Vladislav S. Markin

An intimate collaboration between peroxisomes and lipid bodies

Although peroxisomes oxidize lipids, the metabolism of lipid bodies and peroxisomes is thought to be largely uncoupled from one another. In this study, using oleic acid–cultured Saccharomyces cerevisiae as a model system, we provide evidence that lipid bodies and peroxisomes have a close physiological relationship. Peroxisomes adhere stably to lipid bodies, and they can even extend processes into lipid body cores. Biochemical experiments and proteomic analysis of the purified lipid bodies suggest that these processes are limited to enzymes of fatty acid β oxidation. Peroxisomes that are unable to oxidize fatty acids promote novel structures within lipid bodies (“gnarls”), which may be organized arrays of accumulated free fatty acids. However, gnarls are suppressed, and fatty acids are not accumulated in the absence of peroxisomal membranes. Our results suggest that the extensive physical contact between peroxisomes and lipid bodies promotes the coupling of lipolysis within lipid bodies with peroxisomal fatty acid oxidation.

Membrane Fusion: Stalk Model Revisited

Membrane fusion is believed to proceed via intermediate structures called stalks. Mathematical analysis of the stalk provided the elastic energy involved in this structure and predicted the possible evolution of the overall process, but the energies predicted by the original model were suspiciously high. This was due to an erroneous assumption, i.e., that the stalk has a figure of revolution of a circular arc. Here we abandon this assumption and calculate the correct shape of the stalk. We find that it can be made completely stress free and, hence, its energy, instead of being positive and high can become negative, thus facilitating the fusion process. Based on our new calculations, the energies of hemifusion, of complete fusion, and of the pore in a bilayer were analyzed. Implications for membrane fusion and lipid phase transitions are discussed.

Biophysics and Structure of the Patch and the Gigaseal

Interpreting channel behavior in patches requires an understanding of patch structure and dynamics, especially in studies of mechanosensitive channels. High resolution optical studies show that patch formation occurs via blebbing that disrupts normal membrane structure and redistributes in situ components including ion channels. There is a 1-2 microm region of the seal below the patch where proteins are excluded and this may consist of extracted lipids that form the gigaseal. Patch domes often have complex geometries with inhomogeneous stresses due to the membrane-glass adhesion energy (E(a)), cytoskeletal forces, and possible lipid subdomains. The resting tension in the patch dome ranges from 1-4 mN/m, a significant fraction of the lytic tension of a bilayer ( approximately 10 mN/m). Thus, all patch experiments are conducted under substantial, and uneven, resting tension that may alter the kinetics of many channels. E(a) seems dominated by van der Waals attraction overlaid with a normally repulsive Coulombic force. High ionic strength pipette saline increased E(a) and, surprisingly, increased cytoskeletal rigidity in cell-attached patches. Low pH pipette saline also increased E(a) and reduced the seal selectivity for cations, presumably by neutralizing the membrane surface charge. The seal is a negatively charged, cation selective, space with a resistance of approximately 7 gigohm/microm in 100 mM KCl, and the high resistivity of the space may result from the presence of high viscosity glycoproteins. Patches creep up the pipette over time with voltage independent and voltage dependent components. Voltage-independent creep is expected from the capillary attraction of E(a) and the flow of fresh lipids from the cell. Voltage-dependent creep seems to arise from electroosmosis in the seal. Neutralization of negative charges on the seal membrane with low pH decreased the creep rate and reversed the direction of creep at positive pipette potentials.

Energetics of peptide (pHLIP) binding to and folding across a lipid bilayer membrane

The pH low-insertion peptide (pHLIP) serves as a model system for peptide insertion and folding across a lipid bilayer. It has three general states: (I) soluble in water or (II) bound to the surface of a lipid bilayer as an unstructured monomer, and (III) inserted across the bilayer as a monomeric α-helix. We used fluorescence spectroscopy and isothermal titration calorimetry to study the interactions of pHLIP with a palmitoyloleoylphosphatidylcholine (POPC) lipid bilayer and to calculate the transition energies between states. We found that the Gibbs free energy of binding to a POPC surface at low pHLIP concentration (state I–state II transition) at 37°C is approximately −7 kcal/mol near neutral pH and that the free energy of insertion and folding across a lipid bilayer at low pH (state II–state III transition) is nearly −2 kcal/mol. We discuss a number of related thermodynamic parameters from our measurements. Besides its fundamental interest as a model system for the study of membrane protein folding, pHLIP has utility as an agent to target diseased tissues and translocate molecules through the membrane into the cytoplasm of cells in environments with elevated levels of extracellular acidity, as in cancer and inflammation. The results give the amount of energy that might be used to move cargo molecules across a membrane.

Mimosa pudica: Electrical and mechanical stimulation of plant movements

Thigmonastic movements in the sensitive plant Mimosa pudica L., associated with fast responses to environmental stimuli, appear to be regulated through electrical and chemical signal transductions. The thigmonastic responses of M. pudica can be considered in three stages: stimulus perception, electrical signal transmission and induction of mechanical, hydrodynamical and biochemical responses. We investigated the mechanical movements of the pinnae and petioles in M. pudica induced by the electrical stimulation of a pulvinus, petiole, secondary pulvinus or pinna by a low electrical voltage and charge. The threshold value was 1.3-1.5 V of applied voltage and 2 to 10 microC of charge for the closing of the pinnules. Both voltage and electrical charge are responsible for the electro-stimulated closing of a leaf. The mechanism behind closing the leaf in M. pudica is discussed. The hydroelastic curvature mechanism closely describes the kinetics of M. pudica leaf movements.

