The role of calcium activated and voltage gated potassium channel BK, in glioblastoma multiforme membrane potential.Tools Ratnasingham, Mathuscha (2024) The role of calcium activated and voltage gated potassium channel BK, in glioblastoma multiforme membrane potential. MRes thesis, University of Nottingham.
AbstractGlioblastoma multiforme (GBM), is an aggressive brain tumour that accounts for nearly half of all glial brain tumours. Large conductance voltage and Ca2+-activated potassium channels, BK, are overexpressed in GBM and are thought to play a role in their invasion and migration. Although changes in resting membrane potential modulate these processes, little is known about the origin of it in GBM and the role of BK. I have used cell-attached and whole-cell patch clamp in the glioblastoma cell lines, SF188 and GCE62 to investigate the role of BK in GMB resting membrane potential. Single channel BK currents were measured with cell-attached patch clamp. Pipettes contained 140 mM K+. Currents were measured with holding potentials from 0 mV to -90 mV. The resting membrane potential of intact cells was estimated from the reversal potential of cell-attached single-channel BK current-voltage (I-V) plots. Resting membrane potential was then measured with current clamp immediately after forming the whole-cell configuration. In SF188s, at a pipette potential of 0 mV, BK was spontaneously active in 31 out of 71 patches. Cell-attached I-V analyses indicated voltage-dependent activation of gBK with a bimodal distribution of median slope-conductance of around 124 and 215 pS. Membrane potential estimated from the cell-attached I-V reversal potential, -35±0.18 mV was similar to that subsequently measured under whole-cell current clamp -35±0.18 mV ([Ca2+] = 120 nM). With high [Ca2+] pipette solution (2.5 mM), membrane potential was significantly hyperpolarized in whole-cell current clamp (-44 ± 17 mV) whereas the input resistance, 220 ± 173 M; was similar to that with low pipette [Ca2+]: 396 ± 173 M. In 100% of cell-attached patches, BK activity was abolished by 10 µM paxilline and 200 µM quinine. With low pipette [Ca2+] whole-cell membrane potential was unaffected by 200 µM quinine but was significantly hyperpolarised by 13 mV with 10 µM paxilline and 9 mV with 1 mM TEA. With low pipette [Ca2+] whole-cell membrane potential was unaffected by Cl- free Hanks but was significantly depolarised by 8 mV with K+ Hanks and hyperpolarised by 8 mV with Na+ free Hanks. In GCE62’s, at a pipette potential of 0 mV, BK was spontaneously active in 2 out of 30 patches. In whole cell current clamp there was no significant difference in the membrane potential and input resistance in the high [Ca2+] pipette solution compared to low [Ca2+] in the pipette solution. GBM SF188 exhibit spontaneous K+ channel activity in cell-attached patches, with biophysical and pharmacological properties typical for BK. At low intracellular [Ca2+] BK does not appear to be responsible for the resting membrane potential. The reversal potential of BK in cell-attached patches appears to be an accurate non-invasive measure of the resting membrane potential of SF188 cells. The membrane potential seems to be predominated by potassium with a degree of sodium. Further studies are required to determine what underlies BK activation in cell-attached patches in SF188 and under what conditions is BK activated to contribute to membrane potential in this cell line.
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