life (QOL) issues. In this way, the older person with cancer can be treated in an optimal manner and survival can hopefully be improved in a meaningful way.

Aged↗

PubMed2000

Ca(2+) sensors of L-type Ca(2+) channel.

Ca(2+)-induced inactivation of L-type Ca(2+) is differentially mediated by two C-terminal motifs of the alpha(1C) subunit, L (1572-1587) and K (1599-1651) implicated for calmodulin binding. We found that motif L is composed of a highly selective Ca(2+) sensor and an adjacent Ca(2+)-independent tethering site for calmodulin. The Ca(2+) sensor contributes to higher Ca(2+) sensitivity of the motif L complex with calmodulin. Since only combined mutation of both sites removes Ca(2+)-dependent current decay, the two-site modulation by Ca(2+) and calmodulin may underlie Ca(2+)-induced inactivation of the channel.

Amino Acid Sequence↗

PubMed2000

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.

Amino Acid Sequence↗

PubMed2000

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.

Animals↗

PubMed2000

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.

Amino Acid Sequence↗

PubMed2000

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.

Animals↗

PubMed2000

ATP-induced activation of expressed RyR3 at low free calcium.

Ca(2+) channel properties of the mink ryanodine receptor type 3 (RyR3), expressed in HEK293 cells, were studied in planar lipid bilayers to which RyR3 rich membrane fragments had been fused. RyR3 channels were not active at resting levels of Ca(2+)(free) but were gated by an additional 1 mM ATP, exhibiting long open times. The second major finding was the absence of channel inactivation at millimolar Ca(2+)(free). Insertion of a myc tag at the N-terminus of RyR3 did not affect the channel properties. As to skeletal muscle, the observed type 3 channel properties appear physiologically meaningful by assisting type 1 channels in calcium release.

Adenosine Triphosphate↗

PubMed1998

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.

Calcium Channels↗

PubMed1998

Removing non-random artifacts from patch clamp traces.

Analysis and interpretation of current records from cellular membranes or lipid bilayers is complicated by perturbations. These perturbations can originate from random noise or deterministic artifacts as capacitive currents caused by step depolarization's, drift, 50 Hz pick up or microphonics. Accurate removal of deterministic perturbations is a prerequisite for further analysis of ion channel kinetics. In this report we present two methods developed for parameter estimation of such artifacts in order to remove these perturbations from single channel patch clamp traces. For both methods artifact removal does not require sweeps lacking channel activity. The first method was designed to extract artifacts from sweeps showing moderate channel activity. Within a certain number of sweeps the artifacts should remain rather constant. The second method allows for artifact removal from one individual sweep with channel activity. With the second method, correction for drift, pick up and microphonics is possible without long periods of minor channel activity. To evaluate the correctness of artifact removal a specific test was set up. The methods were carefully examined using simulated records for a wide range of parameters found in single channel experiments. Further, the developed algorithms were applied to original records obtained from cardiac and recombinant L-type Ca2+ channels.

Algorithms↗

PubMed1997

Essential role of the beta subunit in modulation of C-class L-type Ca2+ channels by intracellular pH.

Elevation of intracellular pH (pHi) enhances the activity of native L-type Ca2+ channels in cardiac and smooth muscle. We studied the modulation by pHi of expressed L-type Ca2+ channels comprised of either the alpha1c subunits alone or of alpha1c plus beta2a subunits. Ca2+ channels were expressed in human embryonic kidney cells (HEK 293) and pHi was increased from a basal level of 7.3 to 8.3 by exposure of cells to NH4Cl (20 mM) or by elevation of extracellular pH to 8.5. Elevation of pHi enhanced the activity of Ca2+ channels derived by coexpression of alpah1c and beta2a subunits. This alkalosis-induced stimulation of channel activity was mainly due to an increase in channel availability. Channels derived by expression of alpha1c alone were not affected by intracellular alkalosis. Our results demonstrate that the pHi sensitivity of L-type Ca2+ channels is conferred by the beta subunit of the channel complex.

Ammonium Chloride↗

PubMed1997

Intracellular Ca2+ inactivates L-type Ca2+ channels with a Hill coefficient of approximately 1 and an inhibition constant of approximately 4 microM by reducing channel's open probability.

The patch-clamp technique was used to characterize the mechanism of Ca2+-induced inactivation of cardiac L-type Ca2+ channel alpha(1C-a) + beta3 subunits stably expressed in CHO cells. Single Ca2+ channel activity was monitored with 96 mM Ba2+ as charge carrier in the presence of 2.5 microM (-)BAYK 8644 and calpastatin plus ATP. This enabled stabilization of channel activity in the inside-out patch and allowed for application of steady-state Ca2+ concentrations to the intracellular face of excised membrane patches in an attempt to provoke Ca2+-induced inactivation. Inactivation was found to occur specifically with Ca2+ since it was not observed upon application of Ba2+. Ca2+-dependent inhibition of mean Ca2+ channel activity was characterized by a Hill coefficient close to 1. Ca2+ binding to open and closed states of the channel obtained during depolarization apparently occurred with similar affinity yielding half-maximal inhibition of Ca2+ channel activity at approximately 4 microM. This inhibition manifested predominantly in a reduction of the channel's open probability whereas availability remained almost unchanged. The reduction in open probability was achieved by an increase in first latencies and a decrease in channel opening frequency as well as channel open times. At high (12-28 microM) Ca2+ concentrations, 72% of inhibition occurred due to a stabilization of the closed state and the remaining 28% by a destabilization of the open state. Our results suggest that binding of one calcium ion to a regulatory domain induces a complex alteration in the kinetic properties of the Ca2+ channel and support the idea of a single EF hand motif as the relevant Ca2+ binding site on the alpha1 subunit.