Lateral organization of membranes and cell shapes

The relations among membrane structure, mechanical properties, and cell shape have been investigated. The fluid mosaic membrane models used contains several components that move freely in the membrane plane. These components interact with each other and determine properties of the membrane such as curvature and elasticity. A free energy equation is postulated for such a multicomponent membrane and the condition of free energy minimum is used to obtain differential equations relating the distribution of membrane components and the local membrane curvature. The force that moves membrane components along the membrane in a variable curvature field is calculated. A change in the intramembrane interactions can bring about phase separation or particle clustering. This, in turn, may strongly affect the local curvature. The numerical solution of the set of equations for the two dimensional case allows determination of the cell shape and the component distribution along the membrane. The model has been applied to describe certain erythrocytes shape transformations.

Gating Pore Currents in DIIS4 Mutations of NaV1.4 Associated with Periodic Paralysis : Saturation of Ion Flux and Implications for Disease Pathogenesis

S4 voltage–sensor mutations in CaV1.1 and NaV1.4 channels cause the human muscle disorder hypokalemic periodic paralysis (HypoPP). The mechanism whereby these mutations predispose affected sarcolemma to attacks of sustained depolarization and loss of excitability is poorly understood. Recently, three HypoPP mutations in the domain II S4 segment of NaV1.4 were shown to create accessory ionic permeation pathways, presumably extending through the aqueous gating pore in which the S4 segment resides. However, there are several disparities between reported gating pore currents from different investigators, including differences in ionic selectivity and estimates of current amplitude, which in turn have important implications for the pathological relevance of these aberrant currents. To clarify the features of gating pore currents arising from different DIIS4 mutants, we recorded gating pore currents created by HypoPP missense mutations at position R666 in the rat isoform of Nav1.4 (the second arginine from the outside, at R672 in human NaV1.4). Extensive measurements were made for the index mutation, R666G, which created a gating pore that was permeable to K+ and Na+. This current had a markedly shallow slope conductance at hyperpolarized voltages and robust inward rectification, even when the ionic gradient strongly favored outward ionic flow. These characteristics were accounted for by a barrier model incorporating a voltage-gated permeation pathway with a single cation binding site oriented near the external surface of the electrical field. The amplitude of the R666G gating pore current was similar to the amplitude of a previously described proton-selective current flowing through the gating pore in rNaV1.4-R663H mutant channels. Currents with similar amplitude and cation selectivity were also observed in R666S and R666C mutant channels, while a proton-selective current was observed in R666H mutant channels. These results add support to the notion that HypoPP mutations share a common biophysical profile comprised of a low-amplitude inward current at the resting potential that may contribute to the pathological depolarization during attacks of weakness.

Electrical memory in Venus flytrap.

Electrical signaling, memory and rapid closure of the carnivorous plant Dionaea muscipula Ellis (Venus flytrap) have been attracting the attention of researchers since the XIX century. The electrical stimulus between a midrib and a lobe closes the Venus flytrap upper leaf in 0.3 s without mechanical stimulation of trigger hairs. Here we developed a new method for direct measurements of the exact electrical charge utilized by the D. muscipula Ellis to facilitate the trap closing and investigated electrical short memory in the Venus flytrap. As soon as the 8 microC charge for a small trap or a 9 microC charge for a large trap is transmitted between a lobe and midrib from the external capacitor, the trap starts to close at room temperature. At temperatures 28-36 degrees C a smaller electrical charge of 4.1 microC is required to close the trap of the D. muscipula. The cumulative character of electrical stimuli points to the existence of short-term electrical memory in the Venus flytrap. We also found sensory memory in the Venus flytrap. When one sustained mechanical stimulus was applied to only one trigger hair, the trap closed in a few seconds.

The gibbs free energy of ion transfer between two immiscible liquids

Abstract The modern theories of ion solvation have been considered. A table is presented which lists experimental data on the standard Gibbs free energy of ion resolvation and on the standard potential of ion partitition between water and organic solvents. The contribution of dielectric saturation to the electrostatic part of the Gibbs free solvation energy has been analysed. Calculation results are compared with experimental dependences of the standard Gibbs free solvation energy on the static and optical solvent permittivities, and also on the ion size. An estimate is presented for the solvophobic contribution to the resolvation energy.

Electrical double layers at the oil/water interface

This review presents the historical development and current status of the theory of the electrical double layer at a liquid/liquid interface. It gives rigorous thermodynamic definitions of all basic concepts related to liquid interfaces and to the electrical double layer. The difference between the surface of a solid electrode and the interface of two immiscible electrolyte solutions (ITIES) is analyzed in connection to their electrical properties. The most important classical relationships for the electrical double layer are presented and critically discussed. The generalized adsorption isotherm is derived. After a short review of the classical Gouy-Chapman and Verwey-Niessen models, more recent developments of the double layer theory are presented. These include effects of variable dielectric permittivity, nonlocal electrostatics, hydration forces, the modified Poisson-Boltzmann equation and the ion-dipole plasma. The relative merits of different theories are estimated by comparing them with computer simulation of the ITIES and electrical double layer. Special attention is given to the structure of ITIES and its variation due to adsorption of ions and amphiphilic molecules.