Membrane Lipid Organization Research Articles

DEER/PELDOR Study of the Effect of Extremely Low Concentrations of the Antimicrobial Peptide Chalciporin A on the Membrane Lipid Organization

DEER/PELDOR Study of the Effect of Extremely Low Concentrations of the Antimicrobial Peptide Chalciporin A on the Membrane Lipid Organization

Curvature induced membrane structure from fluorescence.

Intrinsic curvature difference between lipids drive lipid organization in mixed lipid membranes. Steady state fluorescence of bilayer soluble Laurdan and water soluble Fluorescein were able to elucidate the packing heterogeneity and composition dependent shape change of mixed lipid vesicles. Mixtures included gel and liquid phase lipids. Computed second harmonic (SH) spectra of Laurdan, in all cases, revealed two lines. Spectra were fit to two log-normal distributions. The SH of Fluorescein showed three peaks. Areas, peak intensities, positions, and linewidths of the resolved lines were examined as functions of lipid mixture composition. Area of the Laurdan blue line from the more packed lower curvature lower polarity regions, Ab, increased and that of the red line from less packed higher curvature higher polarity regions, Ar, decreased with the higher intrinsic curvature lipid fraction. The red peak shifted to longer wavelengths but the blue peak did not. Area changes indicate increased presence of Laurdan in packed flatter regions. Red-shift in red peak position indicates decrease in packing and increase in polarity of the region of origin of the red emission. Fluorescein being outside the vesicle does not sense changes within the bilayer but line parameters showed evidence of change in vesicle shape due to scattering of the fluorescence. These results are consistent with the model of separation of lipids of dissimilar intrinsic curvatures into domains differing in packing and increase in the extent of the flatter sections to relieve curvature stresses. Growth in packed flat regions together with Laurdan's preference for lower curvature regions accounts for the increase in blue and decrease in red emissions. The added higher curvature lipid enters the higher curvature part of the bilayer and causes a red-shift of the peak from this region, but does not affect the blue peak position.

Visualizing ion channels in lipid nanodomains using live-cell fluorescence lifetime imaging.

Ion channels function in a lipid-membrane nano-environment where the lipid composition and membrane compartmentalization can undergo rapid changes. How transmembrane potential activates voltage-gated ion channels differently in liquid-ordered, cholesterol-rich (Lo, or called “lipid raft”) versus in liquid-disordered, cholesterol-poor membrane (Ld) nanodomains remains less explored in intact cells. Using phasor plot fluorescence-lifetime imaging microscopy (phasor FLIM), combined with Förster resonance energy transfer (FRET) between donor- and acceptor-fluorophores conjugated to cholera toxin subunit B (CTxB) or to short lipidated peptides that specifically recognize Lo and Ld domains, we visualized the membrane domains in different cells, including Xenopus oocytes, cultured primary hepatocytes and dorsal root ganglion (DRG) sensory neurons. We postulate that a large percentage of the plasma membrane could be Lo domain. Extracting cholesterols using β-cyclodextrin was able to decrease the FRET efficiency indicating a reduced size of Lo domains. Interestingly, the measured apparent FRET was low in DRG neurons, consistent with the unique lipid organization of neuronal membranes, in comparison to other cell types. Furthermore, the localization of voltage-gated ion channels in proximity to lipid nanodomains could be examined by either FLIM-FRET or spectral FRET using these fluorescent markers. Moreover, fluorescent noncanonical amino acids (ncAAs) incorporated near the hydrophobic constriction site of voltage sensors of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels showed a similar lifetime as when their free forms were dissolved in hydrophobic solvents; lifetime of the incorporated ncAAs changed upon membrane hyperpolarization. In addition, using the stop-codon suppression strategies and click chemistry via the inverse-electron demand Diels-Alder cycloaddition, a photostable dye was tagged site-specifically to the extracellular region of the HCN voltage sensor. These labeling strategies will facilitate measuring the voltage-sensor conformational dynamics of ion channels in distinct lipid nanodomains using time-resolved phasor FLIM-FRET or single-molecule FRET.

Direct determination of membrane protein-lipid organization from customizable bilayer and its application in synaptic vesicle fusion.

