Összes szerző

Panyi Gyorgy

az alábbi absztraktok szerzői között szerepel:

Gaál Szabolcs
Characterization of a novel mutation in Brugada Syndrome

Aug 30 - szerda

08:45 – 09:00

Membránok és membránfehérjék biofizikája

E20

Characterization of a novel mutation in Brugada Syndrome

Szabolcs Gaál^1,2, Beáta Arnódi-Mészáros¹, István Balogh³, Bálint L. Bálint⁴, Zoltán Csanádi², György Panyi¹, and Tibor G Szántó¹

¹ University of Debrecen, Department of Biophysics and Cell Biology, Debrecen, Hungary

² University of Debrecen, Department of Cardiology and Cardiac Surgery, Debrecen, Hungary

³ University of Debrecen, Department of Human Genetics, Debrecen, Hungary

⁴ University of Debrecen. Faculty of Medicine. Department of Biochemistry and Molecular Biology. Genomic Medicine and Bioinformatic Core Facility, Debrecen, Hungary

Voltage-gated sodium channels (Na_V) play a key role in the initiation and propagation of cardiac action potential essential for the rhythmic beating of the heart. Therefore, alterations of the sodium current (I_Na) in cardiomyocytes can lead to diseases responsible for cardiac arrhythmias, such as Brugada Syndrome (BrS) that has an increased risk for sudden cardiac death due to ventricular fibrillation. The major disease gene for BrS is SCN5A encoding the primary alpha-subunit of the cardiac Na_V1.5 channel. Exploring SCN5A mutations in patients with inherited arrhythmogenic syndromes is critical for the deeper understanding the pathogenesis of BrS.

Accordingly, we aimed at fully characterizing the biophysical properties of Na_V1.5 containing a novel heterozygous mutation of R893C localized in the P-loop of domain II identified in a patient with BrS. We subsequently compared the main gating parameters of R893C channels to wild-type Na_V1.5 channels (WT). The mutation was introduced by site-directed mutagenesis. The channels were transiently expressed in CHO cells and I_Na was measured using the standard whole cell patch-clamp technique.

We found that the peak current density is substantially reduced by the R893C mutation compared to WT channels. We also observed slower activation kinetics of I_Na current in R893C channels, although the mutation had no significant effect on the steady-state activation. All observations confirmed the loss-of-function of R893C channels. Pharmacological studies revealed that the reducing agent Dithiothreitol might restore the normal function of Na_V1.5 containing R893C by reducing the cysteine bridges that may be responsible for the loss of conduction.

Our conclusion is that the analysed mutation in the SCN5A gene result in biophysical changes in the Na_V1.5 current and thus, may be responsible for BrS. Understanding the structure-function relationship of Na_V1.5 will shed new light on exploiting new therapeutic drugs for SCN5A channelopathies.

Ghofrane Medyouni
Role of ion channels in CAR T-cell

Aug 30 - szerda

15:30 – 17:00

II. Poszterszekció

P42

Role of ion channels in CAR T-cell

Ghofrane Medyouni¹, Vivien Jusztus¹, Orsolya Vörös¹, Maria Eduarda Lima^1,2, György Panyi¹, Péter B. Hajdu^{1, 2}

¹ University of Debrecen, Faculty of Medicine, Department of Biophysics and Cell Biology

² University of Debrecen, Faculty of Dentistry, Division of Dental Biochemistry

Cancer immunotherapy partly relies on the reprogramming of host immune cells to eliminate cancer cells. Genetic modification of T cells to express chimeric antigen receptors (CARs) is utilized in the treatment of hematological malignancies. Despite its success, many challenges remain to improve the efficacy and safety of this therapy. Ion channels in T-cells participate in the regulation of Ca²⁺-dependent activation pathway and play a role in various effector functions inevitable for target cell abolition. Hence, modification of ion channels’ function can contribute to successful immune therapy. However, no study has been reported about functional role of CAR T-cell ion channels yet.

