In the present study, the functional role of the inwardly rectifying K+ channel, Kir4.1, and large-conductance Ca2+-activated K+ (BK) channel during cell migration in U251 cell line was investigated. We focused on polarised cells which are positive for the active-Cdc42 migration marker. The perforated patch technique was used to avoid intracellular dialysis and to maintain physiological changes in intracellular calcium. Wound healing was employed to assay migration after 24 h. Polarised cells recorded displayed different hallmarks of undifferentiated glial cells: depolarised resting membrane potential and high membrane resistance. Cells recorded outside wounded area did not display either constitutive inward or outward rectification. After migration, U251 cells were characterised by a constitutively smaller Kir4.1 and larger BK currents with a linearly related amplitude. Menthol modulation increased both currents in a linearly dependent manner, indicating a common mechanism triggered by activation of transient receptor potential melastatin 8 (TRPM8), a Ca2+-permeable non-selective cation channel. We hypothesised that both migration and menthol modulation would share an increase of intracellular calcium triggering the increase in Kir4.1 and BK channels. Immunocytochemistry demonstrated the cytoplasmic expression of both Kir4.1 and BK channels and a mislocation in the nucleus under basal conditions. Before and after migration, polarised cells increased the expression of Kir4.1 and BK channels both in the cytoplasm and nucleus. TEM ultrastructural analysis displayed a different nuclear distribution of Kir4.1 and BK channels. In the present study, the physiological role of Kir4.1 and BK currents at membrane potential, their involvement in migration, and the functional role of nuclear channels were discussed.

Squaring the Circle : A New Study of Inward and Outward-Rectifying Potassium Currents in U251 GBM Cells / D. Ratto, B. Ferrari, E. Roda, F. Brandalise, S. Siciliani, F. De Luca, E.C. Priori, C. Di Iorio, F. Cobelli, P. Veneroni, M.G. Bottone, P. Rossi. - In: CELLULAR AND MOLECULAR NEUROBIOLOGY. - ISSN 0272-4340. - 40:5(2020), pp. 813-828. [10.1007/s10571-019-00776-3]

Squaring the Circle : A New Study of Inward and Outward-Rectifying Potassium Currents in U251 GBM Cells

F. Brandalise;E.C. Priori;
2020

Abstract

In the present study, the functional role of the inwardly rectifying K+ channel, Kir4.1, and large-conductance Ca2+-activated K+ (BK) channel during cell migration in U251 cell line was investigated. We focused on polarised cells which are positive for the active-Cdc42 migration marker. The perforated patch technique was used to avoid intracellular dialysis and to maintain physiological changes in intracellular calcium. Wound healing was employed to assay migration after 24 h. Polarised cells recorded displayed different hallmarks of undifferentiated glial cells: depolarised resting membrane potential and high membrane resistance. Cells recorded outside wounded area did not display either constitutive inward or outward rectification. After migration, U251 cells were characterised by a constitutively smaller Kir4.1 and larger BK currents with a linearly related amplitude. Menthol modulation increased both currents in a linearly dependent manner, indicating a common mechanism triggered by activation of transient receptor potential melastatin 8 (TRPM8), a Ca2+-permeable non-selective cation channel. We hypothesised that both migration and menthol modulation would share an increase of intracellular calcium triggering the increase in Kir4.1 and BK channels. Immunocytochemistry demonstrated the cytoplasmic expression of both Kir4.1 and BK channels and a mislocation in the nucleus under basal conditions. Before and after migration, polarised cells increased the expression of Kir4.1 and BK channels both in the cytoplasm and nucleus. TEM ultrastructural analysis displayed a different nuclear distribution of Kir4.1 and BK channels. In the present study, the physiological role of Kir4.1 and BK currents at membrane potential, their involvement in migration, and the functional role of nuclear channels were discussed.
Glioblastoma; Inward rectifier; Outward rectifier; Perforated patch clamp; TRPM8; U251; Brain Neoplasms; Calcium; Cell Line, Tumor; Glioblastoma; Humans; Large-Conductance Calcium-Activated Potassium Channels; Membrane Potentials; Potassium Channels, Inwardly Rectifying; TRPM Cation Channels; Cell Movement
Settore BIO/09 - Fisiologia
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/878884
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