A, IGR1 cells were grown for 24, 48 and 72 h in normoxia (20% oxygen, □) or hypoxia (3% O₂, ▪). Summarized data showing mean amplitude of the macroscopic K_Ca currents at +50 mV. Also shown are the ratios of the current, the current density, and the cell capacitance. The numbers in parentheses indicate the number of cells (n) measured. B, similar experiments as in A, but IGR1 cells were treated for 24 h with 100 μm CoCl₂ or 100 μm desferrioxamine (DFO) to mimic the effect of hypoxia (controls, □; treated cells, ▪). C, Western blot analysis of HIF-1α in IGR1 cells exposed for 4 and 24 h to hypoxia or treated with 100 μm CoCl₂. After indicated times, cell lysates were prepared, and lysates containing equal amounts of protein were used for Western blot analysis. Blots for β-actin are shown as a gel-loading control.

A, proliferation of IGR1 cells under normoxic (○) or hypoxic conditions (•) during a growth period of 48 h. Proliferation rates were determined as BrdU incorporation (number of cell batches, N = 3) or MTT conversion (N = 4) and normalized to the respective normoxic controls. Mean values for all experiments are shown for the following conditions: control (−), presence of 100 nm apamin (A), 100 nm ChTX (C) or 100 nm of each blocker (AC). Hypoxia (3% O₂) increased the proliferation rate by about 10%. B, up-regulation of apamin-sensitive channels by hypoxia. Mean current amplitudes of K_Ca currents in HEK293 cells expressing rSK2 channels and in IGR1 cells with and without 24-h incubation in 100 μm CoCl₂. The open histogram bars represent the control currents and the shaded bars the currents after application of 100 nm apamin. This procedure blocks almost all of the heterologously expressed rSK2 channels. IGR1 cells under control conditions do not contain apamin-sensitive K_Ca channels. Treatment with CoCl₂ increases K_Ca current magnitude and part of this up-regulated conductance is blocked by apamin. The individual ratios of the apamin effects are: rSK2, 0.12 ± 0.03; IGR1, 1.01 ± 0.02; IGR1 + CoCl₂, 0.71 ± 0.04. C, RT-PCR analysis for IK and SK channel subtypes in IGR1 cells. mRNA levels detected with RT-PCR under control conditions for SK1, SK2, SK3 and IK transcripts. D, relative changes in the concentration of mRNA coding for SK2 and IK as a function of incubation time in 3% O₂ (n = 4–6). Straight lines connect the data points.

A, each six examples of current responses to voltage-ramp stimulation (from −100 to +50 mV) in the absence of Ca²⁺ (left) and with 800 nm free Ca²⁺ in the pipette solution (right). Note that the reversal potential is close to the K⁺ equilibrium potential of about −80 mV, indicating almost no contribution of non-specific leak currents. B, box plot for the distribution of resting membrane potential of the cells described in A. C, influence of intracellular Ca²⁺ on the current amplitude at +50 mV, the current density and the cell capacitance. The panels on the right show the ratios of the respective parameters with indication of the statistical significance (***P < 0.001); the dashed line marks a ratio of one, i.e. no change. The numbers in parentheses in B and C indicate the number of cells (n) measured.

A, IGR1 melanoma cells were transiently transfected with plasmids coding for HIF-1α or HIF-1α-ΔN. Either EGFP or CD8 were cotransfected as a marker. The histogram bars show the effects of HIF transfection on the K_Ca current amplitude (top). The lower panels show the ratios (overexpressed versus control) of the current amplitude, the current density and the cell capacitance. B, IGR1 melanoma cells were transfected with plasmids coding for VHL-IRES (transient expression), VHL-Δβ (transient expression), and VHL19 (stable expression). EGFP was used (either as IRES construct or separately) as a marker. C, IGR1-VHL19 stable cells were transiently transfected with plasmids coding for HIF-1α or HIF-1α-P564A. The panels in B and C are the same as in A.

A, mean K_Ca currents, ratio of currents, current densities and cell capacitances for IGR39 cells (number of cell batches, N = 5) and primary human melanoma metastasis cells (N = 2). The open bars indicate control measurements and the grey bars indicate measurements after 24-h exposure to 100 μm CoCl₂. B, proliferation of IGR39 cells under normoxic (○) or hypoxic conditions (•) during a period of 48 h. Proliferation rates were determined as BrdU incorporation (N = 1) or MTT conversion (N = 4) and normalized to the respective normoxic controls. Mean values of all experiments are shown for the following conditions: control (−), presence of 100 nm apamin (A), 100 nm ChTX (C) or 100 nm of each blocker (AC). C, mean proliferation rates of primary human melanoma cells measured by BrdU incorporation (N = 3). All symbols are the same as in B.