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1.

001-es BibID:BIBFORM049403
Első szerző:Almássy János (élettanász, biológus, angol-magyar szakfordító)
Cím:Investigation of ryanodine receptor using peptide toxins / J. Almassy, S. Sarkozi, I. Jona
Dátum:2010
ISSN:0231-424X 1588-2683
Megjegyzések:Action potential of the skeletal muscle surface membrane leads to Ca2+ release from the sarcoplasmic reticulum evoked by direct coupling between dihydropyridine receptor (DHPR) and Ca2+ release channel (RyR). The structural basis of the allosteric connection is the intracellular loop between the 2nd and 3rd transmembrane repeat of the DHPR ?1 subunit. We previously showed that a ?-scorpion toxin maurocalcine (MCa) that shares primary sequence homology with the amino acid residues of DHPR-loop peptide essential for RyR activation raises single RyR channel open probability and blocks RyR in long lasting subconductive states These long lasting subconductance events characterised by mean event duration, which is about 12 secs for the wild type toxin. Using peptide where one charged amino acid (at a time) were replased with a neutral one ? wew showed that the critical amino acid is the 24th. Furtehrmore the strength of the toxin effect is proportional witht the distance of the muatted and of the 24th AA. The aimed to of this study was to confirm the hypothesis that MCa shares common binding sites with the DHPR loop peptide on RyR, and to characterize the potential effects of other MCa-homologue toxins (such as charybdotoxin (ChTx) and iberiotoxin (IbTx)) on RyR gating. Using single channel electrophysiology we demonstrated a competitive interaction between the loop peptide ? Mca and MCa ? ChTx respectively. We also found that ChTx albeit increased RyR open probability, induced long channel closures in dose-, and membrane potential- and Ca2+-dependent manner, but not in a use dependent fashion, while IbTx in spite of the significant homology was ineffective.
Tárgyszavak:Természettudományok Biológiai tudományok idézhető absztrakt
calcium release
Megjelenés:Acta Physiologica Hungarica. - 97 : 3 (2010), p. 89. -
További szerzők:Sárközi Sándor (1966-) (élettanász) Jóna István (1948-) (élettanász, fizikus)
Pályázati támogatás:81923
OTKA
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2.

001-es BibID:BIBFORM049381
Első szerző:Bárdi Miklós
Cím:The effect of phenol derivatives on the sr Ca2+-atpase / Bardi M., Sarkozi S., Jona I.
Dátum:2012
Megjegyzések:During the contraction of a skeletal muscle fiber an action potential running along the fiber surface membrane results in the conformational change of the dihydropyridine receptors which in turn causes the opening of the sarcoplasmic reticulum (SR) calcium channels (RyR1). Calcium ions - released from the SR through the channels ? increase the myoplasmic calcium concentration that finally evokes the contraction of the fibre. The decrease in the myoplasmic calcium concentration causing relaxation of the fiber is achieved by the action of the SR Ca2+-ATPase (SERCA).It's known from the literature and our earlier results that thymol and its structural analogues ? which are widely used in the food, cosmetic and pharmaceutical industry as preservatives ? have influence on the activity of the RyR1 and SERCA. Continuing our previous work our aim was to study the effect of further phenol derivatives on the SERCA.Light SR (LSR) vesicles were prepared from rabbit skeletal muscle (m. longissimus dorsi) containing the SR Ca2+-pump in their membrane. ATP dependent hydrolytic activity of LSR vesicles was measured using "coupled enzyme assay" at 37oC. Specific activity of SERCA was calculated after determination of the non-specific activity in the presence of 20 ?M cyclopiazonic acid which specifically blocks SERCA.Pump activities were plotted against the concentration values of different drugs, dots were fitted by Hill-equation revealing the following parametes:4-Chloro-meta-crezol IC50=167 ? 8 ?M, nHill ~3;5-Chloro-orho-crezol IC50=554 ? 45 ?M, nHill ~2,4-Chloro-ortho-crezol IC50=1370 ? 88 ?M, nHill ~8.Almost all the compounds investigated here inhibited the pump except cresol which didn't exert any effect in concentration range 0?3 mM. Other compounds inhibited SERCA activity, but affinity and the number of ligands differed from each other.Our results prove that phenol derivative structural analogues have an inhibitory effect on SERCA activity but this effect is significantly modified by the relative position of the different substituent groups and the presence of cloride is also required for inhibition. The alterations in the shape of the pi electron cloud caused by the different substituents can be also involved in the effect of these compounds.Supported by: OTKA 81923
Tárgyszavak:Természettudományok Biológiai tudományok idézhető absztrakt
calcium release
calcium pump
Megjelenés:Acta Physiologica. - 205 : Suppl. 690 (2012), p. P1. -
További szerzők:Sárközi Sándor (1966-) (élettanász) Jóna István (1948-) (élettanász, fizikus)
Pályázati támogatás:81923
OTKA
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3.

