OPTIMIZATION OF THE METHOD OF NUCLEI ISOLATION FOR ELECTROPHYSIOLOGICAL STUDIES OF ION CHANNELS IN THE NUCLEAR MEMBRANE OF THE RAT CARDIOMYOCYTES
O.A. Kotyk, A.B. Kotliarova, S.M. Marchenko
O.O. Bogomoletz Institute of Physiology National Academy of Sciences of Ukraine, Kyiv, Ukraine
DOI: https://doi.org/10.15407/fz64.02.026
Abstract
The conditions for the nuclei isolation from cardiomyocytes
for electrophysiological studies of ion channels in the native
nuclear membrane were optimized. The structural and functional
intactness of isolated nuclei is crucial for investigation
of the biophysical properties of the transport systems in the
native nuclear membrane. The quality of the obtained sample
was evaluated by ion currents recording through the nuclear
membrane in nucleus-attached or excised patch configuration
at voltage-clamp mode of patch-clamp technique. In our study
the solutions with different compositions were tested and the
quality of the samples obtained using different methods of
myocardium homogenization were compared. We found that
the activity of ion channels in the nuclear membrane were
most often registered when myocardium was homogenized
in solution with the next composition (mmol/l): sucrose –
300, KCl – 60, HEPES – 10 (pH 7,2) with protease inhibitors
cocktail, DL-dithiothreitol (1 mmol/l) and glycerol, using glass
homogenizer (Dounce).
Keywords:
nuclear envelope; cardiomyocytes; nuclear fraction isolation; native membrane; patch-clamp; ion channels
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