We established a 3^rd-generation CAR expressing cell line (CD19-CAR cells) from Jurkat cells. We used the whole-cell patch-clamp technique and FURA-2-based Ca²⁺-imaging to determine the biophysical properties of Kv1.3 and Ca²⁺-response of CD19-CAR cells, respectively. We adapted a Calcein Red based killing assay to test CD19-CAR cells’ target cell killing capacity. We assessed the localization of Kv1.3 in standalone and in the CAR-synapse engaged CD19-CAR cells.

We showed that Kv1.3 activation and inactivation kinetics are the same in non-transfected and CD19-CAR cells, while voltage-dependence of activation was different. Thapsigargin-induced Ca²⁺-response of CD19-CAR cells was lower as compared to the control. We showed that Kv1.3 channels are co-localized with CARs in standalone CD19-CAR cells, and they redistribute to the contact region between a CD19-CAR cell and a target cell (Raji B cell). Upon Vm24 addition (specific Kv1.3 inhibitor, 1 nM) the target cell ability of CD19-CAR cells was impaired. Based on these results, we suppose that ion channels can affect the outcome of immunotherapy, and further experiments are needed to clarify their functional role.

Acknowledgment

This work was supported by Stipendium Hungaricum Scholarship to M.G.t and NKFIH (K128525, P.H.).

Jusztus Vivien
Ion channel expression of CD8+ T cells in ovarian cancer

Aug 30 - szerda

15:30 – 17:00

II. Poszterszekció

P43

Ion channel expression of CD8⁺ T cells in ovarian cancer

Vivien Jusztus¹, Ghofrane Medyouni¹, Orsolya Vörös¹, Zsolt Szabó¹, Rudolf Lampé³, György Panyi¹, Orsolya Matolay³, Eszter Maka³, Zoárd Krasznai³, Péter Hajdu^1,2

¹ University of Debrecen, Faculty of Medicine, Department of Biophysics and Cell Biology

² University of Debrecen, Faculty of Dentistry, Division of Dental Biochemistry

³ University of Debrecen, Faculty of Medicine, Department of Gynecology and Obstetrics

The ion channels of T lymphocytes have an important role in effector functions such as activation, cytokine production and tumor cell elimination. T cells recognise and kill cancer cells during continuous monitoring. Although tumor infiltrating lymphocytes (TILs) are able to penetrate the tumor, they cannot fulfil their effector function due to the suppressive nature of the tumour microenvironment. K⁺ channels, such as Kv1.3 and KCa3.1, stabilize the negative membrane potential of T cells to control Ca²⁺-influx through CRAC channels and Ca²⁺-dependent signaling. In the present study, we determined the expression of T cell ion channels from peripheral blood of untreated ovarian cancer patients and healthy donors.

PBMCs were isolated from blood of ovarian patients and healthy donors using Ficoll-Paque density gradient method. Cells were activated with CD3/CD28 antibodies. Whole-cell current was measured in activated CD8⁺ T cells using patch-clamp technique. Ca²⁺ response of CD8⁺ cells was evaluated with FURA-2 Ca²⁺-imaging method.

KCa3.1 expression level in blood CD8⁺ cells from malignant tumor patients were lower than in healthy and benign tumor groups. Contrary, the Kv1.3 conductance of CD8⁺ T cells were significantly higher in malignant tumor patients as compared to other two groups. To asses Ca²⁺ response of cells, we determined the quotient of FURA-2 ratios measured in 2 mM Ca²⁺ and 0 mM Ca²⁺ after thapsigargin addition: there was no differences between the groups.

In summary, we suppose that down-regulation of KCa3.1 expression and Kv1.3 level upregulation in blood CD8⁺ cells could be a reporter on the presence of malignancy, as we reported before for head and neck cancer patients [1].

Acknowledgment

This work was supported by Stipendium Hungaricum Scholarship to M.G. and NKFIH (K128525, P.H.).

