Introduction

Glioblastoma 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 these processes are modulated by changes in resting membrane potential, Vm, little is known about the origin of Vm and the role of BK. We have used cell-attached and whole-cell patch clamp in the glioblastoma cell line, SF188 to investigate the role of BK in GBM Vm. 

Method 

Single-channel BK currents were measured with cell-attached patch-clamp (CA). Pipettes contained 140 mM K⁺. Currents were measured with holding potentials from 0 mV to -90mV. Vm of intact cells were estimated from the reversal potential of CA single-channel BK current-voltage plots ( i-V) . Vm was then also measured with current clamp immediately after forming the whole-cell (WC) configuration. WC current-voltage-relationships (I-V) were characterised with voltage step protocol from a holding potential of -60mV. Data were normally distributed and are expressed as means ± S.D with n the number of cells. Statistical significance is defined as p<0.05 with tests stated. 

Results

At a pipette-potential of 0 mV, KB was spontaneously active in 23 out of 49 CA patches. CA I-V analyses indicated voltage-dependent activation of BK with a median slope-conductance of 202 pS. Vm estimated from the BK I-V reversal potential, -34.9±10.9 mV (n=21) was similar (p=0.9221, Paired t-test) to that subsequently measured under WC current clamp: -29.7±13 mV (n=7) ([Ca²⁺] = 45 nM). With a high [Ca²⁺] pipette solution (1.5 mM) Vm became significantly hyperpolarized in WC current clamp (-46.4±16.3 mV, n=14; p=0.001, Unpaired t-test). With the high pipette [Ca²⁺] the WC input resistance, Rm, was 221±194 MW (n = 8;  p=0.0443, Unpaired t-test); a value significantly smaller (p=0.0443, Unpaired t-test) to that measured with low pipette [Ca²⁺]: 394±194 MW (n=8). In 100% of CA patches, BK activity was abolished following perfusion of either 1 µM paxilline (n=3) or 200 µM quinine (n=3). With a low pipette [Ca²⁺], WC Vm was unaffected by 1 mM TEA (n=15) or 1 µM paxilline (n=7) , but was significantly depolarised by 21±3.3 mV with 200 µM quinine (n=3) relative to perfusion control (n=4; p=0.0091 ANOVA Dunnet’s multiple comparison test). 

Conclusion

GBM SF188 cells exhibit spontaneous K⁺ channel activity in CA patches, with biophysical and pharmacological properties typical for BK. At low intracellular [Ca²⁺]  BK does not appear to be responsible for the resting Vm, however the hyperpolarization of Vm  associated with a decrease in Rm that is seen on  elevation of [Ca²⁺]_i are indicative of BK activation The reversal potential of BK in CA patches appears to be an accurate non-invasive measure of the resting membrane potential of SF188 cells. Further studies are required to determine what underlies the BK activation observed in CA patches on SF188 and to find under what physiological conditions does BK become activated to contribute  to Vm in this cell line.