The standing outward K+ current (IKso) is a major determinant of cerebellar granule neurone (CGN) excitability and is predominantly mediated by an acid-sensitive member of the 2P domain K+ channel family, TASK (Millar et al. 2000). We have recently shown that TASK1 is acutely sensitive to hypoxia in CGN (Plant et al. 2002) and so chose to investigate the effects of chronic hypoxia on this physiologically important current.
Primary cultures of rat cerebellar granule neurones were prepared as previously described from tissue obtained from humanely killed animals (Plant et al. 2002). For amphotericin B perforated patch-clamp recordings, extracellular and pipette solutions used were those detailed in Plant et al. (2001). IKso was measured in cells by holding at a potential of -20 mV and ramping the voltage to -100 mV at a rate of 100 mV s-1. All values are given as means ± S.E.M.
The effects of chronic hypoxia on IKso were measured following incubation of cells for 24 h at 2.5 % O2. Chronic hypoxia significantly increased IKso measured at -20 mV in CGN from 98 ± 0.1 to 164 ± 1.0 pA pF-1 (n = 34 and 10, respectively, P < 0.01, Student’s unpaired t test). This effect could be mimicked by the chronic application of the Alzheimer’s disease peptide amyloid β protein (Aβ) to cells. 24 h incubation with 100 nM Aβ elevated IKso to 170 ± 0.8 pA pF-1 (n = 12, P < 0.01). In both cases (chronic hypoxia and Aβ) the acid-sensitive component of IKso was increased to a similar extent (82.6 ± 4.0 %, n = 3 and 85 ± 4.3 %, n = 3, respectively), indicating that both treatments augmented TASK in these cells. To test whether chronic hypoxia was acting via a change in production of endogenous Aβ, cells were exposed to chronic hypoxia and an increase in Aβ was visualised by immunofluoresence. This hypoxia-induced increase in Aβ immunofluoresence could be prevented by incubation with 2-naphthoyl-VF-CHO (λ-IV, 10 mM), an inhibitor of the λ secretase that cleaves amyloid precursor protein to produce Aβ. Crucially, λ-IV also prevented the hypoxia-induced increase in IKso.
These data indicate that chronic hypoxia upregulates the functional expression of TASK in native neurones. Furthermore, our data indicate that Aβ may represent a key signalling molecule in the modulation of TASK channel activity induced by chronic hypoxia. Such an effect may contribute to the known increased incidence of dementias following hypoxic/ischaemic episodes (Moroney et al. 1997).
This work was supported by the MRC and The Wellcome Trust.
All procedures accord with current UK legislation.