Carlos Gutiérrez-Merino: Synergy of Theory and Experimentation in Biological Membrane Research

Professor Carlos Gutiérrez-Merino, a prominent scientist working in the complex realm of biological membranes, has made significant theoretical and experimental contributions to the field. Contemporaneous with the development of the fluid-mosaic model of Singer and Nicolson, the Förster resonance energy transfer (FRET) approach has become an invaluable tool for studying molecular interactions in membranes, providing structural insights on a scale of 1–10 nm and remaining important alongside evolving perspectives on membrane structures. In the last few decades, Gutiérrez-Merino’s work has covered multiple facets in the field of FRET, with his contributions producing significant advances in quantitative membrane biology. His more recent experimental work expanded the ground concepts of FRET to high-resolution cell imaging. Commencing in the late 1980s, a series of collaborations between Gutiérrez-Merino and the authors involved research visits and joint investigations focused on the nicotinic acetylcholine receptor and its relation to membrane lipids, fostering a lasting friendship

A novel agonist effect on the nicotinic acetylcholine receptor exerted by the anticonvulsive drug Lamotrigine

AbstractThe anticonvulsive drug Lamotrigine (LTG) is found to activate adult muscle nicotinic acetylcholine receptors (AChR). Single-channel patch-clamp recordings showed that LTG (0.05–400 μM) applied alone is able to open AChR channels. [125I]α-bungarotoxin-binding studies further indicate that LTG does not bind to the canonical ACh-binding sites. Fluorescence experiments using the probe crystal violet demonstrate that LTG induces the transition from the resting state to the desensitized state of the AChR in the presence of excess α-bungarotoxin, that is, when the agonist site is blocked. Allosterically-potentiating ligands or the open-channel blocker QX-314 exhibited a behavior different from that of LTG. We conclude that LTG activates the AChR through a site that is different from those of full agonists/competitive antagonists and allosterically-potentiating ligands, respectively

Free cholesterol and cholesterol esters in bovine oocytes: Implications in survival and membrane raft organization after cryopreservation.

Part of the damage caused by cryopreservation of mammalian oocytes occurs at the plasma membrane. The addition of cholesterol to cell membranes as a strategy to make it more tolerant to cryopreservation has been little addressed in oocytes. In order to increase the survival of bovine oocytes after cryopreservation, we proposed not only to increase cholesterol level of oocyte membranes before vitrification but also to remove the added cholesterol after warming, thus recovering its original level. Results from our study showed that modulation of membrane cholesterol by methyl-β-cyclodextrin (MβCD) did not affect the apoptotic status of oocytes and improved viability after vitrification yielding levels of apoptosis closer to those of fresh oocytes. Fluorometric measurements based on an enzyme-coupled reaction that detects both free cholesterol (membrane) and cholesteryl esters (stored in lipid droplets), revealed that oocytes and cumulus cells present different levels of cholesterol depending on the seasonal period. Variations at membrane cholesterol level of oocytes were enough to account for the differences found in total cholesterol. Differences found in total cholesterol of cumulus cells were explained by the differences found in both the content of membrane cholesterol and of cholesterol esters. Cholesterol was incorporated into the oocyte plasma membrane as evidenced by comparative labeling of a fluorescent cholesterol. Oocytes and cumulus cells increased membrane cholesterol after incubation with MβCD/cholesterol and recovered their original level after cholesterol removal, regardless of the season. Finally, we evaluated the effect of vitrification on the putative raft molecule GM1. Cholesterol modulation also preserved membrane organization by maintaining ganglioside level at the plasma membrane. Results suggest a distinctive cholesterol metabolic status of cumulus-oocyte complexes (COCs) among seasons and a dynamic organizational structure of cholesterol homeostasis within the COC. Modulation of membrane cholesterol by MβCD improved survival of bovine oocytes and preserved integrity of GM1-related rafts after vitrification

α7-type acetylcholine receptor localization and its modulation by nicotine and cholesterol in vascular endothelial cells

