Prolonged hypoxia regulates the expression of native ion channels (e.g. Colebrooke et al. 2002). Whether this functional remodelling also occurs in recombinant systems has yet to be addressed. Here, we report the effects of chronic hypoxia on human recombinant large conductance, Ca²⁺-activated K⁺ (maxi-K) channel activity in human embryonic kidney (HEK 293) cells stably expressing the α and β subunits (Ahring et al. 1997).

Cells were cultured as previously described (Lewis et al. 2002) in P_O2 of either 142 mmHg (normoxia) or 18 mmHg (chronic hypoxia) for 72 h. Whole-cell currents were recorded by the patch-clamp technique using standard ‘physiological’ solutions and voltage-clamp protocols with [Ca²⁺]_i buffered to 4 nM, 27 nM, 300 nM or 3 mM. The effect of acute hypoxia was examined by perfusing cells with N₂-bubbled extracellular solution as previously described (Lewis et al. 2002).

Consistent with our data from inside-out patches (Lewis et al. 2002), inhibition of whole-cell K⁺ currents by acute hypoxia was Ca²⁺_i-dependent; at low levels (4 nM) of Ca²⁺_i, K⁺ current displayed no significant acute hypoxic inhibition whilst at higher levels (> 27 nM), acute hypoxia always depressed K⁺ currents. Chronic hypoxia increased K⁺ current density at all [Ca²⁺]_i. For example, at 27 nM, chronic hypoxia caused an increase from 56.0 ± 7.1 pA pF^-1 (mean ± S.E.M., n = 10) to 108.3 ± 14.9 pA pF^-1 (measured at +60 mV; P < 0.005, Student’s unpaired t test). Importantly, chronic hypoxia dramatically modulated the Ca²⁺ sensitivity of the acute hypoxic response such that hypoxic inhibition of K⁺ current could now be seen at low [Ca²⁺]_i. For example, at 4 nM, acute hypoxic inhibition was 1.1 ± 3.4 % (n = 8) in normoxic cells and a 15.5 ± 5.0 % (n = 8, P < 0.05) in chronic hypoxic cells; this functional remodelling was prevented by pretreatment with 5 mg ml^-1 actinomycin D (n = 5). Western blotting showed that α subunit expression was unaffected by chronic hypoxia, whilst β subunit expression was increased by 324.0 ± 42 % (n = 6, P < 0.05); as with the functional assay, this augmentation was prevented by actinomycin D (n = 4). Finally, immunocytochemistry, employing confocal microscopy, showed that chronic hypoxia increased plasma membrane colocalisation of the α and β subunits and strengthens the argument that modulation of the acute hypoxic response of maxi-K channels by chronic hypoxia is due to differential transcriptional upregulation of the β subunit.

This work was funded by British Heart Foundation and The Wellcome Trust.