Fluorescent Membrane Probes Research Articles

Lipid bilayer condition abnormalities following Macrovipera lebetina obtusa snake envenomation

Viper bites is an endemic public health problem in Armenia, even in the cities. Human envenomation is often characterized by clotting disorders, hypofibrinogenemia, and local tissue necrosis. In this original study, we assess some changes of cell membranes plastic properties (namely, its microviscosity, thickness, permeability) in a rat envenomation model using the biophysical approaches. We describe the interaction of Macrovipera lebetina obtusa (MLO) venom with giant unilamellar vesicles (GUVs) composed of the native phospholipid mixtures visualized through fluorescent microscopy. GUVs with a mean diameter of 30 μm have a minimum curvature and mimic cell membranes in this respect.The membrane fluorescence probe, ANS and pyrene, were used to assess the state of membrane and specifically mark the phospholipid domains. Independent of their lipid composition, GUVs were enlarged in size as venom-dependent lipid hydrolysis proceeded. Except of the visible morphological changes, ANS and pyrene also allows us to quantify the fluidity changes in the membrane by measuring of the fluorescence intensity. The presence of viper venom in GUVs media reveals a noticeable decreasing of membrane fluidity compare the control, while the binding of fluorophores with GUVs modified by venom lead to appearance of channel activity. These studies also emphasize the importance of a membrane surface curvature for its interaction with enzymatic components of venom.

Insight into NSAID-induced membrane alterations, pathogenesis and therapeutics: Characterization of interaction of NSAIDs with phosphatidylcholine

Nonsteroidal anti-inflammatory drugs (NSAIDs) are one of the most widely consumed pharmaceuticals, yet both the mechanisms involved in their therapeutic actions and side-effects, notably gastrointestinal (GI) ulceration/bleeding, have not been clearly defined. In this study, we have used a number of biochemical, structural, computational and biological systems including; Fourier Transform InfraRed (FTIR). Nuclear Magnetic Resonance (NMR) and Surface Plasmon Resonance (SPR) spectroscopy, and cell culture using a specific fluorescent membrane probe, to demonstrate that NSAIDs have a strong affinity to form ionic and hydrophobic associations with zwitterionic phospholipids, and specifically phosphatidylcholine (PC), that are reversible and non-covalent in nature. We propose that the pH-dependent partition of these potent anti-inflammatory drugs into the phospholipid bilayer, and possibly extracellular mono/multilayers present on the luminal interface of the mucus gel layer, may result in profound changes in the hydrophobicity, fluidity, permeability, biomechanical properties and stability of these membranes and barriers. These changes may not only provide an explanation of how NSAIDs induce surface injury to the GI mucosa as a component in the pathogenic mechanism leading to peptic ulceration and bleeding, but potentially an explanation for a number of (COX-independent) biological actions of this family of pharmaceuticals. This insight also has proven useful in the design and development of a novel class of PC-associated NSAIDs that have reduced GI toxicity while maintaining their essential therapeutic efficacy to inhibit pain and inflammation.

Molecular events associated with Macrovipera lebetina obtusa and Montivipera raddei venom intoxication and condition of biomembranes

Studies on the interaction of snake venom and organized lipid interfaces have been conducted using a variety of systems, including BLMs, SUVs and GUVs. The present study was undertaken to elucidate how the plastic properties (namely, its microviscosity, thickness, permeability) of model membranes from native lipids of different tissues of rats change in the course of Macrovipera lebetina obtusa (MLO), Montivipera raddei (MR) and Naja kaouthia (NK) venoms processing. The presence of viper venom in organism leads to increasing of the electrical resistance of BLMs from liver and muscle lipids approximately on a sequence, while the BLMs from brain lipids have not shown noticeable differences of plastic properties compared to the control. Giant unilamellar vesicles (GUVs) with a mean diameter of 30μm have a minimum curvature and mimic cell membranes in this respect. Snake venom was added to the sample chamber before the vesicles were formed. The membrane fluorescence probes, ANS and pyrene, were used to assess the state of the membrane and specifically mark the phospholipid domains. Fluorescent spectra were acquired on a Varian fluorometer instrument. ANS and pyrene allow us to quantify the fluidity changes in the membrane by measuring of the fluorescence intensity. The presence of viper venom in GUVs media reveals a noticeable decreasing of membrane fluidity compared to the control, while the binding of fluorophores with GUVs modified by venom leads to the appearance of channel activity. These studies also emphasize the importance of a membrane surface curvature for its interaction with enzymatic components of venom.

