Coupling Optical and Electrical Measurements in Artificial Membranes: Lateral Diffusion of Lipids and Channel Forming Peptides in Planar Bilayers

Planar lipid bilayers (PLB) were prepared by the Montal-Mueller technique in a FRAP system designed to simultaneously measure conductivity across, and lateral diffusion of, the bilayer. In the first stage of the project the FRAP system was used to characterise the lateral dynamics of bilayer lipids with regards to phospholipid composition (headgroup, chain unsaturation etc.), presence of cholesterol and the effect of divalent cations on negatively-charged bilayers. In the second stage of the project, lateral diffusion of two fluorescently-labelled voltage-dependent pore-forming peptides (alamethicin and S4s from Shaker K(+) channel) was determined at rest and in the conducting state. This study demonstrates the feasibility of such experiments with PLBs, amenable to physical constraints, and thus offers new opportunities for systematic studies of structure-function relationships in membrane-associating molecules

Raman spectroscopy of nerve fibers. A study of membrane lipids under steady state conditions

The molecular structures of different nerve fibers kept in good physiological conditions were studied by laser Raman spectroscopy. For myelinated nerves like the rat sciatic nerve, the Raman spectrum is dominated by bands due to the lipid component of the myelin sheath. The temperature dependence of these bands does not reveal any thermotropic phase transition between 0 and 40 degrees C. There is, however, with temperature, a linear increase in the intermolecular disorder that is accompanied by an increase in the number of gauche bonds of the phospholipid acyl chains. For unmyelinated nerves such as the lobster leg nerve, the C-H stretching region of the Raman spectrum is covered by bands arising from the protein component of the axoplasm. However, for the garfish olfactory nerve that has a high density of excitable membranes, phospholipid bands are observed and can be used as intrinsic structural probes of the excitable membranes. The relative intensity of these bands is also temperature dependent

Interplay between Ca2+ release and Ca2+i nflux underlies localized hyperpolarization-induced [Ca2+]i waves in prostatic cells

International audienceCalcium seems to be a major second messenger involved in the regulation of prostatic cell functions, but the mechanisms underlying its control are poorly understood. We investigated spatiotemporal aspects of Ca2+ signals in the LNCaP cell line, a model of androgen-dependent prostatic cells, by using non-invasive external electric field pulses that hyperpolarize the anode facing membrane and depolarize the membrane facing the cathode. Using high-speed fluo-3 confocal imaging, we found that an electric field pulse (10–15 V/cm, 1–5 mA, 5 ms) initiated rapidly, at the hyperpolarized end of the cell, a propagated [Ca2+]i wave which spread through the cell with a constant amplitude and an average velocity of about 20 μm/s. As evidenced by the total wave inhibition either by the block of Ca2+ entry or the depletion of Ca2+ stores by thapsigargin, a specific Ca2+ -ATPase inhibitor, the [Ca2+]i wave initiation may imply a localized Ca2+ influx linked to a focal auto-regenerative process of Ca2+ release. Using different external Ca2+ and Ca2+ entry blockers concentrations, Mn2+ quenching of fluo-3 and fura-2 fluorescence and inhibitors of InsP3 production, we found evidence that the [Ca2+]i wave progression required, in the presence of basal levels of InsP3, an interplay between Ca2+ release from InsP3-sensitive Ca2+ stores and Ca2+ influx through channels possibly activated by the [Ca2+]i rise