Multimeric organization of membrane proteins (MP) and lipids at the cell membrane is fundamental to all living organisms. To study these, we have developed a novel tunable in-vitro nativeMS platform that allows direct detection of the organization of MPs and bound lipids from membranes. Using customizable bilayers, the method can quantitatively report how specific membrane composition and biophysical properties, such as lateral pressure, curvature, and fluidity, modulate the functional organization of MPs.

A multi-view reflector microscope visualizes six-dimensional single-molecule translational and rotational dynamics with isotropic nanoscale resolution.

Quantifying the translational and rotational dynamics of single molecules (SMs) is key to studying the structure and organization of actin filaments, amyloid aggregates, and lipid membranes. However, robustly measuring this high-dimensional information from noisy SM images remains extremely challenging due to the limited number of photons collected from each molecule.

Cannabidiol and cannabinol have distinct effects on membrane structure.

Despite widespread public use, relatively little is known about the compounds cannabidiol (CBD) and cannabinol (CBN), or their interactions with cellular targets. Produced by Cannabis sativa, CBD and CBN have similar chemical structures and are used both pharmaceutically and commercially/recreationally; the former is not psychoactive while the latter is mildly so. Previously CBD was shown to affect the organization of membrane lipids, which indirectly alters sodium channel function (Ghovanloo et al (2021) Cannabidiol inhibits the skeletal muscle Nav1.4 by blocking its pore and by altering membrane elasticity J Gen Physiol; 153 (5): e202012701). Here we use deuterium nuclear magnetic resonance (2H NMR) spectroscopy to investigate the effects of CBD and CBN on 1-palmitoyl-d31-2-oleoyl-sn-glycero-3-phosphocholine (POPC-d31) and POPC-d31/cholesterol multilamellar dispersions as a function of temperature. CBD and CBN affect POPC-d31 membrane organization in a similar way, with increased order in the region of the bilayer near the lipid-water interface and decreased order in the middle of the bilayer. However, CBD and CBN have markedly different effects when added to POPC-d31/cholesterol membranes. Both cannabinoids disorder POPC-d31 in the presence of cholesterol, but CBN's impact on lipid chain organization is significantly greater than that of CBD. These results add support to the theory that CBD and CBN can exert effects on membrane proteins indirectly by altering cell membrane physical properties.

C-Laurdan: Membrane Order Visualization of HEK293t Cells by Confocal Microscopy.

Membrane order is a biophysical characteristic dependent on cellular lipid makeup. Cells regulate the membrane structure as it affects membrane-bound protein activity levels and membrane stability. Spatial organization of membrane lipids, such as lipid rafts, is a proposed theory that has been indirectly measured through polarity-sensitive fluorescent dyes. C-Laurdan is one such dye that penetrates plasma and internal membranes. C-Laurdan is excited by a single 405nm photon and emits in two distinct ranges depending on membrane order. Herein, we present a protocol for staining HEK293t cells with C-Laurdan and acquiring ratiometric images using a revised ImageJ macro and confocal microscopy. An example figure is provided depicting the effects of methyl-β-cyclodextrin, known to remove lipid rafts through cholesterol sequestration, on HEK293t cells. Further image analysis can be performed through region of interest (ROI) selection tools.

Ulf Diederichsen (1963–2021)