Voltage-Clamped Supported Bilayer System to Record Ion Channel Activity

Structure-function studies of voltage-dependent ion channels require controlled electrical stimuli to drive channel activity. Under voltage control, site-directed fluorescence spectroscopy is often used to probe structural rearrangements that underlie channel function. Many voltage-dependent ion channels, such as the KvAP K+ channel, must be purified from bacteria and reconstituted in a lipid membrane. The ensuing (conventional) experiment in a black lipid membrane has several problems: 1) too few channels can be measured to observe gating currents; 2) the bilayer stability is generally poor and inversely related to protein density; 3) fluorescence analysis is difficult due to channel diffusion. To circumvent these problems, we previously developed a system to voltage clamp a supported bilayer with simultaneous fluorescence imaging (Hyde et al, 2010 BPS Meeting). This system employed a 5 mm diameter supported bilayer grown atop a transparent electrode-coated coverslip. We could measure gating currents of small charged molecules and voltage-dependent responses from fluorescent membrane probes; however, we have since found that direct contact with the supporting electrode significantly hinders recordings of functional channels. We have thus introduced a self-assembled monolayer cushion grown atop a gold-coated coverslip as the support electrode. Importantly, the gold-coated coverslip is also designed to enable simultaneous fluorescence imaging of ion channels in the supported bilayer using surface plasmon-assisted microscopy. Preliminary results indicate that in response to an applied transmembrane potential, we can observe voltage-dependent fluorescence changes from S4-labeled KvAP channels consistent with presumptive gating behavior. Support: NIH GM030376; Medical Scientist National Research Service Award 5 T32 GM007281.

Recent developments in molecular dynamics simulations of fluorescent membrane probes.

Due to their sensitivity and versatility, the use of fluorescence techniques in membrane biophysics is widespread. Because membrane lipids are non-fluorescent, extrinsic membrane probes are widely used. However, the behaviour of these probes when inserted in the bilayer is often poorly understood, and it can be hard to distinguish between legitimate membrane properties and perturbation resulting from probe incorporation. Atomistic molecular dynamics simulations present a convenient way to address these issues and have been increasingly used in recent years in this context. This article reviews the application of molecular dynamics to the study of fluorescent membrane probes, focusing on recent work with complex design fluorophores and ordered bilayer systems.

MAL Protein Controls Protein Sorting at the Supramolecular Activation Cluster of Human T Lymphocytes

T cell membrane receptors and signaling molecules assemble at the immunological synapse (IS) in a supramolecular activation cluster (SMAC), organized into two differentiated subdomains: the central SMAC (cSMAC), with the TCR, Lck, and linker for activation of T cells (LAT), and the peripheral SMAC (pSMAC), with adhesion molecules. The mechanism of protein sorting to the SMAC subdomains is still unknown. MAL forms part of the machinery for protein targeting to the plasma membrane by specialized mechanisms involving condensed membranes or rafts. In this article, we report our investigation of the dynamics of MAL during the formation of the IS and its role in SMAC assembly in the Jurkat T cell line and human primary T cells. We observed that under normal conditions, a pool of MAL rapidly accumulates at the cSMAC, where it colocalized with condensed membranes, as visualized with the membrane fluorescent probe Laurdan. Mislocalization of MAL to the pSMAC greatly reduced membrane condensation at the cSMAC and redistributed machinery involved in docking microtubules or transport vesicles from the cSMAC to the pSMAC. As a consequence of these alterations, the raft-associated molecules Lck and LAT, but not the TCR, were missorted to the pSMAC. MAL, therefore, regulates membrane order and the distribution of microtubule and transport vesicle docking machinery at the IS and, by doing so, ensures correct protein sorting of Lck and LAT to the cSMAC.

