Journal of Membrane Biology最新文献

Exosomes are special extracellular vesicles secreted by cells, which are of great significance in the basic research of life science and clinical application and has become a hot research field with rapid development in recent 10 years. Therefore, the isolation and separation of exosomes is particularly important for the research and application of exosomes. This paper aims to review the research progress of exosome isolation and separation methods in recent years, including ultracentrifugation, ultrafiltration, size‑exclusion chromatography, precipitation, immunomagnetic bead capture method, aptamer-based isolation, and isolation methods based on microfluidic technology. It is generally accepted that most of the existing methods have limitations, for example, ultracentrifugation is time-consuming and laborious, and immunomagnetic bead capture method and aptamer-based separation method have small sample processing capacity and high cost. As a result, we also introduce some common situations in which two or more methods are combined for use. Finally, the separation and isolation methods including all those presented in this review were compared and summarized.

Most blockers of both hERG (human ether-à-go-go-related gene) channels and pancreatic ß-cell ATP-sensitive K⁺ (K_ATP) channels access their binding sites from the cytoplasmic side of the plasma membrane. It is unknown whether binding to intracellular components competes with binding of these substances to K⁺ channels. The whole-cell configuration of the patch-clamp technique, a laser-scanning confocal microscope, and fluorescence correlation spectroscopy (FCS) were used to study hERG channels expressed in HEK (human embryonic kidney) 293 cells and K_ATP channels from the clonal insulinoma cell line RINm5F. When applied via the pipette solution in the whole-cell configuration, terfenadine blocked both hERG and K_ATP currents with much lower potency than after application via the bath solution, which was not due to P-glycoprotein-mediated efflux of terfenadine. Such a difference was not observed with dofetilide and tolbutamide. 37-68% of hERG/EGFP (enhanced green-fluorescent protein) fusion proteins expressed in HEK 293 cells were slowly diffusible as determined by laser-scanning microscopy in the whole-cell configuration and by FCS in intact cells. Bath application of a green-fluorescent sulphonylurea derivative (Bodipy-glibenclamide) induced a diffuse fluorescence in the cytosol of RINm5F cells under whole-cell patch-clamp conditions. These observations demonstrate the presence of intracellular binding sites for hERG and K_ATP channel blockers not dialyzable by the patch-pipette solution. Intracellular binding of terfenadine was not influenced by a mutated hERG (Y652A) channel. In conclusion, substances with high lipophilicity are not freely diffusible inside the cell but steep concentration gradients might exist within the cell and in the sub-membrane space.

Pore-forming proteins (PFPs) are produced by various organisms, including pathogenic bacteria, and form pores within the target cell membrane. Streptolysin O (SLO) is a PFP produced by Streptococcus pyogenes and forms high-order oligomers on the membrane surface. In this prepore state, multiple α-helices in domain 3 of each subunit exist as unfolded structures and transiently interact with each other. They subsequently transition into transmembrane β-hairpins (TMHs) and form pores with diameters of 20-30 nm. However, in this pore formation process, the trigger of the transition in a subunit and collaboration between subunits remains elusive. Here, I observed the dynamic pore formation process using high-speed atomic force microscopy. During the oligomer transition process, each subunit was sequentially inserted into the membrane, propagating along the oligomer in a domino-like fashion (chain reaction). This process also occurred on hybrid oligomers containing wildtype and mutant subunits, which cannot insert into the membrane because of an introduced disulfide bond. Furthermore, propagation still occurred when an excessive force was added to hybrid oligomers in the prepore state. Based on the observed chain reactions, I estimate the free energies and forces that trigger the transition in a subunit. Furthermore, I hypothesize that the collaboration between subunits is related to the structure of their TMH regions and interactions between TMH-TMH and TMH-lipid molecules.