“For he was our own! … Fruitful both in counsel and deed; this we experienced and appreciated.” With the death of Ulf Diederichsen on November 11, 2021, after a short but severe illness, we lost a great scientist in chemical biology and good friend. He was a generous and extremely gifted man whose strength was to tackle essential biological problems at a molecular level with simple model systems and to draw insightful conclusions realizing a hallmark of chemical biology by addressing biological challenges with chemical approaches derived from simple principles. Ulf Diederichsen was born on October 7, 1963, in Munich and grew up in Göttingen, enjoying excellent scholar education at the Max-Planck Gymnasium. He moved to Freiburg im Breisgau in 1983 to study Chemistry at the Albert-Ludwigs-University. For his doctorate he moved to Zurich in 1988 to work with Prof. Eschenmoser at the ETH on the question why Nature chose pentose—but not hexose nucleic acids. He received the Dr. rer. nat. in 1993 on the Base pairing of hypoxanthine in HOMO DNA oligonucleotides and the question of the pairing behavior of glucopyranosyl oligonucleotides. He then joined the group of Prof. Dennis P. Curran at the University of Pennsylvania (Pittsburgh/USA) as postdoctoral researcher (1993–1994) investigating radical chemistry. Back to Europe he started his habilitation on “Linear nucleic acid analogues with peptidic backbones” under the mentorship of Prof. Horst Kessler at the Technical University of Munich, which was finished in 1999. He then joined the University of Würzburg as Professor of Organic Chemistry, and finally, in 2001, the University of Göttingen. He served as Dean (2005 to 2007) and Vice President for Research (2015 to 2021). In addition, he was Guest Professor at the Ludwig-Maximilians-University in Munich (1998–1999) and Goering Visiting Professor at the University of Wisconsin, USA (2000). He was elected member of the Academy of Sciences in Göttingen in 2012 and became its president in 2020. His scientific interest was mainly centered on the non-covalent interaction of peptide helices with respect to the incorporation, organization, and dynamics in lipid membranes. He successfully employed spin-labeled peptides for distance measurements. Similarly, he addressed the membrane attachment and fusion processes of SNARE proteins with model systems (conjugates of transmembrane peptides with coiled coil peptides or peptide nucleic acids). He was interested in functionalized β-peptides as artificial matrices for aggregation and modification of model membranes. The excellent results of this outstanding research were summarized in largely over 100 publications in highly renowned international journals. Besides his exceptional scientific achievements and admirable mentorship for a large number of doctoral students and postdocs, he deserves high recognition for his excellent research management particularly as Vice President of the University of Göttingen. He also served as a highly esteemed reviewer in various panels, including the German Research Foundation. The large experience collected in this important activity, particularly also for the German peptide community, he adapted in a very successful manner also in his editorial engagement as Deputy Editor of the Journal of Peptide Science since 2012, where he was supposed to take over the full responsibility as the Editor-in-Chief from 2022. Those of us who knew him are saddened by his sudden passing away and mourn the great loss of a dear friend and colleague. He will always be remembered as an inspiring teacher and mentor, as an enthusiastic scientist, but first and foremost as a warm and sincere human being, as a “feiner Kerl.”

Membrane lipid organization and nicotinic acetylcholine receptor function: A two-way physiological relationship

Nicotinic acetylcholine receptors (nAChRs) are involved in a great range of physiological and pathological conditions. Since they are transmembrane proteins, they interact strongly with the lipids surrounding them. Thus, the plasma membrane composition and heterogeneity play an essential role for the correct nAChR function, on the one hand, and the nAChR influences its immediate lipid environment, on the other hand. The aim of this work was to investigate in more detail the role of the biophysical properties of the membrane in nAChR function and vice versa, focusing on the relationship between Chol and nAChRs. To this end, we worked with different model systems which were treated either with (i) more Chol, (ii) cholesteryl hemisuccinate, or (iii) the enzyme cholesterol oxidase to generate different membrane sterol conditions and in the absence and presence of γTM4 peptide as a representative model of the nAChR.Fluorescence measurements with crystal violet and patch-clamp recordings were used to study nAChR conformation and function, respectively. Using confocal microscopy of giant unilamellar vesicles we probed the membrane phase state/order and organization (coexistence of lipid domains) and lipid-nAChR interaction. Our results show a feedback relationship between membrane organization and nAChR function, i.e. whereas the presence of a model of nAChRs conditions membrane organization, changing its lipid microenvironment, membrane organization and composition perturb nAChRs function. We postulate that nAChRs have a gain of function in disordered membrane environments but a loss of function in ordered ones, and that Chol molecules at the outer leaflet in annular sites and at the inner leaflet in non-annular sites are related to nAChR gating and desensitization, respectively. Thus, depending on the membrane composition, organization, and/or order, the nAChR adopts different conformations and locates in distinct lipid domains and this has a direct effect on its function.