Labeling the Plasma Membrane with TMA-DPH

INTRODUCTION TMA-DPH (trimethylamine-diphenylhexatriene) is a fluorescent membrane probe that has classically been used to label the outer leaflet of a membrane bilayer, to label the outer leaflet of the plasma membrane in cells, and to report on membrane dynamics using the techniques of fluorescence polarization and/or fluorescence lifetime. This probe has also been used to follow exocytosis and endocytosis of labeled plasma membranes. The interaction of the aqueous environment with mitochondrial inner membrane dynamics has also been studied following the fluorescence polarization and the lifetime of TMA-DPH. This protocol describes the use of TMA-DPH to label the plasma membrane.

Abstract 690: Selection of resistance by anticancer drugs is not responsible for alterations in membrane micro-fluidity: A study by Fluorescence Correlation Micro-Spectroscopy

Abstract Expression of P-glycoprotein (P-gp), the multidrug resistance (MDR) 1 gene product, can lead to MDR in tumors. Previous studies on multidrug-resistant cells have suggested changes in membrane fluidity associated with P-gp expression in drug-selected resistant cells. However, Aleman and co-workers have shown that, in MDR1 transfected cells, expression of P-gp has little effect on membrane fluidity and the changes in this parameter observed in drug-selected cells must reflect other host adaptations to drug selection. In this study and the other reported on drug-selected resistant cells, membrane fluidity has been measured using fluorescent membrane probes and fluorescence anisotropy or electron spin resonance on cell population samples (Aleman et al. Cancer Res. 2003 63:3084-91). Here, we propose to explore the plasma membrane microfluidity by using the fluorescent membrane probe DiA and fluorescence correlation micro-spectroscopy (FCM-S) on a single living cell and nanometer scale. By using the fluorescent membrane probe DiA and FCM-S to explore the plasma membrane micro-fluidity, we have previously shown a decrease in plasma membrane micro-fluidity in LR73R cells (the Chinese hamster ovarian LR73 cells transfected with MDR1 cDNA; Boutin et al., J Biomed Opt. 2009 14: 034030). Moreover, a strategy using flow cytometry and calcein-AM functional fluorescence-activated cell sorting permitted us to select two cell populations from LR73R cells expressing distinct levels of P-gp function. In this case, we were able to observe distinct level of membrane micro-fluidity in the two populations. Cells showing high level of calcein-AM uptake presented a higher membrane micro-fluidity. In contrast, the FCM-S yielded opposite data when we compared the membrane micro-fluidity of several parental cell lines (MCF7, KB3.1, LR73, ME-SSA) and their resistant counterparts which have been selected in the presence of anticancer drugs. Our data are not in agreement with previous published data and demonstrates clearly that alterations that can affect drug-selected cells do not induce any changes in membrane fluidity. Moreover, micro-analysis of cell membrane could allow to more precise measurements of membrane fluidity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 690. doi:10.1158/1538-7445.AM2011-690

Relationships Between Bilayer Phase and Equilibration Rates of Patman and Laurdan

Patman is a fluorescent membrane probe related to laurdan. The structural distinctions between the two probes are the lengths of the aliphatic tails (eleven carbons in laurdan and fifteen in patman) and the presence of a trimethylammonium forming a positively-charged head on patman. Studies exploring patman as a probe to detect membrane properties during apoptosis revealed that the two edges of the emission spectrum (435 and 500 nm) stabilize at different rates as the probe binds to the cell membrane. To test whether these differences represent dissimilarities in probe binding to ordered and disordered domains, experiments were conducted to monitor patman equilibration with bilayers composed of various mixtures of saturated and unsaturated phosphatidylcholines at temperatures above, at, and below the main thermotropic phase transition. In general, patman equilibrated more rapidly with bilayers in the liquid disordered phase than in the solid ordered phase. With solid phase membranes, the fluorescence stabilized faster at 435 nm than at 500 nm. Similar yet more subtle results occurred in the lipid disordered phase. In contrast, the situation was reversed at the phase transition temperature; equilibration was faster at 500 nm than at 435 nm. To determine whether these results reflected specific properties of patman, the experiments were repeated with laurdan, and several distinctions were observed. First, equilibration with solid phase lipids was faster than for patman and not different from equilibration with the fluid phase. Second, differences in rates between the two wavelengths were less than with patman for solid phase membranes but greater than with patman for melted bilayers. Third, at the phase transition temperature, equilibration rates favored 435 nm over 500 nm, the opposite of the result obtained with patman. Computer simulations were used to assist with interpretation of these results.

