The A-type K+ current (IKA) is functionally increased in cerebellar granule neurones (CGN) by exogenous treatment with 10-100 nM amyloid β protein 1-40 (Aβ; Ramsden et al. 2001), the Alzheimer’s disease-related peptide that is constitutively cleaved from its precursor protein by β- and λ-secretases. In agreement with this, inhibitors of β- and λ-secretase reduce endogenous Aβ levels in CGN and significantly diminish IKA. This reduction in current is rescued by co-application of 1 nM Aβ (Plant et al. 2002). Here we use Western blotting techniques to investigate the effects of exogenous Aβ treatment and λ-secretase inhibition on the expression levels of Kv4.2 and Kv4.3, the K+ channel α-subunits thought to mediate IKA.
CGN were isolated and cultured from humanely killed 6-day-old neonatal rats as previously described (Ramsden et al. 2001). Whole cell lysates were prepared after 7 days and Western blot analysis of 10 % SDS-PAGE gels was performed using polyclonal antibodies raised against Kv4.2 and Kv4.3 α-subunits. Quantification of the bands was performed by laser scanning densitometry and data are given as means ± S.E.M.
Following 24 h treatment with 100 nM Aβ there was a significant increase in the expression of Kv4.2 and Kv4.3 by 49 ± 11 % (P < 0.02, Student’s paired t test, n = 4) and 45 ± 20 % (P < 0.05, n = 5), respectively. Kv4.2 expression showed a significant increase of 45 ± 11 % (P < 0.05, n = 4) as rapidly as 2 h after Aβ treatment but no significant effects were seen following 48 h treatment (-24 ± 19 %, n = 5). In contrast to its effects on IKA (Plant et al. 2002), the λ-secretase inhibitor 2-naphthoyl-VF-CHO (λ-IV, 10 µM) had no significant effect on the expression of Kv4.2 (protein expression was 96 ± 11 % of control levels, n = 5). Furthermore, at a concentration of Aβ (1 nM) that can negate the reduction in current observed following treatment with λ-IV (Plant et al. 2002), there was no significant effect of Aβ on the protein expression levels of Kv4.2 channels (protein expression was 100 ± 24 % of controls in the presence of λ-IV and 1 nM Aβ, n = 5). These results were confirmed qualitatively by immunofluorescent labelling of permeabilised cells with an antiKv4.2 polyclonal antibody.
Thus, exogenous Aβ causes a rapid transient increase in Kv4.2 expression that mirrors changes seen in IKA. Block of endogenous Aβ production reduces IKA but has no effect on Kv4.2 protein expression, suggesting an effect that may be mediated by changes in trafficking of Kv subunits. Our data provide further support for a physiological role for Aβ in regulating K+ channel function in the central nervous system.
This work was supported by The Wellcome Trust.