A 5-color flow cytometry panel to assess plasma membrane integrity, acrosomal status, membrane lipid organization and mitochondrial activity of boar and stallion spermatozoa following liquid semen storage

For a more practically applicable analysis of different sperm characteristics, this study aimed to develop a 5-color flow cytometry (FC) panel to concurrently analyze four sperm parameters in liquid boar and stallion semen, using also a DNA-marker for selecting sperm cell events. From each of thirty extended boar semen doses and twelve stallion semen doses, six aliquots were taken. For evaluating mitochondrial activity (A), degree of lipid disorder of plasma membrane (B), integrity of plasma membrane (C), acrosomal status (D) and marking DNA (E), five aliquots were individually stained with Rhodamine 123, Merocyanine 540, Propidium Iodide, PNA-Alexa Fluor 647, and Hoechst 33342, respectively. The sixth aliquot was stained with all the five fluorochromes simultaneously, whereas spectral overlap was corrected by a compensation matrix. Strong correlations were found between the single and 5-color staining assays for boar sperm (A: 0.99, B: 0.96, C: 0.93, D: 0.98, E: 0.99; P < 0.01). Furthermore, moderate and substantial Concordance Correlation Coefficients (CCC) were presented by all these parameters (0.99, 0.96, 0.92, 0.98, and 0.99, respectively). For stallion sperm, the correlation coefficients between the assays were also strong (A: 0.99, B: 0.98, C: 0.99, D: 0.99, E: 0.95; P < 0.01) and substantial CCC were observed for all of them (0.99, 0.97, 0.99, 0.99, and 0.90, respectively). For both species, the mean difference between the methods (d̅) did not overtake 0.84. The results confirmed that this 5-color panel could be successfully implemented for analyzing boar and stallion sperm quality in a single, practical and quick FC assay.

Fluorescent Probe for the Monitoring of Plasma Membrane Hydration

AbstractSubcellular microenvironment plays a crucial role in maintaining cell homeostasis being hydration an essential parameter to monitor the variations of lipid organization in biological membranes. Fluorescent microenvironment‐sensitive probes are stimuli‐responsive molecules and able to change their fluorescence profile according to the microenvironment properties (polarity, hydration, microviscosity and tension). Here we present an organic fluorescent probe, NPM‐1, as a new microenvironmental dye with selective plasma membrane localization in live cells. Using high‐resolution confocal microscopy and fluorescence spectroscopy, we explored the hydration and polarity variations during the formation of apoptotic bodies in real time. To have a clear understanding of the cell microenvironment variations, an analogue polarity probe able to localize the cytosol and nucleoli was also synthetized, CellPol‐1. Then, we were able to monitor the subcellular hydration variations during the apoptotic body dynamics.

Cholesterol Transport to the Endoplasmic Reticulum.

Most cholesterol in mammalian cells is stored in the plasma membrane (PM). Cholesterol transport from the PM to low-sterol regulatory regions of the endoplasmic reticulum (ER) controls cholesterol synthesis and uptake, and thereby influences the rates of cholesterol flux between tissues of complex organisms. Cholesterol transfer to the ER is also required for steroidogenesis, oxysterol and bile acid synthesis, and cholesterol esterification. The ER-resident Aster proteins (Aster-A, -B, and -C) form contacts with the PM to move cholesterol to the ER in mammals. Mice lacking Aster-B have low adrenal cholesteryl ester stores and impaired steroidogenesis because of a defect in cholesterol transport from high-density lipoprotein (HDL) to the ER. This work reviews the molecular characteristics of Asters, their role in HDL- and low-density lipoprotein (LDL)-cholesterol movement, and how cholesterol transferred to the ER is utilized by cells. The roles of other lipid transporters and of membrane lipid organization in maintaining aspects of cholesterol homeostasis are also highlighted.

Direct determination of oligomeric organization of integral membrane proteins and lipids from intact customizable bilayer

Direct determination of oligomeric organization of integral membrane proteins and lipids from intact customizable bilayer