The neuronal-type 7 nicotinic acetylcholine receptor (7AChR) is also found in various non-neural tissues, including vascular endothelium, where its peculiar ionotropic properties (high Ca2+ permeability) and its supervening Ca2+-mediated intracellular cascades may play important roles in physiology (angiogenesis) and pathology (inflammation and atherogenesis). Changes in molecular (up-regulation, affinity and conformational states) and cellular (distribution, association with membranes) properties of the 7AChR related to angiogenesis (wound-repair cell migration) and atherogenesis (alterations in cholesterol content) were studied in living endothelial cells, with the aim of determining whether such changes constitute early markers of inflammatory response. The combination of pharmacological, biochemical and fluorescence microscopy tools showed that 7AChRs in rat arterial endothelial (RAEC) and human venous endothelial (HUVEC) cells occur at extremely low expression levels (~50 fmol/mg protein) but undergo agonist-induced up-regulation at relatively high nicotine concentrations (~300-fold with 50 M ligand), increasing their cell-surface exposure. When analyzed in terms of cold Triton X-100 solubility and subcellular distribution, 7AChRs occur in the "non-raft" subcellular membrane fractions.Acute cholesterol depletion reduced not only cholesterol levels but the number of cell-surface 7AChRs. Nicotine exposure markedly stimulated cell migration and accelerated wound repair, which drastically diminished in cells deprived of the sterol. The angiogenic effect of nicotine appears to be synergistic with cholesterol content. Finally, the apparent KD of 7AChRs for the open-channel blocker crystal violet was found to be ~600-fold lower in receptor-enriched membranes obtained from up-regulated HUVEC.Fil: Ayala Peña, Victoria Belen. Consejo Nacional de Investigaciones Cientificas y Técnicas. Centro Científico Tecnológico Bahia Blanca. Instituto de Investigaciones Bioquímicas Bahia Blanca (i); Argentina. UNESCO Chair of Biophysics & Molecular Neurobiology; ArgentinaFil: Bonini, Ida Clara. Consejo Nacional de Investigaciones Cientificas y Técnicas. Centro Científico Tecnológico Bahia Blanca. Instituto de Investigaciones Bioquímicas Bahia Blanca (i); Argentina. UNESCO Chair of Biophysics & Molecular Neurobiology; ArgentinaFil: Antollini, Silvia Susana. Consejo Nacional de Investigaciones Cientificas y Técnicas. Centro Científico Tecnológico Bahia Blanca. Instituto de Investigaciones Bioquímicas Bahia Blanca (i); Argentina. UNESCO Chair of Biophysics & Molecular Neurobiology; ArgentinaFil: Barrantes, Francisco Jose. Consejo Nacional de Investigaciones Cientificas y Técnicas. Centro Científico Tecnológico Bahia Blanca. Instituto de Investigaciones Bioquímicas Bahia Blanca (i); Argentina. UNESCO Chair of Biophysics & Molecular Neurobiology; Argentin

The position of the double bond in monounsaturated free fatty acids is essential for the inhibition of the nicotinic acetylcholine receptor

Abstract: Free fatty acids (FFAs) are non-competitive antagonists of the nicotinic acetylcholine receptor (AChR). Their site of action is supposedly located at the lipid-AChR interface. To elucidate the mechanism involved in this antagonism, we studied the effect that FFAs with a single double-bond at different positions (ω6, ω9, ω11 and ω13 cis-18:1) have on different AChR properties. Electrophysiological studies showed that only two FFAs (ω6 and ω9) reduced the duration of the channel open-state. The briefest component of the closed-time distribution remained unaltered, suggesting that ω6 and ω9 behave as allosteric blockers. Fluorescence resonance energy transfer studies indicated that all FFAs locate at the lipid-AChR interface, ω6 being restricted to annular sites and all others occupying non-annular sites. The perturbation of the native membrane order by FFAs was evaluated by DPH (1,6-diphenyl-1,3,5-hexatriene) and Laurdan fluorescence polarization studies, with the greatest decrease observed for ω9 and ω11. AChR conformational changes produced by FFAs present at the lipid bilayer were evaluated by fluorescence quenching studies of pyrene-labeled AChR and also using the AChR conformational-sensitive probe crystal violet. All cis-FFAs produced AChR conformational changes at the transmembrane level, but only ω9, ω11 and ω13 perturbed the resting state. Thus, the position and isomerism of the torsion angle of unsaturated FFAs are probably a key factor in terms of AChR blockage, suggesting that FFAs with a unique cis double bond at a superficial position inside the membrane directly inhibit AChR function by perturbing a potential conserved core structure for AChR gating at that level

Oleic Acid Could Act as a Channel Blocker in the Inhibition of nAChR: Insights from Molecular Dynamics Simulations

Obiol DJ, Amundarain MJ, Zamarreno F, Vietri A, Antollini SS, Costabel MD. Oleic Acid Could Act as a Channel Blocker in the Inhibition of nAChR: Insights from Molecular Dynamics Simulations. Journal of Physical Chemistry B: Biophysics, Biomaterials, Liquids, and Soft Matter. 2024.The activation of the muscular nicotinic acetylcholine receptor (nAChR) produces the opening of the channel, with the consequent increase in the permeability of cations, triggering an excitatory signal. Free fatty acids (FFA) are known to modulate the activity of the receptor as noncompetitive antagonists, acting at the membrane-AChR interface. We present molecular dynamics simulations of a model of nAChR in a desensitized closed state embedded in a lipid bilayer in which distinct membrane phospholipids were replaced by two different monounsaturated FFA that differ in the position of a double bond. This allowed us to detect and describe that the cis-18:1omega-9 FFA were located at the interface between the transmembrane segments of alpha2 and gamma subunits diffused into the channel lumen with the consequent potential ability to block the channel to the passage of ions