Real-Time Single-Cell Probing of the Action Mechanism of Amphotericin B Ion Channels---From Extracellular to Intracellular

The polyene antibiotic amphotericin B (AmB) has been considered the standard in antifungal therapy since its release in the 1950s. AmB's antifungal activity is most intimately related to its ability to form hydrophilic aqueous pores via binding to ergosterol in the fungal cell membrane. Pore formation not only promotes plasma membrane destabilization, but allows leakage of intracellular components such as potassium ions, responsible for cell lysis, ultimately resulting in rapid cell death. On the other hand, AmB has been reported to have a substantially higher affinity for ergosterol than cholesterol, namely, AmB interacts more rapidly with fungal cells than human cells. Recently, it has been recognized that the extract of Taiwanofungus camphorates possesses extensive biological activity, such as hepatoprotective, antihypertensive, anti-hyperlipidemic, immuno-modulatory, antioxidant, anti-inflammatory and anticancer activities. In our preliminary experiments, we found that treating cancer cells with the ethanolic extract of Taiwanofungus camphorates might increase the susceptibility of the plasma membrane of cancer cells to AmB. In addition, pretreatment with the ethanolic extract of Taiwanofungus camphorates followed by AmB dramatically inhibited cell proliferation in HT29 cells, which implies that the substitution of cholesterol, the dominant sterol in mammalian cell membranes, with ergosterol has occurred. This project will primarily focus on three specific aims, including Aim 1: Investigating the membrane fluidity in the absence and presence of ergosterol; Aim 2: Investigating the relationship between AmB ion channels and intracellular cytoskeleton dynamics, and Aim 3: Identifying and quantifying the relationship between AmB ion channels and MAPK signal transduction pathways. We anticipate the proposed bottom-up biomechanical approaches, together with the platform at single-cell level, could be applied to validate whether the AmB ion channels could replace damaged aquaporin (AQP) family of membrane water channels, where AQP water channels not only facilitate the diffusion of water but enhance cell migration and wound healing.