Harántcsíkolt izom SR kalcium csatornájának modulációja egészséges és kóros körülmények között = Modulation of the SR calcium channels of striated muscle in health and disease

Az izom összehúzódás kiváltásában centrális szerepet játszó elektromechanikai csatolás kulcs-molekuláit tanulmányoztuk. Megállapítottuk, hogy a gadolinium ion mind a cisz mind a transz oldalról hasonlóan gátolja a csatornát (Ki=5,38+-0,63microM és Ki=5,72+-0,84microM, Ncis=4,21+-0,47, Ntrans=4,5+-0,41), feltehetően a kötőhely a csatorna vezető pórusának belsejében található, és a két oldal felől egyformán érhető el. Az európium a cis oldalról kvalitatíve hasonló módon gátol (Ki=107+-17nM), míg trans oldalról hatástalan. Megállapítottuk, hogy a timol gátolja a kalcium pumpát (K i=253+-4,7 microM, NHill=1,62+-0,05) és a csatorna aktiválásával gyors kalcium felszabadulást vált ki (Vmax=0,47+-0,04 nmol/s, EC50=258+-21 nM, NHill=3,0+-0,54). Szintetizáltuk a DHP receptor adott szakaszait, és megállapítottuk, hogy a maurocalcin (MCa) két módon is hat a RyR1 kapuzására, "hosszú fél-nyitott" (LLSS) állapotokat indukál, és az LLSS időszakok között növeli a csatorna nyitvatartási valószínűségét. A toxin pontmutánsai segítségével kimutattuk, hogy a 31 aminosavból álló teljes szekvenciának a hatás szempontjából legkritikusabb tagja a 24-es aminosav. A toxinmolekulát a 23, 22 20 és 8-as pozícióban alaninra mutálva a hatás erőssége attól függött, hogy térbelileg milyen messzire van a mutációs pont a kritikus 24-es pozíciótól. A MCa hatása polaritás- és potenciálfüggő | Key molecules playing central role on the muscle contraction have been studied. We have shown using bilayer technique that Gadolinium inhibits the calcium channel from both sides (Ki=5.38+-0.63microM and Ki=5.72+-0.84 microM, Ncis=4.21.+-0.47, Ntrans=4.5+-0.41) showing, that the Gd3+ binding site is located in the middle region of the channel pore equally accessible from both sides while Eu3+ inhibited the channel only from the cis side (Ki=107+-17nM). We have shown, that thymol inhibits the SR calcium pump (K i=253+-4.7 microM and NHill=1.62+-0.05) and evokes calcium release (Vmax=0.47+-0.04 nmol/s, EC50=258+-21 nM, NHill=3.0+-0.54). The effect of thymol on RyR1 and RyR2 is similar. Analogous sequence to the different segments of the II-III loop of the DHPR were synthesized followed by the demonstration that maurocalcin (MCa) alters the gating of the RyR1 and RyR2. It induces long lasting subconductance events (LLSS) which might last even for several seconds, and it increases the open probability between the LLSS events too. Using different point mutations of the 31 residue peptide it was shown, that the position 24 is critical for the toxin effect. Alanine mutations in the position of 8, 9, 20, 22, 23 resulted partial LLSS effect according to the three dimensional distance of the given amino acid from the crucial 24 residue. The MCa effect found to be strongly polarity and slightly potential dependent

Bile acids activate ryanodine receptors in pancreatic acinar cells via a direct allosteric mechanism

The earliest critical event of pancreatitis is a long lasting high amplitude rise of intracellular Ca(2+) concentration of the acinar cell, which can be triggered by high concentration of bile acids. Although, Ca(2+)-release through ryanodine receptors (RyR) is involved in the process, the significance and the exact mechanism of bile acid's action on RyR has not been fully elucidated yet. Therefore, we aimed to test with various techniques and aspects whether bile acids exert a direct effect on RyR and SERCA pump. Our data show that taurocholic acid (TCA)-induced Ca(2+) release in pancreatic acinar cells was significantly reduced by the RyR antagonist dantrolene. Further, we show that TCA enhanced RyR's (3)H-ryanodine binding and triggered robust Ca(2+)-release from RyR-enriched vesicles in the pathologically relevant concentration range. RyR single channel current analysis demonstrated that 200μM TCA induced a 5-fold increase in the channel's open probability and caused a significant lengthening of the mean open time. TCA also suppressed Ca(2+)-uptake rate and ATP-ase activity of SERCA-enriched vesicles, but interestingly, failed to decrease Ca(2+) elimination rate in intact cells. Overall, our results strongly suggest that TCA opens RyR by an allosteric mechanism, which contribute significantly to bile acid-induced pathologic Ca(2+)-leak from the endoplasmic reticulum in pancreatic acinar cells

A kalcium transzport szerepe a harántcsíkolt izom jelátvitelében = The role of calcium transport in signal transduction of striated muscle

