Cholesterol and nicotinic acetylcholine receptor: An intimate nanometer-scale spatial relationship spanning the billion year time-scale

Once the sterol biosynthetic machinery had progressed over the course of several million years to yield cholesterol, this neutral lipid became an omnipresent and essential component of biomembranes in Eukaryotes. The hopanoids in Prokaryotesand eukaryotic sterols share the ability to provide stability and domain compartmentalization in membranes. Even more important is the intimate association of cholesterol with a wide range of cell-surface membrane proteins, probably responsible for its modulatory effects on neurotransmitter and hormone receptors and ion channels. These effects appear to be exerted via the membrane-embedded segments of essentially all members of the pentameric ligand-gated ion channel and G-protein-coupled receptor superfamilies, which possess consensus linear arrays of amino acid residues recognizing cholesterol with relativelyhigh affinity and specificity, an early evolutionary acquisition already present in ancient bacteria, conserved, and further improved in Eukaryotes. This review focuses on the long-term relationship between cholesterol and these functionally important membrane protein superfamilies, and the ability of cholesterol to induce lateral segregation and ordered domain formation at the nanoscale in cell membranes.Fil: Barrantes, Francisco Jose. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; Argentin

Structural biology of coronavirus ion channels

Viral infection compromises specific organelles of the cell and readdresses its functional resources to satisfy the needs of the invading body. Around 70% of the coronavirus positive-sense single-stranded RNA encodes proteins involved in replication, and these viruses essentially take over the biosynthetic and transport mechanisms to ensure the efficient replication of their genome and trafficking of their virions. Some coronaviruses encode genes for ion-channel proteins - the envelope protein E (orf4a), orf3a and orf8 - which they successfully employ to take control of the endoplasmic reticulum-Golgi complex intermediate compartment or ERGIC. The E protein, which is one of the four structural proteins of SARS-CoV-2 and other coronaviruses, assembles its transmembrane protomers into homopentameric channels with mild cationic selectivity. Orf3a forms homodimers and homotetramers. Both carry a PDZ-binding domain, lending them the versatility to interact with more than 400 target proteins in infected host cells. Orf8 is a very short 29-amino-acid single-passage transmembrane peptide that forms cation-selective channels when assembled in lipid bilayers. This review addresses the contribution of biophysical and structural biology approaches that unravel different facets of coronavirus ion channels, their effects on the cellular machinery of infected cells and some structure-functional correlations with ion channels of higher organisms.Fil: Barrantes, Francisco Jose. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; Argentin

Nanoscale interactions between the nicotinic acetylcholine receptor and cholesterol

Cholesterol is a major lipid in biological membranes. It not only plays a structural role but also modulates a wide range of functional properties of neurotransmitter and hormone receptors and ion channels. The membraneembedded segments of the paradigm neurotransmitter receptor for acetylcholine (nAChR) contain linear sequences of amino acids with the capacity to recognize cholesterol. These cholesterol consensus domains have been designated as "CARC" and its mirror sequence "CRAC". CARC preferentially occurs in the exoplasmic-facing membrane leaflet, and CRAC, in the cytoplasmic-facing hemilayer. Both motifs are highly conserved among ion-channel and neurotransmitter receptor proteins in vertebrate nervous systems, where they recognize cholesterol, and in prokaryotic homologues in bacteria, where they recognize hopanoids. This phylogenetically conserved trait is an indication that the hopanoids in some bacteria and cholesterol in eukaryotes subserve analogous functions, probably contributing to the stability of membrane-embedded protein domains. Structural studies from our laboratory using superresolution optical microscopy ("nanoscopy") have disclosed other interrelated functional and structural properties exerted by cholesterol on the nAChR. The neutral lipid content at the cell surface influences both the macromolecular organization of the receptor and its translational mobility (diffusion) in the plane of the membrane.Fil: Barrantes, Francisco Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; Argentin

The Contribution of Biophysics and Structural Biology to Current Advances in COVID-19

Critical to viral infection are the multiple interactions between viral proteins and host-cell counterparts. The first such interaction is the recognition of viral envelope proteins by surface receptors that normally fulfil other physiological roles, a hijacking mechanism perfected over the course of evolution. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of coronavirus disease 2019 (COVID-19), has successfully adopted this strategy using its spike glycoprotein to dock on the membrane-bound metalloprotease angiotensin-converting enzyme 2 (ACE2). The crystal structures of several SARS-CoV-2 proteins alone or in complex with their receptors or other ligands were recently solved at an unprecedented pace. This accomplishment is partly due to the increasing availability of data on other coronaviruses and ACE2 over the past 18 years. Likewise, other key intervening actors and mechanisms of viral infection were elucidated with the aid of biophysical approaches. An understanding of the various structurally important motifs of the interacting partners provides key mechanistic information for the development of structure-based designer drugs able to inhibit various steps of the infective cycle, including neutralizing antibodies, small organic drugs, and vaccines. This review analyzes current progress and the outlook for future structural studies.Fil: Barrantes, Francisco Jose. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; Argentin