References

[1] Chimote AA, Balajthy A, Arnold MJ, Newton HS, Hajdu P, Qualtieri J, et al. (2018) Sci Signal. 11(527): 1–12

Kashmala Shakeel
Isolation of novel peptide toxins from the venom of the scorpion Centruroides bonito which block Kv1.2 ion channel with picomolar affinity

Aug 30 - szerda

15:30 – 17:00

II. Poszterszekció

P44

Isolation of novel peptide toxins from the venom of the scorpion Centruroides bonito which blocks Kv1.2 ion channel with picomolar affinity 

Kashmala Shakeel¹, Muhammad Umair Naseem¹, Lourival D Possani², Gyorgy Panyi¹

¹ Department of Biophysics and Cell Biology, Faculty of Medicine, University of Debrecen, Hungary

² Departamento de Medicina Molecular y Bioprocesos, Universidad Nacional Autónoma de México, Mexico

Seven new peptide toxins named as CboK1 to CboK7 were isolated from the venom of the Mexican scorpion Centruroides bonito by liquid chromatography. The primary structure of these peptides were determined by Edman degradation. Mass spectrometry analysis was used to determine the molecular weights which range between 3760.4 Da to 4357.9 Da, comprising 32 to 39 amino acid residues cross-linked with three tightly folded disulfide-bridges. The amino acid sequence alignment with known potassium scorpion toxins (KTx) and phylogenetic tree analysis unveiled that CboK1 (α-KTx 10.5) and CboK2 (α-KTx 10.6) belong to α-KTx 10 subfamily, whereas CboK3 (α-KTx 2.22), CboK4 (α-KTx 2.23), CboK6 (α-KTx 2.21), CboK7 (α-KTx 2.24) bears more than 95% amino acid similarity with the members of α-KTx 2 subfamily, and CboK5 is 100% identical with previously described Ce3 toxin (α-KTx 2.10). The electrophysiological assays (whole-cell patch clamp) revealed that except CboK1, all other six peptide toxins blocked the voltage-gated potassium channel Kv1.2 with high affinity, having Kd values in the picomolar range (24-763 pM) and inhibited the Kv1.3 ion channel with comparatively less potency (Kd values between 20-171 nM). Moreover, CboK2 and CboK3 inhibited ~10% and CboK7 inhibited ~50% of Kv1.1 currents at 100 nM concentration. Among all CboK7 (α-KTx 2.24) has the highest affinity for Kv1.2 ion channel with Kd value of 24 pM, and reasonable selectivity over Kv1.3 (~1000-fold) and Kv1.1 (~6000-fold) ion channels. These distinguishable characteristics of the CboK7 toxin may provide a framework for developing tools to treat Kv1.2-related gain of function channelopathies.

Muhammad Umair Naseem
Cm39 (α-KTx 4.8): A novel scorpion toxin that inhibits voltage-gated K+ channel Kv1.2 and small- and intermediate-conductance Ca2+-activated K+ channels KCa2.2 and KCa3.1

Aug 30 - szerda

15:30 – 17:00

II. Poszterszekció

P48

Cm39 (α-KTx 4.8): A novel scorpion toxin that inhibits voltage-gated K⁺ channel Kv1.2 and small- and intermediate-conductance Ca²⁺-activated K⁺ channels KCa2.2 and KCa3.1

Muhammad Umair Naseem¹, Georgina Gurrola-Briones², Margarita R. Romero-Imbachi³, Jesus Borrego¹, Edson Carcamo-Noriega², José Beltrán-Vidal³, Fernando Z. Zamudio², Kashmala Shakeel¹, Lourival D. Possani², Gyorgy Panyi¹

¹ University of Debrecen, Faculty of Medicine, Department of Biophysics and Cell Biology, Hungary

² Departamento de Medicina Molecular y Bioprocesos, Universidad Nacional Autónoma de México, Mexico

³ Departamento de Biología, Facultad de Ciencias Naturales, Universidad del Cauca, Colombia

