Basic Science and Pathogenesis

The roles of Aβ in the pathogenesis of Alzheimer 's disease (AD) include disruption of synaptic communication/function and synaptic plasticity mechanisms thought to underlie learning and memory. Exactly how these abnormal processes arise is incompletely understood, but evidence suggests that dysregulation of intracellular Ca2+ levels is involved in alterations of neuronal excitability, synaptic remodeling, and neurodegeneration in AD. Our lab has focused on the potential involvement of voltage-gated potassium channels (VGKCs) in these processes, particularly Kv1.x family members as they regulate Ca2+ influx, and their inhibition may lead to synapto- and neuro-toxicity through hyperexcitability and excess glutamate release. We have previously observed rapid and robust Aβ(1-42)-suppression (∼50% in 30 min) of macroscopic currents mediated by homomeric Kv1.1, Kv1.2, and heteromeric Kv1.1/1.2 channels. BAPTA-AM treatment, as well as Cyclosporin A (CsA), resulted in significantly less (∼20%) Aβ-suppression, implicating Ca2+-dependent PP2B as critical. Additionally, the broad spectrum PTK inhibitor genistein completely eliminated suppression by Aβ(1-42). Here we sought to understand the role of tyrosine residue Y132 in Kv1.2 in the suppression by Aβ.

The Y132F mutant was generated from a WT KV1.2-pGEMHE plasmid using a PCR-based site-directed mutagenesis kit (NEB), and an invitro transcription reaction was utilized to generate both mutant and WT cRNA. Stage V/VI Xenopus laevis oocytes were injected with ∼1ng of WT Kv1.2 cRNA or Y132F cRNA. The effects of bath application of 1 μM of Aβ(1-42) were evaluated using two-electrode voltage clamp electrophysiology (TEVC), with recordings 24-48 hrs post-injection.

Baseline currents were unaffected by the Y132F mutation in kinetics or voltage-dependency compared to WT controls. Similarly, neither WT nor mutant channels significantly differed in their response to 0.01% DMSO with no more than ∼5% suppression observed (n = 15, p >0.05). However, Y132F exhibited greater resistance to suppression by Aβ(1-42), with 17% suppression (n = 10) compared to the WT 50% 30 minutes post exposure (n = 10, p < 0.007).

Phosphorylation at site Y132 may be a critical mediator of Aβ based suppression of Kv1.2 current, particularly through endocytotic methods. Suppression of these channels may lead to increased excitotoxicity via increased Ca2+ influx into excitatory neurons and glutamate release.