Breast cancer cells and fibroblasts in co-culture: reciprocal influences on cell adhesion, membrane fluidity and migration

The growing role of the reciprocal interaction between epithelial and stromal cells in the development and progression of breast cancer has been recognized. In particular, the migratory/invasive behaviour of tumor cells seems to be strongly influenced by their dialogue with neighbouring stromal cells. To verify if this cross-talk may affect some molecular and functional aspects of the cell biology correlated with the metastasizing vocation of the tumor cells (i.e. adhesion molecule expression, membrane fluidity, migration), we co-cultured estrogen receptor (ER)-positive, poorly invasive and low metastasizing (MCF-7) or ER-negative, highly invasive and metastatic (MDA-MB-231) breast cancer cells with fibroblasts isolated from breast healthy skin (normal fibroblasts, NFs) or from breast tumor stroma (cancer associated fibroblasts, CAFs) in monolayer or in a three-dimensional system (nodules). We previously reported the ability of NFs and CAFs to respectively induce or inhibit the epithelial adhesion molecule, E-cadherin, expression in MCF-7 cells. In the present study, the expression of the mesenchymal adhesion protein N-cadherin (N-cad) was investigated by confocal immunofluorescence microscopy on frozen nodule sections. An increase in N-cad levels was observed in CAFs, but not in NFs, as a result of the interaction with both kinds of epithelial cancer cells. CAFs, in turn, promoted an increase in N-cad level of MDA-MB-231 cells and induced its expression in MCF-7 cells, originally negative for N-cad. Two-photon microscopy imaging of cells labeled with Laurdan, a membrane fluorescent probe, was used to investigate fluidity changes in plasma membranes of all the cell types in monolayer cultures. Tumor cell/fibroblast interaction enhanced fluidity of cancer cell membrane while tumor cells generally promoted an increase in fibroblast membrane packing density. Cell tracking by confocal microscopy demonstrated that the interaction of mammary cancer cells with NFs or CAFs determined a definite increment in tumor cell migration velocity, even with a marked enhancement of the migration directionality induced by CAFs. Our results demonstrate a reciprocal influence of mammary cancer and stromal cells on various adhesiveness/invasiveness features. In particular, an overall pro-tumoral/-invasive effect of CAFs on both well- and poorly differentiated mammary cancer cells was exteriorized by reduction of cell adhesion, induction of membrane fluidity, and migration velocity and directionality, along with a promotion of epithelial-mesenchymal transition.

Electronically excited states of membrane fluorescent probe 4-dimethylaminochalcone. Results of quantum chemical calculations

Quantum-chemical calculations of ground and excited states for membrane fluorescent probe 4-dimethylaminochalcone (DMAC) in vacuum were performed. Optimized geometries and dipole moments for lowest-lying singlet and triplet states were obtained. The nature of these electronic transitions and the relaxation path in the excited states were determined; changes in geometry and charge distribution were assessed. It was shown that in vacuum the lowest existed level is of (n, π*) nature, and the closest to it is the level of (π, π*) nature; the energy gap between them is narrow. This led to an effective (1)(π, π*) →(1)(n, π*) relaxation. After photoexcitation the molecule undergoes significant transformations, including changes in bond orders, pyramidalization angle of the dimethylamino group, and planarity of the molecule. Its dipole moment rises from 5.5 Debye in the ground state to 17.1 Debye in the (1)(π, π*) state, and then falls to 2 Debye in the (1)(n, π*) state. The excited (1)(n, π*) state is a short living state; it has a high probability of intersystem crossing into the (3)(π, π*) triplet state. This relaxation path explains the low quantum yield of DMAC fluorescence in non-polar media. It is possible that (3)(π, π*) is responsible for observed DMAC phosphorescence.

