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Most significant results of the Institute of Molecular Physiology and Genetics, SAS published in 2015

Basic research: Gating of T-type calcium channels is controlled by the voltage sensor in domain I

Application type: Molecular mechanisms affecting treatment of AML and MDS

International scientific projects: Ryanodine receptor controls formation of calcium waves in cardiac myocytes

Basic research:

Gating of T-type calcium channels is controlled by the voltage sensor in domain I

Authors: M. Karmažínová, K. Jašková, P. Griač, E. Perez-Reyes, Ľ. Lacinová

Low-voltage calcium channels of the T-type (Ca_V3.n) have a low activation threshold (about -60 mV) that enables their participation in the initiation of the action potential. It is not clear why they can activate much more easily than the high-voltage calcium channels (HVCCs). Opening of the channel pore, which allows passion of ion currents, is preceded by activation of the voltage sensor that can be measured as the gating current. There are four independent voltage sensors in the structure of voltage-dependent calcium channels, one in each of the four domains of the channel. Accorgind to the classical model, channel opening follows activation of all four voltage sensors. We have shown that in contrast to HVCCs, the voltage dependence gating current amplitude does not precede the voltage dependence of ion current, but crosses it. It means that activation of Ca_V3.n channels does not require activation of all four voltage sensors. Based on our results we have proposed a hypothesis that the decisive step on T-type channel activation id activation of the voltage sensor in the first domain of the channel. They differ from HVCCs also by the higher effectiveness of coupling between activation of the voltage sensor and opening of the conducting pore of the channel.

Schematic description of the structure of a  Ca_V3.n channel. Green cylinders represent α-helices of the conducting pored, orange cylinders are the positively charged S4 segments, the movement of which (step 1) corresponds to activation of the voltage sensor and generates the gating current I_G. Step 2 represents the resultant opening of the conducting pore, through which the ion current I_Ca flows.

Project: VEGA 2/0044/13

Publication: KARMAŽÍNOVÁ, Mária – JAŠKOVÁ, Katarína – GRIAČ, Peter – PEREZ-REYES, Edward – LACINOVÁ, Ľubica. Contrasting the roles of the I-II loop gating brake in CaV3.1 and CaV3.3 calcium channels. In Pflugers Archiv-European Journal of Physiology, 2015, vol. 467, no. 12, p. 2519–2527. (4.101 – IF2014). ISSN 0031-6768.

Aplication type:

Molecular mechanisms affecting treatment of AML and MDS.

Authors: D. Imrichová, L. Messingerová, M. Šereš, H. Kavcová, L. Pavlíková, M. Cocuľová, K. Turáková, A. Jonášová, M. Barančík, L. Poleková, L. Gibalová, A. Breier, Z. Sulová.

Myelodysplastick syndrome (MDS) and acute myeloid leukemia (AML), which may develop from MDS,  are characteristic by the presence of weakly differentited blood cells, the so called myeloblasts with surface expression of the CD33 marker, which is not present in normal blood stem cells and in non-myeloid tissue. Compounds with hypomethylation effect, e.g.  azacytidine (AzaC), or with immunomodulatory effects, e.g. lenalidomide (LEN) may be effective in treatment of  MDS and AML, but resistance to these drugs develops in some patients. Mechanisms of development of resistance to these drugs are not sufficiently understood. By repeated cultivation in media with increasing concentration of AzaC, LEN, mitoxantrone and vincristine we developed resistant variants of AML lines SKM-1 and MOLM-13 derived from patients, in which AML developed from MDS. Both cell lines showed  phenotype of multidrug resistance upon selection pressure of the compounds, with a characteristic overexpression of P-glycoprotein (P-gp), glutathione S-transferase (GST), and surprisingly with redused expression of CD33 marker. This marker is used in targeting the therapy by humanized antibodies with bound drug. The results suggest that development of drug resistance in AML cells may lead not only to primary resistance against drugs used for therapy, but also to secondary resistance to substrates of P-gp a GST and to CD33-targeted immunotherapy.

Development of multidrug resistance in  AML cells SKM-1 and MOLM-13 induced by drugs leads to development of resistance with a characteristic increase of  P-gp and GST expression and with a decrease of CD33 expression. This leads to complex resistance to several chemotherapeutic procedures.

