KCa3.1 channels are absent in adult’s healthy brain and cerebellum, but are highly expressed in glioblastoma tumor samples and derived primary cell lines.

<p>(<b>A</b>) Real-time PCR on three primary human cell lines of glioblastomas (CRL8, lane 1; FCN9, lane 2; MZC12, lane 3) demonstrated that KCa3.1 transcripts expression is higher compared to NHA (lane 4). (<b>B–G</b>) Immunohistochemical staining on normal human brain tissue revealed KCa3.1 protein presence only in endothelial cells of blood vessels (<b>B</b>) while we observed a diffuse staining in high grade tumors (CRL8, <b>C</b>; FCN9, <b>D</b>, MZC12, <b>E</b>) and a KCa3.1 signal only in neo-vascularisation area (glio low grade, F). As positive control we used physiological lung tissue (<b>G</b>). (<b>H</b>) Typical time course of the current recorded at −40 mV from a FCN9 cell by applying repetitive (every 5s) voltage ramps from −100 to +100 mV. 3 mM TEA and 1 mM octanole were added to block the BK and gap junctional channel, respectively, usually co-expressed with KCa3.1 channels in glioblastoma cells (21; 22). DC-EBIO (100 µM)+ ionomycin (0.5 µM) and DC-EBIO+ion +3 µM TRAM-34 were applied in succession to verify the functional expression of KCa3.1 currents (cf text). (<b>I</b>) Representative I–V relationships in presence of DC-EBIO+ionomycin, and DC-EBIO+ ionomycin +TRAM-34. Data in panel (<b>H</b>) and (<b>I</b>) are from the same experiment. <i>Inset</i>: I-V relationship of the KCa3.1 current obtained by subtracting the current ramps recorded in DC-EBIO+ion+TRAM-34 from that recorded in DC-EBIO+ion. (<b>L</b>) Plot reporting the KCa3.1 current density at 0 mV (assessed as in panel <b>I</b>) measured in the three primary glioblastoma cell lines. Since in several cells a voltage-gated K current activating at membrane potentials higher than −20 mV was present, in these cases measurements of the KCa3.1 current density were performed at −40 mV, and the assessed current density was then extrapolated at 0 mV by assuming a linear current-voltage relationship. *ANOVA test, p<0.05.