Interactions of egg yolk lipoprotein fraction with boar spermatozoa assessed with a fluorescent membrane probe.

The interactions of a fluorescent membrane probe, 1-anilinonaphthalene-8-sulfonic acid (1,8-ANS), with boar spermatozoa were followed through the use of lipoprotein fraction of ostrich egg yolk (LPFo). Semen samples, extended in Kortowo 3 (K3) extender, were supplemented with 2% or 5% LPFo and stored for 3h at 16 degrees C. Additionally, cold shock-treated spermatozoa (1h at 4 degrees C) were stored in K3 extender supplemented with LPFo for 3h at 16 degrees C. In each boar, the fluorescent enhancement of ANS was observed in K3-extended semen supplemented with LPFo, prior to storage. Following storage, there was a significant increase in LPFo-ANS fluorescence, particularly in the sperm membrane overlying the head and midpiece regions. There were significant differences among the boars with respect to the sperm populations defined by the LPFo-ANS fluorescence. Sperm viability was not significantly affected during the storage period. Furthermore, the proportions of spermatozoa defined by the different patterns of LPFo-ANS fluorescence were low and remained unchanged after storage of cold shock-treated spermatozoa with 2% or 5% LPFo, suggesting irreversible damage to the sperm membrane architecture. These findings indicate that the ANS fluorescent probe could be used to shed more light on the nature of the interactions between LPFo and sperm membrane following semen preservation. Such valuable information could contribute to the development of an optimal protocol for cryopreservation of boar semen.

Interactions of egg yolk lipoprotein fraction with boar spermatozoa assessed with a fluorescent membrane probe.

Interactions of egg yolk lipoprotein fraction with boar spermatozoa assessed with a fluorescent membrane probe.

Experimental study on allografts of amniotic epithelial cells in calcaneal tendon lesions of sheep

An experimental protocol was designed to study the survival and behaviour of an allograft of amniotic epithelial cells (AECs) in an ovine model. The study was conducted on three healthy adult sheep. A core lesion was created in both calcaneal tendons under ultrasound (US) guidance by injecting 400 UI of Type 1A collagenase diluted in 0.6 ml saline. The AECs were obtained from a 60-80-day-old fetus and cultured under standard conditions. After 15 days of collagenase treatment, 2 x 10(6) AECs stained with a vital membrane fluorescent probe (PHK26) were injected under US guidance in 500 microl saline solution into the lesion of one limb. The contralateral untreated limb was used as a control. Animals were euthanatized 7 (1) and 30 (2) days later. Histological analyses performed on explanted tendons clearly demonstrate that AECs survived for at least 1 month inside the lesion without any adverse reactions. The damaged tissue of the treated tendons showed a high number of reparative cells in active proliferation that were accumulating collagen within the extracellular matrix. In addition, after 1 month, the neo-collagen began to be organized into parallel arrays of fibers oriented along the longitudinal axis of the tendon.

Abstract 3537: P-glycoprotein expression level is responsible for alterations in plasma membrane microfluidity as probed by fluorescence correlation spectroscopy

Abstract Expression of P-glycoprotein (P-gp), the multidrug resistance (MDR) 1 gene product, leads to MDR in tumors. By using the fluorescent membrane probe DiA and Fluoresence Correlation Spectroscopy (FCS) to explore the plasma membrane microfluidity, we have previousely shown a showed a decrease in plasma membrane microfluidity in LR73R cells (the Chinese hamster ovarian LR73R cells transfected with MDR1 cDNA) (1). In the present study, and in order to study the relationship between membrane microfluidity and the level of P-gp expression, a strategy using flow cytometry and calcein-AM functional fluorescence-activated cell sorting permitted us to select two cell populations from LR73R cells expressing distinct levels of P-gp (P4 “lower calcein-AM uptake” and P5 “higher calcein-AM uptake”). Data obtained on the cytotoxic effects of doxorubicin (DOX) showed clearly that P4 cells were 10-fold resistant to DOX than P5 cells. A direct correlation between the sensitivity of the cells to DOX and the calcein-AM uptake was observed. In addition, intranuclear DOX measurements using microspectrofluorometry showed a decrease in nuclear accumulation of DOX in P4 cells when compared to P5 cells. This was in agreement with the functional calcein-AM uptake assay. By using the fluorescent membrane probe DiA FCS analysis, we explored the membrane microfluidity in P4 and P5 cells. We show that P5 cells presented an increase in membrane fluidity when compared to P4 cells. LR73 cells presented the highest microfluidity when compared to P4 and P5 cells. This was in agreement with the previous published data (1) and demonstrate also that the microfluidity of the plasma membrane is strongly related to the level of Pgp expression. (1) Boutin C, Roche Y, Millot C, Deturche R, Royer P, Manfait M, Plain JM, Jeannesson P, Millot JM, Jaffiol R. High heterogeneity of plasma membrane microfluidity in multidrug-resistant cancer cells. J Biomed Opt. 2009 14(3): 034030. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3537.

