Squaring the Circle: A New Study of Inward and Outward-Rectifying Potassium Currents in U251 GBM Cells
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ORIGINAL RESEARCH
Squaring the Circle: A New Study of Inward and Outward‑Rectifying Potassium Currents in U251 GBM Cells Daniela Ratto1 · Beatrice Ferrari1 · Elisa Roda1,2 · Federico Brandalise3 · Stella Siciliani1 · Fabrizio De Luca1 · Erica Cecilia Priori1 · Carmine Di Iorio1 · Filippo Cobelli1 · Paola Veneroni1 · Maria Grazia Bottone1 · Paola Rossi1 Received: 29 August 2019 / Accepted: 9 December 2019 © Springer Science+Business Media, LLC, part of Springer Nature 2019
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 C a2+-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. Keywords Glioblastoma · Inward rectifier · Outward rectifier · TRPM8 · Perforated patch clamp · U251
Introduction Most primary brain tumours in adult humans arise from glial cells. These neoplasms carry a very poor prognosis largely due to the diffuse invasiveness into normal brain that Daniela Ratto and Beatrice Ferrari are co-first authors. * Paola Rossi [email protected] 1
Department of Biology and Biotechnology “L. Spallanzani”, University of Pavia, 27100 Pavia, Italy
2
Toxicology Unit, Laboratory of Clinical & Experimental Toxicology, Pavia Poison Centre, National Toxicology Information Centre, ICS Maugeri SpA, IRCCS Pavia, 27100 Pavia, Italy
3
Department of Fundamental Neurosciences (NEUFO), University of Ge
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