P1-162: suppressive effects of amyloid peptide fragments on macro- and micro-scopic kv1.1 channel activity

Past studies have linked the beta amyloid peptide (Aβ) to the disruption of Ca2+ homeostasis, synaptic communication, and long-term potentiation (LTP) in rodent models of Alzheimer's Disease (AD) but the mechanism(s) underlying these effects is/are still largely unclear. Because Kv1.1 and related channels are activated during an action potential, regulate depolarization-produced Ca2+ influx, and inhibition of Kv1 channels can be neurotoxic, we have proposed that Aβ-suppression of Kv1 channels may be an early critical step in AD pathogenesis. Stage V and VI Xenopus laevis oocytes [Ecocyte Bioscience (Austin, TX)] were injected with Kv1.1 cRNA. The effects of bath application of Aβ fragments (1-42) and (25-35) on macroscopic currents were assessed using standard two electrode voltage-clamp (TEVC) methods, whereas direct single channel effects were assessed using patch clamp and artificial membrane techniques [“tip-dip” and black lipid membrane (BLM)]. Bath application of both 1 μM Aβ(1-42) and Aβ(25-35) produced ∼45% suppression of macroscopic Kv1.1 current within 30 min. Aβ suppression of Kv1.1 was partially Ca2+- and PP2B-dependent, with only ∼25% Aβ suppression occurring when cells were loaded with BAPTA-AM or exposed to the PP2B-inhibitor cyclosporine A (CsA). Reduction in macroscopic currents was unlikely to reflect a reduction in the number of channels present on the plasmamembrane, as western blot analyses of plasmamembrane protein failed to reveal any detectable decreases in Kv1.1 channel band intensities following acute or prolonged exposure to Aβ. Application of Aβ to the intracellular face of Kv1.1 channels in both patch clamp and tip dip experiments produced dramatic reductions in p(open), with no observable current ∼2 min post-addition. BLM experiments also showed reductions in p(open) in response to intra- and extra-cellular Aβ application but did not fully eliminate channel activity (∼45% reduction). Suppression of Kv1.1 and related K+ channels presynaptically could lead to larger and longer action potentials, thus allowing a greater influx of Ca2+ and subsequent increase in glutamate release at excitatory synapses. Postsynaptically, Aβ-produced suppression of dendritic Kv channels and the increased glutamate release, may contribute to excitotoxicity through activation of AMPA and NMDA receptors.