Animals↗

PubMed1997

Estimating the number of channels in patch-clamp recordings: application to kinetic analysis of multichannel data from voltage-operated channels.

Important kinetic information of voltage-operated ion channels can be obtained by estimating the open probability, the availability, and the first latency, and by applying run analysis. In the case of multichannel patches, estimation of the number of available channels is a prerequisite for the above analysis. Here we describe a method for calculation of the a posteriori probability of the number of available channels in each sweep by using the Bayes formula. This probability serves as a measure for the number of channels and allows for first latency determination and run analysis. The methods described were applied to simulated and experimental data obtained from L-type Ca2+ channel recordings.

Bayes Theorem↗

PubMed1997

Intracellular Ca2+ inhibits smooth muscle L-type Ca2+ channels by activation of protein phosphatase type 2B and by direct interaction with the channel.

Modulation of L-type Ca2+ channels by tonic elevation of cytoplasmic Ca2+ was investigated in intact cells and inside-out patches from human umbilical vein smooth muscle. Ba2+ was used as charge carrier, and run down of Ca2+ channel activity in inside-out patches was prevented with calpastatin plus ATP. Increasing cytoplasmic Ca2+ in intact cells by elevation of extracellular Ca2+ in the presence of the ionophore A23187 inhibited the activity of L-type Ca2+ channels in cell-attached patches. Measurement of the actual level of intracellular free Ca2+ with fura-2 revealed a 50% inhibitory concentration (IC50) of 260 nM and a Hill coefficient close to 4 for Ca2+- dependent inhibition. Ca2+-induced inhibition of Ca2+ channel activity in intact cells was due to a reduction of channel open probability and availability. Ca2+-induced inhibition was not affected by the protein kinase inhibitor H-7 (10 microM) or the cytoskeleton disruptive agent cytochalasin B (20 microM), but prevented by cyclosporin A (1 microg/ ml), an inhibitor of protein phosphatase 2B (calcineurin). Elevation of Ca2+ at the cytoplasmic side of inside-out patches inhibited Ca2+ channels with an IC50 of 2 microM and a Hill coefficient close to unity. Direct Ca2+-dependent inhibition in cell-free patches was due to a reduction of open probability, whereas availability was barely affected. Application of purified protein phosphatase 2B (12 U/ml) to the cytoplasmic side of inside-out patches at a free Ca2+ concentration of 1 microM inhibited Ca2+ channel open probability and availability. Elevation of cytoplasmic Ca2+ in the presence of PP2B, suppressed channel activity in inside-out patches with an IC50 of approximately 380 nM and a Hill coefficient of approximately 3; i.e., characteristics reminiscent of the Ca2+ sensitivity of Ca2+ channels in intact cells. Our results suggest that L-type Ca2+ channels of smooth muscle are controlled by two Ca2+-dependent negative feedback mechanisms. These mechanisms are based on (a) a protein phosphatase 2B-mediated dephosphorylation process, and (b) the interaction of intracellular Ca2+ with a single membrane-associated site that may reside on the channel protein itself.

Calcineurin↗

PubMed1996

A type 2A phosphatase-sensitive phosphorylation site controls modal gating of L-type Ca2+ channels in human vascular smooth-muscle cells.

The patch-clamp technique was employed to investigate phosphorylation/dephosphorylation-dependent modulation of L-type Ca2+ channels in smooth-muscle cells isolated from human umbilical vein. Okadaic acid, an inhibitor of phosphoprotein phosphatases type 1 (PP1) and 2A (PP2A), increased the probability of channels being in the open state (Po) in intact cells. This increase in Po was due mainly to promotion of long-lasting channel openings, i.e. promotion of 'mode 2' gating behaviour. Exposure of the cytoplasmic side of excised patches of membrane to the purified catalytic subunit of PP2A (PP2Ac) resulted in the opposite modulation of channel function. PP2Ac (0.2 unit/ml) reduced the Po of Ca2+ channels mainly via suppression of 'mode 2' gating. This effect of PP2Ac was completely prevented by 1 microM okadaic acid. The catalytic subunit of PPI (0.2 unit/ml), however, barely affected channel activity. Our results provide evidence for a PP2A-sensitive regulatory site that controls modal gating of L-type Ca2+ channels in smooth muscle.

Calcium Channels↗

PubMed1996

Inhibition of a store-operated Ca2+ entry pathway in human endothelial cells by the isoquinoline derivative LOE 908.

1. The novel cation channel blocker, LOE 908, was tested for its effects on Ca2+ entry and membrane currents activated by depletion of intracellular Ca2+ stores in human endothelial cells. 2. LOE 908 inhibited store-operated Ca2+ entry induced by direct depletion of Ca2+ stores with 100 nM thapsigargin or 100 nM ionomycin with an EC50 of 2 microM and 4 microM, respectively. 3. LOE 908 did not affect thapsigargin- or ionomycin-induced Ca2+ release from intracellular stores up to concentrations of 3 microM. 4. LOE 908 reversibly suppressed thapsigargin- as well as ionomycin-induced whole-cell membrane currents. 5. The LOE 908-sensitive membrane conductance corresponded to a cation permeability of 5.5 and 6.9 fold selectivity for Ca2+ over K+ in the presence of thapsigargin and ionomycin, respectively. 6. Our results suggest that the isoquinoline, LOE 908 is a novel, potent inhibitor of the store-operated (capacitive) Ca2+ entry pathway in endothelial cells.

Acetamides↗