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UNIVERSITY OF BUCHAREST
FACULTY OF PHYSICS Guest
2024-11-22 1:39 |
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Conference: Bucharest University Faculty of Physics 2013 Meeting
Section: Biophysics; Medical Physics
Title:
Electrophysiology and pharmacology study of a human neuroblastoma cell line
Authors:
Fedinand Halitzchi, Lelioara Jianu, Bodgan Amuzescu
Affiliation:
Dept. Biophysics & Physiology, Faculty of Biology, University of Bucharest
E-mail
fhalitzchi@yahoo.com
Keywords:
SH-SY5Y neuroblastoma cell line, whole-cell patch-clamp, delayed rectifier K current, voltage-dependent Na current, steady-state inactivation, recovery from inactivation
Abstract:
The current experiments were carried out in order to develop experimental protocols suitable for characterization of bone-marrow-derived mesenchymal stromal cells. SH-SY5Y cells cultured in standard conditions in 35-mm Petri dishes were approached by whole-cell patch-clamp in Tyrode external solution. The pharmacological compounds applied were 4-AP 3 mM, TEA 5 mM, and nifedipine 100 {micro}M, freshly prepared from aqueous stock solutions (4-AP and TEA) or ethanol (nifedipine). Electrical access was very good, with resistances often below 10 Mohm, and seals were stable over the entire duration of experiments in 69% of cases. The approached cells were small, with a membrane capacitance Cm = 16.2 +/- 9.3 pF (mean +/- SD, n = 49) and a mean membrane resistance Rm = 558.2 +/- 553.3 Mohm. Using a complex intracellular solution (130 K aspartate, 25 KCl, 5.5 MgATP, 0.1 LiGTP, 1 EGTA, 5 HEPES, pH 7.21) we recorded delayed rectifier K currents insensitive to the compounds tested. In a unique experiment we recorded BK-type single-channel currents, activated upon return to -80 mV following depolarisations larger that 20 mV. In a majority of cells we recorded voltage-dependent Na currents of TTX-s type, using a CsCl pipette solution to block most outward K current components. The proof of a genuine Na current is the reversible disparition upon Na replacement with an impermeant organic cation, NMDG. By applying specific protocols we obtained a steady-state inactivation curve (h infinity) that was fitted with a Boltzmann-type function, yielding a half-activatory potential V1/2 = -84.8 mV and a slope factor = 5.6 mV/e-fold. The recovery from inactivation time constant was in the range of 12.4-17.5 ms. In conclusion, in the SH-SY5Y cell line in undifferentiated form several voltage-dependent ion channels of neuronal type are expressed (INa-TTXs, delayed rectifier IK, and possibly BK), while Ito and If are absent.
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