Peptide-membrane binding is not enough to explain bioactivity: A case study

Membrane-active peptides are a promising class of antimicrobial and anticancer therapeutics. For this reason, their molecular mechanisms of action are currently actively investigated. By exploiting Electron Paramagnetic Resonance, we study the membrane interaction of two spin-labeled analogs of the antimicrobial and cytotoxic peptide trichogin GA IV (Tri), with opposite bioactivity: Tri(Api8), able to selectively kill cancer cells, and Tri(Leu4), which is completely nontoxic. In our attempt to determine the molecular basis of their different biological activity, we investigate peptide impact on the lateral organization of lipid membranes, peptide localization and oligomerization, in the zwitter-ionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) model membrane We show that, despite their divergent bioactivity, both peptide analogs (i) are membrane-bound, (ii) display a weak tendency to oligomerization, and (iii) do not induce significant lipid rearrangement. Conversely, literature data show that the parent peptide trichogin, which is cytotoxic without any selectivity, is strongly prone to dimerization and affects the reorganization of POPC membranes. Its dimers are involved in the rotation around the peptide helix, as observed at cryogenic temperatures in the millisecond timescale. Since this latter behavior is not observed for the inactive Tri(Leu4), we propose that for short-length peptides as trichogin oligomerization and molecular motions are crucial for bioactivity, and membrane binding alone is not enough to predict or explain it. We envisage that small changes in the peptide sequence that affect only their ability to oligomerize, or their molecular motions inside the membrane, can tune the peptide activity on membranes of different compositions.

Membrane Cholesterol Content and Lipid Organization Influence Melittin and Pneumolysin Pore-Forming Activity.

Melittin, the main toxic component in the venom of the European honeybee, interacts with natural and artificial membranes due to its amphiphilic properties. Rather than interacting with a specific receptor, melittin interacts with the lipid components, disrupting the lipid bilayer and inducing ion leakage and osmotic shock. This mechanism of action is shared with pneumolysin and other members of the cholesterol-dependent cytolysin family. In this manuscript, we investigated the inverse correlation for cholesterol dependency of these two toxins. While pneumolysin-induced damage is reduced by pretreatment with the cholesterol-depleting agent methyl-β-cyclodextrin, the toxicity of melittin, after cholesterol depletion, increased. A similar response was also observed after a short incubation with lipophilic simvastatin, which alters membrane lipid organization and structure, clustering lipid rafts. Therefore, changes in toxin sensitivity can be achieved in cells by depleting cholesterol or changing the lipid bilayer organization.

Oxidized Lipids Control Lipid Order and Phospholipase A2 Activity in Model Membranes

Oxidative stress is an important etiologic factor in the pathogenesis of various diseases. The formation of oxidized phospholipid species in vivo induces membrane remodelling with direct pathological implications with a prominent inflammatory component. Secretory phospholipases A2 (sPLA2) are involved in the regulation of inflammation and immune response. Their activity is highly dependent on the lipid membrane composition, structure and organization. In this work, we studied the impact of oxidized phosphatidylcholines (OxPCs) on the membrane lipid order and the sPLA2 activity. The effects of two of the most physiologically active OxPCs, 1-palmitoyl-2-(5′-oxo-valeroyl)-sn-glycero- 3-phosphocholine (POVPC) and 1-palmitoyl-2-glutaroyl-sn-glycero-3- phosphocholine (PGPC) were compared using 1-palmitoyl-2-oleoyl-sn-glycero- 3-phosphocholine (POPC) vesicles. Both OxPCs reduce the membrane lipid order and sPLA2 activity at physiological temperature. Moreover, these changes depend on the chemical nature of the oxidized chains.

A Quantitative Native Mass Spectrometry Platform for Deconstructing Hierarchical Organization of Membrane Proteins and Lipids

HypothesisHierarchical organization of protein and lipids at the cell membrane is essential for regulating a myriad of cellular signaling. The lateral heterogeneity of the biological membrane, both in terms of its lipid composition, as well as biophysical properties such as lateral pressure, curvature, fluidity has been shown to play regulatory role towards maintaining these assemblies of membrane proteins. This in turn imposes a challenging experimental prerequisite towards studying macromolecular assemblies of proteins and lipids at the membrane. It demands experimental approaches that (1) can unambiguously identify the associated proteins and lipids, along with their oligomeric states and binding stoichiometry, and (2) can be directly applied to a lipid‐bilayer environments that mimics the lipid composition and critical biophysical properties of a target physiological membrane.MethodsAddressing this need, we have developed lipid vesicle native mass spectrometry (nMS). This comprehensive tunable in vitro platform enables us to construct lipid bilayer, mimicking a target physiological membrane in terms of its composition and biophysical properties and directly subject it to nMS and MS/MS analysis to determine the oligomeric organization of the proteins present, identity of the bound lipids, and their binding stoichiometry; all directly from the membrane.Preliminary DataFirst, to establish the broad applicability of our platform we chose a diverse set of membrane proteins with their oligomeric states ranging from monomer to pentamer, masses 12kDa‐226kDa, and number transmembrane‐helices 1‐30. We further reconstituted them in a diverse set of lipid vesicles that are mimics of the eukaryotic ER, PM, Mitochondria, Golgi, and bacterial plasma membranes, as well as in vesicles of specific membrane curvature. In each of these cases, by directly subjecting these native‐like vesicles to nMS we can directly determine the physiological oligomeric state of the proteins in the bilayer. Further, using the top‐down MS/MS capability of our platform, we can unambiguously determine which of the membrane lipid associate with target membrane protein. Having established the broad and general applicability of our platform to study membrane proteins directly from a bilayer that can be customized to represent a target physiological membrane, we focused on applying this to two specific problems. First, we applied our approach to synaptic vesicle proteins and we show that through specific lipid binding VAMP2 induces clustering of specific lipid around its transmembrane and regulates the temporal scale of fusions. Next, applying our platform to a sugar transporter protein we quantitatively show that increase in cardiolipin % in the membrane leads to direct increase in functional oligomeric population.ConclusionOur work presents a broad and generally applicable nMS platform that enables quantitative determination of molecular organization of protein and lipids directly from lipid membranes of desired composition and characteristics, identify of the specifically bound lipids, and determine how these specific bindings in turn regulate protein oligomerization.