Projects: APVV-02-90-10, VEGA 2/0100/12, 2/0182/13, 2/0028/15, ITMS 26240220071.

Publication: IMRICHOVÁ, Denisa – MESSINGEROVÁ, Lucia – ŠEREŠ, Mário – KAVCOVÁ, Helena – PAVLÍKOVÁ, Lucia – COCUĽOVÁ, Martina – BREIER, Albert – SULOVÁ, Zdena. Selection of resistant acute myeloid leukemia SKM-1 and MOLM-13 cells by vincristine-, mitoxantrone- and lenalidomide-induced upregulation of P-glycoprotein activity and downregulation of CD33 cell surface exposure. In European Journal of Pharmaceutical Sciences, 2015, vol. 77, no., p. 29-39. (3.350 – IF2014). (2015 – Current Contents). ISSN 0928-0987.

International scientific projects:

Ryanodine receptor controls formation of calcium waves in cardiac myocytes

Authors: P. Petrovič, I. Valent, E. Cocherová, J. Pavelková, A. Zahradníková

Počas srdcového sťahu (v systole) sa cez otvorené ryanodínové receptory (RyR) vyplaví z vnútrobunkových zásobníkov do cytoplazmy veľké množstvo vápnika. Pri uvoľnení sťahu (v diastole) je potrebné vyplavovanie vápnika zastaviť, teda zatvoriť RyR. Preto otváranie a zatváranie ryanodínových receptorov podlieha presnej regulácii. Mnohé srdcové ochorenia sa vyznačujú zvýšeným uvoľňovaním vápnika do cytoplazmy počas diastoly, čo vedie k tvorbe vápnikových vĺn a následným srdcovým arytmiám. Odhalili sme, že poruchy regulácie RyR vedú k tvorbe váp­nikových vĺn,keď sa zvýši pravdepodobnosť, že spontánna aktivácia RyR na jednom mieste vyvolá aktiváciu iného RyR na susediacom mieste. Tvorba vĺn závisí strmo aj na koncentrácii iónov vápnika v lumene sarko­plazma­tického retikula. Zistili sme, že uľahčenie väzby vápnikových iónov na cyto­zolické aktivačné miesto RyR sa môže paradoxne pre­ja­vo­vať ako zvýšenie citlivosti RyR na lumi­nálnu koncentráciu vápnika. Ukázali sme, že poruchy rôznych regu­lačných miest ryanodínového receptora je možné od­strániť znížením prav­de­podobnosti spontánnej, na vápniku nezávislej aktivácie RyR. Spontánna akti­vácia ryanodínového receptora by teda mohla byť vhodným cieľom pre farmako­terapiu relevantných typov arytmií.

Schéma vzniku vápnikových vĺn. Za nor­mál­nych podmienok sa vápnikové vlny netvoria (horný obrázok) a koncentrácia vápnika v lumene sa nemení (čierna čiara). Zvýšenie luminálneho vápnika vedie k tvorbe vĺn (horný obrázok) a k do­časnému vyprázdňovaniu vápnikového zásobníka (červená čiara). Ak je väzba Ca²⁺ na cytozolické aktivačné väzbové miesto RyR uľahčená, vlny sa tvoria už pri zníženej luminálnej koncentrácii Ca²⁺ (dolný obrázok, modrá čiara).

Projekty:LSHM-CT-2005-018802/CONTICA, LSHM-CT-2005-018833/EUGeneHeart, ESF program FUNCDYN, APVV-0721-10, VEGA 2/0148/14

Publikácia: PETROVIČ, Pavol – VALENT, Ivan* – COCHEROVÁ, Elena* – PAVELKOVÁ, Jana – ZAHRADNÍKOVÁ, Alexandra. Ryanodine receptor gating controls generation of diastolic calcium waves in cardiac myocytes. In Journal of General Physiology, 2015, vol. 145, no. 6, p. 489-511. (4.788 – IF2014). (2015 – Current Contents). ISSN 0022-1295

*Pracovníci prijatí na ÚMFG SAV v rámci projektov CONTICA a EUGeneHeart