Kv3.1 potassium channels are expressed in neurons that fire action potentials at high frequency (Perney et al. 1992), and also in PC12 (Conforti & Millhorn, 1997) and pulmonary artery smooth muscle cells (Osipenko et al. 2000), both of which display an oxygen-sensitive K⁺ current. Recent studies showed that in an in vitro expression system, hypoxia inhibits whole-cell Kv3.1b current and single-channel activity (Osipenko et al. 2000). This study investigated the underlying role of mitochondria in the modulation of Kv3.1b channels by hypoxia.

HEK293 cells stably expressing mouse Kv3.1b (mKv3.1b) DNA were used to record whole-cell currents. Bath and internal (pipette) solutions were based on Osipenko et al. (2000). Hypoxic solutions were prepared by bubbling with 100 % N₂, giving giving 7.1 ± 0.8 % O₂ (mean ± S.E.M., n = 7) (control = 20.3 ± 0.5 %, n = 7). A mitochondrial DNA (mtDNA)-depleted cell line (τ⁰) was generated from the transfected HEK293 cells (τ⁺) by culturing for 3-6 weeks in the presence of 50 ng ml^-1 ethidium bromide in medium supplemented with uridine and pyruvate (King & Attardi, 1989). Single-cell PCR demonstrated that mtDNA had been depleted in the cells used for current recording. Mitochondrial membrane potential was detected by confocal microscopy of cells incubated with 100 nM tetramethylrhodamine ethyl ester (TMRE). Statistical comparisons employed Student’s two-tailed, unpaired t test, P < 0.05 being considered significant.

The mKv3.1b current displayed an activation threshold between -20 and -30 mV (n = 31) and amplitude of 157 ± 12 pA pF^-1 at 20 mV. When exposed to hypoxia for 10 min the current at 20 mV was reduced by 24 ± 2 % (n = 17). Mitochondrial inhibition caused by either rotenone (1 µM, n = 7), an electron transport chain inhibitor, or carbonyl cyanide p-(trifluomethoxy) phenylhydrazone (FCCP, 1 µM, n = 7), an uncoupler of oxidative phosphorylation, had no effect on Kv3.1b current under control conditions, but abolished the hypoxic inhibition. The effect of hypoxia on Kv3.1b current was also essentially abolished in τ⁰ cells, with a reduction of only 2.5 ± 0.4 % (n = 8, P < 0.05 compared with the control cells) at 20 mV. Measurements of TMRE fluorescence revealed a lower intensity in τ⁰ cells (117 ± 9 units, n = 9) compared with τ⁺ cells (150 ± 10 units, n = 9, P < 0.05), although both displayed similar, punctate staining that was depleted by 1 µM FCCP. This suggests a reduced mitochondrial membrane potential in τ⁰ cells.

These results suggest that mitochondria play a vital role in mediating the inhibition of Kv3.1b channels by hypoxia.