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1.

001-es BibID:BIBFORM069847
Első szerző:Hsu, Eric J.
Cím:Regulation of Na+ channel inactivation by the DIII and DIV voltage-sensing domains / Hsu Eric J., Zhu Wandi, Schubert Angela R., Voelker Taylor, Varga Zoltan, Silva Jonathan R.
Dátum:2017
ISSN:0022-1295
Tárgyszavak:Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban
Megjelenés:Journal Of General Physiology 149 : 3 (2017), p. 389-403. -
További szerzők:Zhu, Wandi Schubert, Angela R. Voelker, Taylor Varga Zoltán (1969-) (biofizikus, szakfordító) Silva, Jonathan R.
Pályázati támogatás:KTIA_NAP_13-2-2015-0009
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2.

001-es BibID:BIBFORM060424
Első szerző:Rudokas, Michael W.
Cím:The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry / Rudokas Michael W., Varga Zoltan, Schubert Angela R., Asaro Alexandra B., Silva Jonathan R.
Dátum:2014
ISSN:1940-087X
Megjegyzések:The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion channels in oocytes. Recordings from the cut-open setup are particularly useful for resolving low magnitude gating currents, rapid ionic current activation, and deactivation. The main benefits over the two-electrode voltage clamp (TEVC) technique include increased clamp speed, improved signal-to-noise ratio, and the ability to modulate the intracellular and extracellular milieu. Here, we employ the human cardiac sodium channel (hNaV1.5), expressed in Xenopus oocytes, to demonstrate the cut-open setup and protocol as well as modifications that are required to add voltage clamp fluorometry capability. The properties of fast activating ion channels, such as hNaV1.5, cannot be fully resolved near room temperature using TEVC, in which the entirety of the oocyte membrane is clamped, making voltage control difficult. However, in the cut-open technique, isolation of only a small portion of the cell membrane allows for the rapid clamping required to accurately record fast kinetics while preventing channel run-down associated with patch clamp techniques. In conjunction with the COVG technique, ion channel kinetics and electrophysiological properties can be further assayed by using voltage clamp fluorometry, where protein motion is tracked via cysteine conjugation of extracellularly applied fluorophores, insertion of genetically encoded fluorescent proteins, or the incorporation of unnatural amino acids into the region of interest(1). This additional data yields kinetic information about voltage-dependent conformational rearrangements of the protein via changes in the microenvironment surrounding the fluorescent molecule.
Tárgyszavak:Orvostudományok Klinikai orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban
Xenopus Oocyte
Vaseline Gap
Fluorometry
Megjelenés:Journal of Visualized Experiments. - 2014 : 85 (2014). -
További szerzők:Varga Zoltán (1969-) (biofizikus, szakfordító) Schubert, Angela R. Asaro, Alexandra B. Silva, Jonathan R.
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3.

001-es BibID:BIBFORM060421
Első szerző:Varga Zoltán (biofizikus, szakfordító)
Cím:Direct Measurement of Cardiac Na+ Channel Conformations Reveals Molecular Pathologies of Inherited Mutations / Varga Zoltan, Zhu Wandi, Schubert Angela R., Pardieck Jennifer L., Krumholz Arie, Hsu Eric J., Zaydman Mark A., Cui Jianmin, Silva Jonathan R.
Dátum:2015
ISSN:1941-3149 1941-3084
Megjegyzések:BACKGROUND:-Dysregulation of voltage-gated cardiac Na+ channels (NaV1.5) by inherited mutations, disease-linked remodeling, and drugs causes arrhythmias. The molecular mechanisms whereby the NaV1.5 voltage-sensing domains (VSDs) are perturbed to pathologically or therapeutically modulate Na+ current (INa) have not been specified. Our aim was to correlate INa kinetics with conformational changes within the four (DI-DIV) VSDs to define molecular mechanisms of NaV1.5 modulation.METHOD AND RESULTS:-Four NaV1.5 constructs were created to track the voltage-dependent kinetics of conformational changes within each VSD, using voltage-clamp fluorometry (VCF). Each VSD displayed unique kinetics, consistent with distinct roles in determining INa. In particular, DIII-VSD deactivation kinetics were modulated by depolarizing pulses with durations in the intermediate time domain that modulates late INa. We then used the DII-VSD construct to probe the molecular pathology of two Brugada Syndrome (BrS) mutations (A735V and G752R). A735V shifted DII-VSD voltage-dependence to depolarized potentials, while G752R significantly slowed DII-VSD kinetics. Both mutations slowed INa activation, even though DII-VSD activation occurred at higher potentials (A735V) or at later times (G752R) than ionic current activation, indicating that the DII-VSD allosterically regulates the rate of INa activation and myocyte excitability.CONCLUSIONS:-Our results reveal novel mechanisms whereby the NaV1.5 VSDs regulate its activation and inactivation. The ability to distinguish distinct molecular mechanisms of proximal BrS mutations demonstrates the potential of these methods to reveal how inherited mutations, post-translational modifications and anti-arrhythmic drugs alter NaV1.5 at the molecular level.
Tárgyszavak:Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban
Brugada syndrome
ion channel
sodium channels
Megjelenés:Circulation-Arrhythmia and Electrophysiology. - 8 : 5 (2015), p. 1228-1239. -
További szerzők:Zhu, Wandi Schubert, Angela R. Pardieck, Jennifer L. Krumholz, Arie Hsu, Eric J. Zaydman, Mark A. Cui, Jianmin Silva, Jonathan R.
Pályázati támogatás:Bolyai Fellowship awardee
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4.

