Electrochemical studies of the mitochondrial ROMK2 potassium channel activity reconstituted into the free-standing and tethered bilayer lipid membranes.

The renal-outer-medullary­potassium (ROMK2) channel modulates potassium transport in the kidney. It has been postulated that the ROMK2 is the pore-forming subunit of the mitochondrial ATP-sensitive potassium channel as a mediator of cardioprotection. In this study, cell-free synthesis of the ROMK2 was performed in presence of membrane scaffold protein (MSP1D1) nanodiscs. Activity measurements were achieved after channel reconstitution into the planar lipid bilayer and tethered bilayer lipid membranes. Both methods allowed for monitoring of channel function, verified with channel blocking and activation/re-activation experiments. The primary function of the mitochondrial potassium channels is to regulate the potential of the mitochondrial membrane, which allows them to play an important role in cytoprotection. This work focuses on obtaining the ROMK2 using a cell-free expression system, followed by the incorporation of the channel protein into the lipid bilayer and studying the influence of voltage changes and molecular modulators on channel activity. Channel activity was measured after its reconstitution into two models of lipid bilayers - BLM (Bilayer Lipid Membrane) and tBLM (Tethered Bilayer Lipid Membrane) deposited on a solid gold electrode. These two model membranes and electrochemical measurements made it possible to measure the flux of K+ ions in the presence of channel modulators.

Methods of Measuring Mitochondrial Potassium Channels: A Critical Assessment.

In this paper, the techniques used to study the function of mitochondrial potassium channels are critically reviewed. The majority of these techniques have been known for many years as a result of research on plasma membrane ion channels. Hence, in this review, we focus on the critical evaluation of techniques used in the studies of mitochondrial potassium channels, describing their advantages and limitations. Functional analysis of mitochondrial potassium channels in comparison to that of plasmalemmal channels presents additional experimental challenges. The reliability of functional studies of mitochondrial potassium channels is often affected by the need to isolate mitochondria and by functional properties of mitochondria such as respiration, metabolic activity, swelling capacity, or high electrical potential. Three types of techniques are critically evaluated: electrophysiological techniques, potassium flux measurements, and biochemical techniques related to potassium flux measurements. Finally, new possible approaches to the study of the function of mitochondrial potassium channels are presented. We hope that this review will assist researchers in selecting reliable methods for studying, e.g., the effects of drugs on mitochondrial potassium channel function. Additionally, this review should aid in the critical evaluation of the results reported in various articles on mitochondrial potassium channels.

Polyvinylpyrrolidone (PVP) hydrogel coating for cylindrical polyurethane scaffolds.

The presented study describes a method for the preparation and modification of cylindrical polyurethane structures with polyvinylpyrrolidone (PVP) hydrogel coating. The modified polyurethane scaffolds were fabricated using the phase-inversion technique and intended to be used as a vascular prosthesis. The proposed modification method involves a two-step Fenton-type reaction. Physicochemical analysis (Fourier transform infrared spectroscopy, scanning electron microscopy) confirmed the presence of the hydrogel coating. The influence of PVP and polymerization initiator (cumene hydroperoxide) concentrations on hydrogel's properties were examined. The higher concentrations of reagent were used, the thicker coating was obtained. After modification, the material's surface becomes more hydrophilic in comparison to pristine polyurethane. Cytotoxicity assay (MTT test) confirmed that PVP-coating is not toxic. The introduction of hydrogel coating resulted in a significant decrease in the fibrinogen adsorbed to the material's surface as compared to a non-modified polymer. Platelet adhesion assay demonstrated almost no platelet adhesion to the modified surfaces.