A novel peptide toxin, Cm39, was identified in the venom of the Colombian scorpion Centruroides margaritatus. It is composed of 37 amino acid residues with a MW of 3980.2 Da and folded by three disulfide bonds. The Cm39 sequence also contains the Lys-Tyr (KY) functional dyad required to block voltage-gated K⁺ (Kv) channel. Amino acid sequence comparison with previously known K⁺ channel inhibitor scorpion toxins (KTx) and phylogenetic analysis revealed that Cm39 is a new member of α-KTx 4 family and registered with systematic number of α-KTx4.8. The full chemical synthesis and proper folding of Cm39 was obtained. The pharmacological properties of the synthetic peptide were determined using patch-clamp electrophysiology. Cm39 inhibits the voltage-gated K⁺ channel hKv1.2 with high affinity (K_d = 65 nM). The conductance-voltage relationship of Kv1.2 was not altered in the presence of Cm39, the analysis of the toxin binding kinetics was consistent with a bimolecular interaction between the peptide and the channel, and therefore the pore blocking mechanism is proposed for the toxin-channel interaction. Cm39 also inhibits the Ca²⁺-activated KCa2.2 and KCa3.1 channels, with K_d = 575 nM, and K_d = 59 nM, respectively, however, the peptide does not inhibit hKv1.1, hKv1.3, hKv1.4, hKv1.5, hKv1.6, hKv11.1, mKCa1.1 potassium channels or the hNav1.5 and hNav1.4 sodium channels at 1 µM concentration. Understanding the unusual selectivity profile of Cm39 motivates further experiments to reveal novel interactions with the vestibule of toxin-sensitive channels [1].

References

[1] Naseem MU, Gurrola-Briones G, Romero-Imbachi MR, Borrego J, Carcamo-Noriega E, Beltrán-Vidal J, Zamudio FZ, Shakeel K, Possani LD and Panyi G, (2023) Toxins 15(1), p.41.

Papp Ferenc
A synthetic flavonoid derivate modulates the fluorescent signal of voltage-gated proton channels

Aug 30 - szerda

15:30 – 17:00

II. Poszterszekció

P49

A synthetic flavonoid derivate modulates the fluorescent signal of voltage-gated proton channels

Zoltán Pethő^1,2, Gilman E. S. Toombes, Dávid Pajtás¹, Martina Piga³, Zsuzsanna Magyar⁴, Nace Zidar³, György Panyi¹, Zoltán Varga¹ and Ferenc Papp¹

¹ Department of Biophysics and Cell Biology,Faculty of Medicine, University of Debrecen,

² Institut für Physiologie II, Münster, Germany

³ Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Ljubljana

⁴ Department of Physiology, Faculty of Medicine, University of Debrecen

The voltage-sensing domain (VSD) of voltage-gated proton channel (Hv1) serves as a pore for protons as well as a voltage sensor, which makes this channel unique among voltage-gated channels. Natural flavonoids, which are widely distributed and act as chemical messengers and physiological regulators in plants, modulate the function of some voltage-gated ion channels (EAG1, HCN2. etc.) in animal cells. We have designed synthetic flavonoid derivatives to inhibit the current of Hv1. We produced and tested tens of flavonoid derivatives on Ciona intestinalis Hv1 using the voltage-clamp fluorometry (VCF). The most potent compound, molecule #109, changed the originally negative VCF signal to positive and altered the biphasic VCF signal shape to monophasic. Also, this molecule caused a rightward shift in the conductance-voltage relationship in a concentration dependent manner. This flavonoid derivative quenched the TAMRA-MTS fluorescence in cuvette and on frog oocytes decreasing the baseline fluorescence, independently the oocyte expressed Hv1 or not. Furthermore, #109 could stain the membrane of HEK cells. These results indicate that #109 binds not directly to CiHv1 but to the cell membrane and in this way, it indirectly modifies the gating of Hv1 and the VCF signal, as a strong quenching molecule in close vicinity of the fluorophore. The latter was confirmed by our model calculations, in which we assumed that molecule #109, as a strong quencher, is embedded in the cell membrane close to TAMRA and during the conformational change due to depolarization, TAMRA is continuously moving away from this strong quencher molecule. Therefore, the VCF signal becomes a continuously increasing fluorescence change from the originally complex shape, which was overall a decreasing fluorescence change.

Acknowledgment

This work was supported by OTKA Bridging Fund 1G3DBKB0BFPF247 (FP); by János Bolyai Research Scholarship of the Hungarian Academy of Sciences (BO/00355/21/8) (FP); by the ÚNKP-21-5-DE-460 New National Excellence Program of the Ministry for Innovation and Technology (FP); OTKA 132906 (ZV). This study was also funded by the Slovenian Research Agency (Grant No. P1-0208) (NZ).