FE65 is an adaptor protein that interacts with the cytoplasmic tail of the amyloid precursor protein (APP). In cultured nonneuronal cells, the formation of the FE65-APP complex is a key element for the modulation of APP processing, signalling and b-amyloid (Ab) production. The functions of FE65 in vivo, including its role in the metabolism of neuronal APP, remain to be investigated. In this study, transgenic mice expressing human FE65 were generated and crossbred with APP transgenic mice, known to develop Ab deposits at 6 months of age. A multimeric protein network centred on the cytoplasmic domain of APP has been described Abbreviations used: Ab, b-amyloid peptide; AD, Alzheimer's disease; AICD, APP intracellular domain; APP, amyloid precursor protein; CTFs, carboxyl terminal fragments; hFE65, human FE65; LRP, low-density lipoprotein receptor-related protein; PBS, phosphate-buffered saline; PBST, phosphate-buffered saline plus Tween 20; PTB2, second phospho-tyrosine binding site; sAPP, secreted APP.

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Inwardly rectifying I K1 potassium currents of the heart control the resting membrane potential of ventricular cardiomyocytes during diastole and contribute to their repolarization after each action potential. Mutations in the gene encoding K ir 2.1 channels, which primarily conduct ventricular I K1 , are associated with inheritable forms of arrhythmias and sudden cardiac death. Therefore, potential iatrogenic inhibition of K ir 2.1-mediated I K1 currents is a cardiosafety concern during new drug discovery and development. K ir 2.1 channels are part of the panel of cardiac ion channels currently considered for refined early compound risk assessment within the Comprehensive in vitro Proarrhythmia Assay initiative. In this study, we have validated a cell-based assay allowing functional quantification of K ir 2.1 inhibitors using whole-cell recordings of Chinese hamster ovary cells stably expressing human K ir 2.1 channels. We reproduced key electrophysiological and pharmacological features known for native I K1 , including current enhancement by external potassium and voltage- and concentration-dependent blockade by external barium. Furthermore, the K ir inhibitors ML133, PA-6, and chloroquine, as well as the multichannel inhibitors chloroethylclonidine, chlorpromazine, SKF-96365, and the class III antiarrhythmic agent terikalant demonstrated slowly developing inhibitory activity in the low micromolar range. The robustness of this assay authorizes medium throughput screening for cardiosafety purposes and could help to enrich the currently limited K ir 2.1 pharmacology.

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