A DHPR II-III hurokkal homológ szekvenciájú maruokalcin (MCa) hatását vizsgáltuk a RyR1 és a RyR2 csatornára. Megállapítottuk, hogy a MCa a RyR2-őt gyengébben befolyásolja, mint a RyR1-et, a gyengébb RyR2-MCa kölcsönhatás miatt. A RyR-MCa kölcsönhatásért a peptid 8, 22, 23, 24-es aminosavai által alkotott pozitív töltésű ?felület? a felelős, bár a kölcsönhatásban a membránpotenciál is fontos szerepet játszik. Patkányokon előidézett miokardiális infarktust (MI) követően kialakuló vázizom-gyengeségben tanulmányoztuk a RyR1 működésének megváltozását. Megállapítottuk, hogy az infarktus után 24 héttel a kalciumcsatornák mintegy 1/3-a a fiziológiással ellentétes áramirány esetén a szokásos 525pS-el szemben 750pS vezetőképességet mutat, míg ugyanezen csatornák a fiziológiás áramirány esetén is a szokásos 525-550 pS vezetőképességűek. A gyógyszerjelölt K201 molekula nem okozott szignifikáns változást a csatorna paramétereiben annak ellenére, hogy a MI-al egyszerre alkalmazva kivédi a vázizom-gyengeség kialakulását, így valamilyen poszt-transzlációs változás áll a kialakuló vázizom-gyengeség hátterében. A TPEN-ről kimutattuk, hogy gátolja a pumpát és aktiválja a csatornát, így kardioprotektív hatású. Kimutattuk, hogy az európium és a szamárium is gátolja a RyR1-et, de ? ellentétben a gadolíniummal kapott eredményekkel ? a cis és trans oldali hatás több nagyságrenddel eltér egymástól. Az európium fél-gátló koncentrációja 167?5nM a cis oldalon, míg 4,7?0,1?M a transz oldalon. | The effect of Maurocalcin (MCa) ? which is homologous to the II-III loop of the DHPR ? were investigated on the RyR1 & RyR2. The modulatory effect of MCa was more pronounced on RyR1 than on RyR2 due to the weaker interaction. The interaction is determined by the 8. 22, 23, 24 AA residues, which form a positive charged area on the MCa surface, and also influenced by the membrane potential. The effect of myocardial infarct (MI) on the function of RyR1 were also studied using rat post MI (PMI) model. It was shown, that 24 weeks after MI about 1/3 of the channels showed abnormally high conductance (750 pS instead of 525pS) at reverse calcium flux, while the forward conductances has not been altered. K-201 - a potential therapeutic agent did not alter the conductivity pattern, even though administering the drug at the onset of the MI resulted in the protection of skeletal muscles, patronizing that a kind of post-translational change is involved in the phenomena. TPEN is cardioprotective because it inhibits the calcium pump and activates the calcium. It was shown that Europium and Samarium inhibits RyR1 with substantial side preference ? in contrast to Gadolinium. Half inhibitory concentration of Eu(3+) is 167?5nM at cis side, while 4,7?0,1?M at the trans side

Laser induced calcium oscillations in fluorescent calcium imaging

Phototoxicity is the most common problem investigators may encounter when performing live cell imaging. It develops due to excess laser exposure of cells loaded with fluorophores and can lead to often overlooked but significant artifacts, such as massive increase of intracellular Ca2+ concentration, which would make data interpretation problematic. Because information about laser- and dye-related changes in cytoplasmic calcium concentration is very limited, we aimed to describe this phenomenon to help investigators using laser scanning confocal microscopy in a non-invasive way. Therefore, in the present study we evaluated fluorescent fluctuations, which evolved in Fluo- 3/4/8 loaded mouse pancreatic acinar cells during very low intensity laser excitation. We demonstrate that after standard loading procedure (2 μM Fluo-3/4/8-AM, 30 min @ room temperature), applying 488 nm laser at as low as ca. 10 μW incident laser power (0.18 μW/μm2) at 1 Hz caused repetitive, 2-3 fold elevations of the resting intracellular fluorescence. The first latency and the pattern of the fluorescence fluctuations were laser power dependent and were related to Ca2+-release from intracellular stores, as they were abolished by BAPTA-AM treatment in Ca2+- free medium, but were not diminished by the ROS scavenger DMPO. Worryingly enough, the qualitative and quantitative features of the Ca2+-waves were practically indistinguishable from the responses evoked by secretagogue stimulation. Since using similar imaging conditions, a number of other cell types were reported to display spontaneous Ca2+ oscillations, we propose strategies to distinguish the real signals from artefacts

Lanthanides Report Calcium Sensor in the Vestibule of Ryanodine Receptor

Ca2+ regulates ryanodine receptor's (RyR) activity through an activating and an inhibiting Ca2+-binding site located on the cytoplasmic side of the RyR channel. Their altered sensitivity plays an important role in the pathology of malignant hyperthermia and heart failure. We used lanthanide ions (Ln3+) as probes to investigate the Ca2+ sensors of RyR, because they specifically bind to Ca2+-binding proteins and they are impermeable to the channel. Eu3+'s and Sm3+'s action was tested on single RyR1 channels reconstituted into planar lipid bilayers. When the activating binding site was saturated by 50 μM Ca2+, Ln3+ potently inhibited RyR's open probability (Kd Eu3+ = 167 ± 5 nM and Kd Sm3+ = 63 ± 3 nM), but in nominally 0 [Ca2+], low [Eu3+] activated the channel. These results suggest that Ln3+ acts as an agonist of both Ca2+-binding sites. More importantly, the voltage-dependent characteristics of Ln3+'s action led to the conclusion that the activating Ca2+ binding site is located within the electrical field of the channel (in the vestibule). This idea was tested by applying the pore blocker toxin maurocalcine on the cytoplasmic side of RyR. These experiments showed that RyR lost reactivity to changing cytosolic [Ca2+] from 50 μM to 100 nM when the toxin occupied the vestibule. These results suggest that maurocalcine mechanically prevented Ca2+ from dissociating from its binding site and support our vestibular Ca2+ sensor-model further