Mapping the functional expression of auxiliary subunits of KCa1.1 in glioblastoma

Figures

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  Figure 1. KCa1.1 is a key K+ ion channel in glioblastoma cell membrane Current density–voltage relationship of whole-cell currents in primary glioblastoma cells. Current densities (J, pA/pF) at the indicated membrane potentials were calculated by dividing the peak current by the cell membrane capacitance. Data were obtained with intracellular solutions containing zero Ca2+ (black, n = 11) or 1 µM Ca2+ (blue, n = 5). (B) Current density–voltage relationship of whole-cell currents in primary glioblastoma cells (GBM, orange, n = 5) and in the U-87 MG glioblastoma cell line (U-87, green, n = 5). (C) Confocal microscopic images of a primary GBM cell. GFAP (top left) was labeled with anti-mouse Alexa 555, the KCa1.1 alpha subunit was labeled with anti-rabbit Alexa 647, the overlay of the two images is in the top right. The bottom right panel depicts the punctate staining pattern of KCa1.1 at higher magnification. (D) Representative whole-cell current traces recorded in a primary glioblastoma cell (GBM, left) and in a U-87 MG cell (U-87 MG, right). Currents were evoked by repeated depolarizations to + 100 mV from a holding potential of -100 mV (see voltage pulse above the raw current traces)…

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  Figure 2. Pharmacological modulators affect β subunit-associated KCa1.1 channels in glioblastoma. (A) The effect of lithocholic acid (LCA, 75 µM) on primary GBM (orange bar, n = 16) and U-87 MG cells (green bar, n = 16). The current recorded during LCA application was normalized to the current recorded in control solution (dotted line). (B) The effect of arachidonic acid (AA, 30 µM) on primary GBM (orange bar, n = 14) and U-87 MG (green bar n = 16) cells. The current recorded during AA application was normalized to the current recorded in control solution (dotted line). (C) Channel inactivation in a 100 ms time interval was determined by the current ratio It1/It0, where t0 was the current amplitude at the beginning, and t1 was the current amplitude at the end of the 100 ms time interval for primary GBM cells (orange bar, n = 33) and for U-87 cells (green bar, n = 19). The pipette filling solution contained 1 µM Ca2+ concentration. (D) The effect of iberiotoxin (IbTx, 23 nM, purple bars, for GBM n = 18, for U-87 n = 8) and paxilline (Pax, 1 µM, red bars, for GBM n = 20, for U-87 n = 13) on the KCa1.1 current in primary GBM and in U-87 cells. Black symbols indicate remaining current fraction…

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  Figure 3. PCR, pharmacology and gene silencing is consistent with the expression of the KCa1.1 β3 subunit in U-87 MG cells. (A) Results of qPCR experiments assessing the relative expression of the β subunits in the U-87 MG cell line compared to the pore-forming α subunit (black bar). β1, β2 and β3 mRNA levels divided by the expression of the KCa1.1 channel α-subunit are represented with green, blue and purple bars, respectively (N = 3, n = 3). (B) Western blot of the untreated and gene-silenced U-87 MG cells. The populations were tested in duplicate, the left two lanes are the untreated U-87 MG cells and the right two lanes are untreated (non-transfected and not gene silenced) CHO cells. The middle lane pairs are for the KCNMB1 (β1), KCNMB2 (β2) and KCNMB3 (β3) silenced populations of U-87 MG, from top to bottom, respectively. The thick band in the top box at 42 kDa corresponds to actin and the 22 kDa marker to the β1 subunit (KCNMB1 gene product). In the second box (middle) the β2 band (27 kDa marker, KCNMB2 gene product), and in the third box (bottom) the β3 band (KCNMB3 gene product) are shown (31 kDa marker). (C to E) Pharmacological studies after gene silencing (NSilence = 3).…

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  Figure 4. KCa1.1 β3 subunit is functionally expressed in U-87 MG cells (A) Representative current trace recorded in a U-87 MG cell using the voltage protocol shown on the top and 10 µM free Ca2+ in the pipette filling solution. The red rectangle highlights current inactivation whereas the blue rectangle marks the current at − 100 mV in the absence of DTT. (B) Inactivation of the current (red rectangle in panel A) at an increased resolution. Continuous black line is the measured current, dashed red line is the fitted trace using a single exponential decay model. (C) The current ratio I10ms/Ipeak denotes the ratio of the current at 10 ms following the start of the depolarization over the peak current. (D) Scatter plot of the inactivation time constants (tau) determined from fitting single-exponential decaying function to the currents. (E) Instantaneous (tail) current (blue rectangle) in a U-87 MG cell treated with 20 mM DTT for 20 min. The current was recorded using the pulse protocol in panel A. (F) Peak currents at − 100 mV for control U-87 MG cells in the absence of DTT (No DTT, black dots) and following 20 min application of 20 mM DTT (20 mM DTT, purple dots). Throughout the figure dots…

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  Figure 5. KCa1.1 β3 is involved in the Ca2+ response of U87-MG cells (A) Representative intracellular Ca2+ measurements, F340/F380 ratio reports the intracellular Ca2+ concentration. Vertical dashed line indicates the beginning of carbachol (10 µM, left, black) or 10 µM carbachol + 1 µM paxilline (right, red) superfusion after a 2 min superfusion with control Ringer's solution (B) Heat map depicting the F340/F380 ratio response as a function of time for each individual cell measured. Left: 10 µM carbachol (N = 5, n = 120); right: 10 µM carbachol + 1 µM paxilline (N = 5, n = 91). Pseudocolor code shows increasing Ca2+ concentrations with more magenta-toned color. Green rectangle confines cells showing > 20% increase in F340/F380 ratio after carbachol superfusion. (C) Peak of the 10 µM carbachol-induced F340/F380 ratio (see A and B for details) of control superfused cells (n = 59, N = 3) and 1 µM paxilline-treated cells (n = 49, N = 3). (D) Carbachol-elicited intracellular Ca2+ response of cells after silencing of GAPDH (green, siGAPDH, N = 3, n = 39, F340/F380 = 1.1 ± 0.06), KCa1.1 (purple, siKCNMA1, N = 3, n = 51, F340/F380 = 0.8 ± 0.04) and KCa1.1 β3 (black, siKCNMB3, N = 3, n = 30,…

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  Figure 6. KCa1.1 expression, but not the β3 subunit function shows cell cycle-dependence (A) Flow cytometry of colchicine treated (4 µg/ml, 24 h; in orange), starving (serum free DMEM, 24 h; in red) and untreated U-87 MG cells (in blue), where propidium iodide (PI) intensity is shown as a function of cell count. (B) Representative patch-clamp traces of the serum starved (red), colchicine-treated (orange) and untreated (blue) populations, respectively. The displayed currents were recorded at + 80 mV depolarizing pulse with 1 µM free Ca2+ in the pipette-filling solution. (C) Current amplitude (I, nA) as a function of membrane potential (mV) for control (untreated, blue, n = 20), for colchicine-treated (orange n = 19), and starving cells (n = 9). Whole-cell currents were obtained as in Fig. 1A (1 µM free Ca2+ in the pipette-filling solution). (D) Current density (J, pA/pF) as a function of membrane potential (mV) was calculated for n = 18 control (blue), n = 18 colchicine-treated (orange) and n = 7 starving cells (red) from the data in panel C. In panels C and D, asterisks indicate the significant difference between the colchicine treated and the untreated cells. (E) Effect of 1 µM paxilline…