An Antimicrobial Peptide-Mimetic Methacrylate Random Copolymer Induces Domain Formation in a Model Bacterial Membrane.

To address the emerging issue of drug-resistant bacteria, membrane-active synthetic polymers have been designed and developed to mimic host-defense antimicrobial peptides (AMPs) as antibiotic alternatives. In this study, we investigated the domain formation induced by synthetic polymer mimics of AMPs using model membranes to elucidate the biophysical principles that govern their membrane-active mechanisms. To that end, lipid vesicles mimicking Escherichia coli(E. coli) membrane were prepared using an 8:2 (molar ratio) mixture of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol), sodium salt (POPG). Our studies using differential scanning calorimetry (DSC) and fluorescence microscopy indicated that cationic amphiphilic methacrylate random copolymers induced the phase separation to form POPE- or POPG-rich domains. A rhodamine-labeled polymer also showed the binding to separated domains in the membrane. Based on these results, we propose the mechanism that the copolymers induce domain formation by clustering of anionic POPG lipids similar to natural AMPs. In addition, the time-course of polymer binding to the GUV membrane was sigmoidal, suggesting the positive feedback loop in the membrane binding. We also hypothesize that this cooperative binding of the polymer is driven by the domain formation. This study demonstrates the potential of the amphiphilic copolymers to modulate the lipid organization of cell membranes, which may provide a new strategy to design membrane-active antimicrobial agents.

A tunable lipid bilayer native MS platform for direct determination of hierarchical organization of membrane proteins and lipids at the membrane

Multimeric organization of membrane proteins (MP) and lipids at the cell membrane is fundamental to all living organisms. A comprehensive analysis of MPs and their complexes thus demands a platform that allows nativeMS analysis of the MPs directly from their lipid bilayer environment. Addressing this, we have developed a novel tunable in-vitro nativeMS platform that allows direct detection of organization of MPs and bound lipids from membranes that can be customized to mimic any target physiological membrane, both in terms of its lipid composition as well as biophysical properties, such as curvature, fluidity, and tension.

A Note of Caution: Gramicidin Affects Signaling Pathways Independently of Its Effects on Plasma Membrane Conductance.

Gramicidin is a thoroughly studied cation ionophore widely used to experimentally manipulate the plasma membrane potential (PMP). In addition, it has been established that the drug, due to its hydrophobic nature, is capable of affecting the organization of membrane lipids. We have previously shown that modifications in the plasma membrane potential of epithelial cells in culture determine reorganizations of the cytoskeleton. To elucidate the molecular mechanisms involved, we explored the effects of PMP depolarization on some putative signaling intermediates. In the course of these studies, we came across some results that could not be interpreted in terms of the properties of gramicidin as an ionic channel. The purpose of the present work is to communicate these results and, in general, to draw attention to the fact that gramicidin effects can be misleadingly attributed to its ionic or electrical properties. In addition, this work also contributes with some novel findings of the modifications provoked on the signaling intermediates by PMP depolarization and hyperpolarization.