Impact of membrane-anchored fluorescent probes on the mechanical properties of lipid bilayers

Fluorescent probes are used in membrane biophysics studies to provide information about physical properties such as lipid packing, polarity and lipid diffusion or to visualize membrane domains. However, our understanding of the effects the dyes themselves may induce on the membrane structure and properties are sparse. As mechanical properties like bending elasticity were already shown to be highly sensitive to the addition of “impurities” into the membranes, we have investigated the impact of six different commonly used fluorescent membrane probes (LAURDAN, TR-DPPE, Rh-DPPE, DiIC18, Bodipy-PC and NBD-PC) on the bending elasticity of dye containing POPC GUVs as compared to single component POPC GUVs. Small changes in the membrane bending elasticity compared to single POPC bilayers are observed when 2 mol% of Rh-DPPE, Bodipy-PC or NBD-PC are added in POPC membranes. These binary membranes are showing non reproducible mechanical properties attributed to a photo-induced peroxidation processes that may be controlled by a reduction of the fluorescent dye concentration. For TR-DPPE, a measurable decrease of the bending elasticity is detected with reproducible bending elasticity measurements. This is a direct indication that this dye, when exposed to illumination by a microscope lamp and contrary to Rh-DPPE, does not induce chemical degradation. At last, LAURDAN and DiIC18 probes mixed with POPC do not significantly affect the bending elasticity of pure POPC bilayers, even at 2 mol%, suggesting these latter probes do not induce major perturbations on the structure of POPC bilayers.

Direct calculation of Förster orientation factor of membrane probes by molecular simulation

Fluorescence is a major tool in biophysics, in particular in membrane studies. However, because lipids are intrinsically nonfluorescent, fluorescence studies of membranes depend on the use of extrinsic probes, whose properties and behavior inside the bilayer are often poorly understood. One important parameter in fluorescence spectroscopy for which there is no experimental technique suited for its determination is the Förster resonance energy transfer (FRET) orientation factor, or κ 2. Its theoretical value for the so-called dynamic isotropic limit is usually used (2/3), but this approximation is debatable because of the intrinsic anisotropic nature of bilayers. Atomistic molecular dynamics simulations were performed in this work in order to determine the distribution and average value of the FRET orientation factor for homotransfer between fluorescent membrane probes 1-palmitoyl,2-(6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino] hexanoyl)- sn-glycero-3-phosphocholine (C6-NBD-PC) and 1-palmitoyl,2-(12-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]dodecanoyl)- sn-glycero-3-phosphocholine (C12-NBD-PC) in a fluid bilayer of 1,2-dipalmitoyl- sn-3-glycerophoshocholine (DPPC, T = 323 K). Although the distributions generally resemble the analytical solutions for isotropic dipoles, the average value for C12-NBD-PC is ∼30% higher than the 2/3 dynamic isotropic limit value. This application illustrates the utility of the present methodology for obtaining accurate estimates of the FRET orientation factor.

