Flow cytometry method to quantify the formation of beta-amyloid membrane ion channels.

Abstract

It is accepted that the cytotoxicity of beta-amyloid is mediated by its oligomers. Amyloid peptides can form ion channels in cell membranes and allow calcium and other ions to enter cells. In this project, we developed a technique to quantify the appearance of calcium in liposomes and applied this technique to study the effect of amyloid peptides on the permeability of membranes.Calcium influx was monitored in liposomes made of phosphatidylcholine (PC) or phosphatidylserine (PS) with an addition of a lipid-soluble dye DiD and containing fluorescent calcium-sensitive probe Fluo-3. The intensity of fluorescence of individual liposomes was measured using a flow cytometer. Calcium ionophore ionomycin served as a positive control. The addition of micromolar concentrations of short fragments of amyloid-beta (Aβ25–35) permeabilized a significant number of PS liposomes. This effect was not observed in PC liposomes. Our data supports the hypothesis that the ion channel formation by amyloid peptide is dependent on electrostatic interactions.High concentrations of Aβ25–35 (above 20 μM) increased signal intensity in a recording channel corresponding to the calcium-sensing probe. However, this phenomenon was also observed in Ca2+-free conditions and even in liposomes without Fluo-3, so we interpreted it as an artifact.Using the described technique, we were not able to detect the formation of calcium channels by several other amyloid peptides. Considering that liposomes appeared resistant to reasonable concentrations of solvents, we expect that described flowmetric technique can be used in high-throughput screening applications.