Studies have shown an increased incidence of Alzheimer’s disease (AD) in individuals who have suffered an episode of cerebral ischaemia (Moroney et al. 1996). The AD related peptide amyloid β protein (Aβ) disrupts cellular ion homeostasis and is increased following hypoxia in PC12 cells (Taylor et al. 1999). In addition, we have reported an increase in potassium channel currents in cerebellar granule neurons following application of Aβ (Ramsden et al. 2001). Here, we investigated the effects of chronic hypoxia on potassium channel currents in primary cultures of cerebellar granule neurones and examined the potential involvement of Aβ in these effects.

The whole-cell configuration of the patch-clamp technique was used to measure K⁺ channel currents. Cells held at -70 mV were prepulsed for 200 ms to -140 or -50 mV before stepping to test potentials between -60 and +70 mV. Untreated cells in addition to those cells treated with the gamma-secretase inhibitor, λ-IV, were incubated in a humidified atmosphere in either normoxic (21 % O₂) or hypoxic (2.5 % O₂) air, at 37 °C for 24-48h. All current values were normalised to whole-cell capacitance and data presented as means ± S.E.M. Western blotting and immunohistochemical staining for Aβ and Kv subunits used the monoclonal antibody 3D6 and polyclonal antibodies, respectively. Statistical comparisons were made using Student’s unpaired t test.

Chronic hypoxia caused a significant increase in K⁺ channel current density when compared to controls. At the +50 mV test potential peak K⁺ channel current was 766 ± 50 pA pF^-1 in normoxic cells (n = 34) compared to 961 ± 69 pA pF^-1 in hypoxia treated cells (n = 36), following a prepulse to -140 mV (P < 0.05). The gamma secretase inhibitor, λ-IV (3 µM), had no effect on K⁺ current under normoxic conditions (797 ± 94 pA pF^-1, n = 25); however, it caused a reversal of the hypoxia-induced increase in potassium current (860 ± 121 pA pF^-1, n = 24). Immunofluorescence imaging revealed an increase in Aβ production following chronic hypoxia when compared to normoxic controls. This increase in Aβ was prevented following application of λ-IV. Western blot and immunofluorescence imaging of Kv4.2 and 4.3 suggested that changes in I_KA were due to an increase in Kv expression.

These data suggest that the hypoxia-induced increase in K⁺ currents in cultured cerebellar granule neurons is, at least in part, due to the increased production of Aβ following chronic hypoxia.