001-es BibID:BIBFORM049379
Első szerző:Lukács Balázs (élettanász)
Cím:Effect of maurocalcine on the skeletal type ryanodine receptor / Lukacs B., Sarkozi S., Almassy J., Jona I.
Dátum:2012
Megjegyzések:Maurocalcine (MCa) is a 33 amino acid long scorpion toxin which shows high homology with dihydropyridine receptor constituent peptide-A (pepA). The latter is believed to directly interact with ryanodine receptor (RyR1) and plays important role in the electromechanical coupling. The position and the positive charge of given amino acids residues of MCa are determinative in this interaction.We studied the effect of pepA and potassium ion on the MCa evoked Long Lasting Subconductance State (LLSS) type RyR1 operation assuming that both of them may have access to MCa binding sites of the channel. To investigate the binding of these peptides to RyR1, we used heavy sarcoplasmic reticulum vesicles (HSR) and CHAPS solubilized ryanodine receptor complex of rabbit skeletal muscle. Gating of RyR1 was monitored on channels incorporated into a planar lipid bilayer under voltage clamp conditions. Ca2+-release measurements were performed on HSR, where changes in extravesicular Ca2+ concentration were followed as changes in the absorption of APIII Ca2+-sensitive dye (?=710 nm).In single channel experiments LLSS type RyR1 gating was evoked applying of 5 and 10 nM MCa in the cytoplasmic side of the channel. The length and frequency of the characteristic subconductance states gradually ceased by consecutively added K+. The half effective concentration of K+ was higher at 10 nM MCa concentration which refers to a possible competition between MCa and potassium ion in biding to the same site on RyR1. A similar competitive-like effect of pepA was observed, when in the presence of 26 ?M pepA, much higher concentration of MCa was able to evoke LLSSs. In Ca2+ release measurements 5 nM MCa induced Ca2+-release at 100 mM K+, but release was completely eliminated at 250 mM K+. High concentration of K+inhibited only the MCa induced Ca2+-release but had no effect on the 4-CMC induced Ca2+-release suggesting specific effect of K+ on MCa-RyR1 interaction. Suppression of release in the presence of 250 mM K+ was inhibited by addition of higher concentration of MCa suggesting charge driven interaction between MCa and RyR1.Our data put forward a possible mode of MCa action with 3 binding sites at the cytosolic side on RyR1. The first binding site located on the surface of the channel, and is responsible for the Po increase at low MCa concentration. The second binding site in the pore of the channel induces potential- and voltage dependent LLSS-s at higher toxin concentration. Occupy of third one which located presumably in the pore, close to the selectivity filter results in closed states of RyR1.Supported by: OTKA 81923
Tárgyszavak:Természettudományok Biológiai tudományok idézhető absztrakt
calcium release
Megjelenés:Acta Physiologica. - 205 : Suppl. 690 (2012), 26. p. -
További szerzők:Sárközi Sándor (1966-) (élettanász) Almássy János (1981-) (élettanász, biológus, angol-magyar szakfordító) Jóna István (1948-) (élettanász, fizikus)
Pályázati támogatás:81923
OTKA
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4.

001-es BibID:BIBFORM049380
Első szerző:Sárközi Sándor (élettanász)
Cím:Altered modulation of the skeletal type ryanodine receptor / calcium release channel by ATP in malignant hyperthermia / Sarkozi S., Lukacs B., Bardi M., Jona I.
Dátum:2012
Megjegyzések:We have shown previously that the activity of the Ryanodine Receptor / Calcium release channel (RyR/CRC) is modulated by ATP. Increasing ATP concentration results in an increase of open probability of the RyR/CRC and the dose response curve of the phenomena is biphasic having two distinctive activatory processes. The amplitude ratio of the two steps are 1:3 suggesting strong cooperation between the ATP binding sites and independent but cooperative binding of the ATP on the RyR1 monomers. It was also shown by several laboratories that mutations leading to malignant hyperthermia (MH) also increases the calcium sensitivity of the channel. Our aim was to test whether the ATP pharmacology is affected in MH and if this is the case in what way.Heavy SR vesicle was prepared as described previously using longissimus dorsi of a swine (Pietrin strain), which carries an MH causing homozygous arg615cysmutation. Following CHAPS+lipid solubilization, the functional RyR1 tetramer - the channel complex - was incorporated into a lipid bilayer. The bathing medium contained symmetrical 250 mM KCl ? 20mM PIPES ? pH:7.4. Free (ionized) calcium concentration was established using Ca-EGTA calcium buffer, calculated by Fabiato's method. Under voltage clamp conditions the channel current was recorded and the channel parameters were determined: such as open probability (Po), mean open time and specific conductance. The ATP pharmacology of the RyR/CRC was determined using 50 ?M Ca2+ free trans and 472 nM Ca2+ free cis, applying increasing Na2ATP concentration on the cis side.The mutant channel showed higher open probability compared to the wild type even in the absence of ATP. The mean open time was slightly higher in the mutant, but not significantly different from the wt. The ATP pharmacology of the mutant channel was different from the wt: the pronounced two phases disappeared from the ATP dependence of the open probability function. The increase of the open probability has two components: the mean open time increased significantly above 100 ?M, and the number of open events increased even more pronounced above 150 ?M ATP. The majority of the Po increase was attributed to the increase of the number of open events. All point histograms showed clearly two peaks without a trace of subconductance state: meaning that the synchrony of the four RyR1 monomers has not been changed due to the given mutation.Supported by: OTKA 81923
Tárgyszavak:Természettudományok Biológiai tudományok idézhető absztrakt
calcium release
Megjelenés:Acta Physiologica. - 205 : Suppl. 690 (2012), p. L9. -
További szerzők:Lukács Balázs (1978-) (élettanász) Bárdi Miklós Jóna István (1948-) (élettanász, fizikus)
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