001-es BibID:BIBFORM073723
Első szerző:Zhu, Wandi
Cím:Mechanisms of noncovalent β subunit regulation of NaV channel gating / Wandi Zhu, Taylor L. Voelker, Zoltan Varga, Angela R. Schubert, Jeanne M. Nerbonne, Jonathan R. Silva
Dátum:2017
ISSN:0022-1295 1540-7748
Megjegyzések:Voltage-gated Na+ (Na) channels comprise a macromolecular complex whose components tailor channel function.Key components arethe non-covalently bound ?1and ?3subunits that regulatechannel gating, expression,andpharmacology.Here,we probethe molecular basis of this regulationby applying voltage clamp fluorometrytomeasurehow the ?subunits affectthe conformational dynamics of the cardiacNaVV channel (Na1.5) voltage-sensingdomains (VSDs). The pore-formingNa1.5 ? subunit contains four domains (DI?DIV), each with aVSD. Our results show that ?1 regulates NaVV1.5 by modulating the DIV-VSD, whereas ?3 alters channel kineticsmainly through DIII-VSD interaction. Introduction of a quenching tryptophan into the extracellular region of the?3 transmembrane segment inverted the DIII-VSD fluorescence. Additionally, a fluorophore tethered to ?3 at thesame position produced voltage-dependent fluorescence dynamics strongly resembling those of the DIII-VSD.Together, these results provide compelling evidence that ?3 binds proximally to the DIII-VSD. Molecular-level differences in ?1 and ?3 interaction with the ? subunit lead to distinct activation and inactivation recovery kinetics, significantly affecting Na channel regulation of cell excitability.
Tárgyszavak:Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban
Voltage-gated Na
Megjelenés:Journal Of General Physiology. - 149 : 8 (2017), p. 813-831. -
További szerzők:Voelker, Taylor Varga Zoltán (1969-) (biofizikus, szakfordító) Schubert, Angela R. Nerbonne, Jeanne M. Silva, Jonathan R.
Pályázati támogatás:KTIA_ NAP_13-2-2015-0009
MTA
Bolyai fellowship
MTA
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5.

001-es BibID:BIBFORM062510
Első szerző:Zhu, Wandi
Cím:Molecular motions that shape the cardiac action potential : insights from voltage clamp fluorometry / Wandi Zhu, Zoltan Varga, Jonathan R. Silva
Dátum:2016
ISSN:0079-6107
Megjegyzések:Very recently, voltage-clamp fluorometry (VCF) protocols have been developed to observe the membrane proteins responsible for carrying the ventricular ionic currents that form the action potential (AP), including those carried by the cardiac Na+ channel, NaV1.5, the L-type Ca2+ channel, CaV1.2, the Na+/K+ ATPase, and the rapid and slow components of the delayed rectifier, KV11.1 and KV7.1. This development is significant, because VCF enables simultaneous observation of ionic current kinetics with conformational changes occurring within specific channel domains. The ability gained from VCF, to connect nanoscale molecular movement to ion channel function has revealed how the voltage-sensing domains (VSDs) control ion flux through channel pores, mechanisms of post-translational regulation and the molecular pathology of inherited mutations. In the future, we expect that this data will be of great use for the creation of multi-scale computational AP models that explicitly represent ion channel conformations, connecting molecular, cell and tissue electrophysiology. Here, we review the VCF protocol, recent results, and discuss potential future developments, including potential use of these experimental findings to create novel computational models.
Tárgyszavak:Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban
Voltage clamp fluorometry
Action potential modeling
Sodium channels
Calcium channels
Potassium channels
Megjelenés:Progress In Biophysics & Molecular Biology 120 : 1-3 (2016), p. 3-17. -
További szerzők:Varga Zoltán (1969-) (biofizikus, szakfordító) Silva, Jonathan R.
Pályázati támogatás:TAMOP-4.2.2.D-15/1/KONV-2015-0016
TÁMOP
KTIA_NAP_13-2-2015-0009
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