Voltage Clamping a Supported Bilayer

Supported bilayer has many advantages over conventional black lipid bilayer. A supported bilayer is highly stable and can be made with a large surface area. Membrane proteins can be incorporated in very large numbers without disruption of the bilayer, thereby allowing robust macroscopic recordings. A very important advantage is that incorporated proteins are immobile. This lack of mobility is essential for the study of conformational changes with single molecule fluorescence. We are interested in studying gating charge movement and conformational changes in voltage dependent membrane proteins such as a voltage dependent K+ channel. For this purpose, we have developed an essentially electrostatic voltage clamp system for a supported bilayer that allows measurement of intramembrane transient currents but not DC currents. The supported bilayer is formed by liposome fusion on top of a semiconductor substrate that serves as the reference electrode. Electrolyte solution is present above the bilayer where an Ag/AgCl electrode serves as the active electrode. The electrode pair is connected to conventional voltage clamp electronics that imposes the voltage and measures the current. We verified supported bilayer formation by the decrease of the total capacitance. Furthermore, we have verified that a voltage is imposed across the bilayer by using voltage dependent fluorescent membrane probes. The electrolyte/supported bilayer/electrode system is essentially linear across a voltage range of −300 to +300 mV. We have seen that direct incorporation of the voltage dependent protein KvAP into the supported bilayer modifies the kinetics of the transient currents as well as the voltage dependence of charge movement. This method opens the possibility for studies of simultaneous gating charge movement electrically and voltage dependent conformational change spectroscopically in purified membrane proteins. Support: NIH GM030376.

Spontaneous oscillations in intracellular Ca2+ concentration via purinergic receptors elicit transient cell swelling in rat parotid ducts

Using multiphoton microscopy, we established that rat parotid ductal cells exhibit spontaneous oscillations in intracellular Ca(2+) concentration ([Ca(2+)](i)). These oscillatory Ca(2+) responses were observed during continuous perfusion with a physiological salt solution at 37 degrees C in the absence of calcium mobilizing agonist stimulation. The timing and patterns of these spontaneous Ca(2+) oscillations varied among individual ductal cells, and the average number of Ca(2+) responses in a single responding ductal cell was 2.1 in a 10-min recording period. High-speed scanning (0.6 s/image) revealed that most spontaneous elevations in [Ca(2+)](i) were initiated at the luminal side of ductal cells and spread toward the basal side within 2 s. Electron microscopic analysis after Ca(2+) imaging indicated that spontaneously oscillating ducts contained numerous granules at the luminal side, which is characteristic of granular ducts. These Ca(2+) oscillations were completely blocked by the purinergic receptor inhibitors 4-[[4-formyl-5-hydroxy-6-methyl-3-[(phosphonooxy)methyl]-2-pyridinyl]azo]-1,3-benzenedisulfonic acid (PPADS) and suramin but were not blocked by the muscarinic antagonist atropine or the alpha-adrenergic antagonist phentolamine. Simultaneous observation of fura-2 fluorescence and differential interference contrast (DIC) images showed that spontaneous elevations of [Ca(2+)](i) were well correlated with changes in shape of ductal cells. Using a plasma membrane fluorescence probe, SynaptoGreen C4, we found that the changes in DIC images reflected spontaneous cell swelling of ductal cells. Our findings present the possibility that purinergic receptors mediate spontaneous Ca(2+) oscillations in parotid ductal cells and regulate electrolyte reabsorption from the primary saliva in the resting state.

Fluorescent membrane probes' behavior in lipid bilayers: insights from molecular dynamics simulations.

Fluorescence spectroscopy and microscopy have been used as tools to study membrane biophysics for decades now. Because phospholipids are non-fluorescent, the use of extrinsic membrane probes in this context is commonplace. Two major points of concern arise regarding this matter, namely the incomplete understanding of the probe behavior inside the bilayer and the perturbation of the latter resulting from probe incorporation. To this effect, molecular dynamics (MD) simulations, by providing detailed atomic-scale information, represent a valuable way to characterize the location and dynamics of bilayer-inserted membrane probes, as well as the magnitude of perturbation they induce on the host lipid structure, and several important classes of reporter molecules have been studied in recent years. This article reviews the state of the art of MD simulations of bilayer-inserted fluorescent probes, focusing on the information that has been obtained from previous studies and hinting at future perspectives in this rapidly emerging field.