Cell-surface translational dynamics of nicotinic acetylcholine receptors

Synapse efficacy heavily relies on the number of neurotransmitter receptors available at a given time. In addition to the equilibrium between the biosynthetic production, exocytic delivery and recycling of receptors on the one hand, and the endocytic internalization on the other, lateral diffusion and clustering of receptors at the cell membrane play key roles in determining the amount of active receptors at the synapse. Mobile receptors traffic between reservoir compartments and the synapse by thermally driven Brownian motion, and become immobilized at the peri-synaptic region or the synapse by: (a) clustering mediated by homotropic inter-molecular receptor-receptor associations; (b) heterotropic associations with non-receptor scaffolding proteins or the subjacent cytoskeletal meshwork, leading to diffusional "trapping," and (c) protein-lipid interactions, particularly with the neutral lipid cholesterol. This review assesses the contribution of some of these mechanisms to the supramolecular organization and dynamics of the paradigm neurotransmitter receptor of muscle and neuronal cells -the nicotinic acetylcholine receptor (nAChR). Currently available information stemming from various complementary biophysical techniques commonly used to interrogate the dynamics of cell-surface components is critically discussed. The translational mobility of nAChRs at the cell surface differs between muscle and neuronal receptors in terms of diffusion coefficients and residence intervals at the synapse, which cover an ample range of time regimes. A peculiar feature of brain a7 nAChR is its ability to spend much of its time confined peri-synaptically, vicinal to glutamatergic (excitatory) and GABAergic (inhibitory) synapses. An important function of the a7 nAChR may thus be visiting the territories of other neurotransmitter receptors, differentially regulating the dynamic equilibrium between excitation and inhibition, depending on its residence time in each domain.Fil: Barrantes, Francisco Jose. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires"; Argentin

Cholesterol modulates acetylcholine receptor diffusion by tuning confinement sojourns and nanocluster stability

Translational motion of neurotransmitter receptors is key for determining receptor number at the synapse and hence, synaptic efficacy. We combine live-cell STORM superresolution microscopy of nicotinic acetylcholine receptor (nAChR) with single-particle tracking, mean-squared displacement (MSD), turning angle, ergodicity, and clustering analyses to characterize the lateral motion of individual molecules and their collective behaviour. nAChR diffusion is highly heterogeneous: subdiffusive, Brownian and, less frequently, superdiffusive. At the single-track level, free walks are transiently interrupted by ms-long confinement sojourns occurring in nanodomains of ~36 nm radius. Cholesterol modulates the time and the area spent in confinement. Turning angle analysis reveals anticorrelated steps with time-lag dependence, in good agreement with the permeable fence model. At the ensemble level, nanocluster assembly occurs in second-long bursts separated by periods of cluster disassembly. Thus, millisecond-long confinement sojourns and second-long reversible nanoclustering with similar cholesterol sensitivities affect all trajectories; the proportion of the two regimes determines the resulting macroscopic motional mode and breadth of heterogeneity in the ensemble population.Fil: Mosqueira, Alejo. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; ArgentinaFil: Camino, Pablo A.. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; ArgentinaFil: Barrantes, Francisco Jose. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; Argentin

How cholesterol interacts with membrane proteins: an exploration of cholesterol-binding sites including CRAC, CARC, and tilted domains

The plasma membrane of eukaryotic cells contains several types of lipids displaying high biochemical variability in both their apolar moiety (e.g., the acyl chain of glycerolipids) and their polar head (e.g., the sugar structure of glycosphingolipids). Among these lipids, cholesterol is unique because its biochemical variability is almost exclusively restricted to the oxidation of its polar −OH group. Although generally considered the most rigid membrane lipid, cholesterol can adopt a broad range of conformations due to the flexibility of its isooctyl chain linked to the polycyclic sterane backbone. Moreover, cholesterol is an asymmetric molecule displaying a planar α face and a rough β face. Overall, these structural features open up a number of possible interactions between cholesterol and membrane lipids and proteins, consistent with the prominent regulatory functions that this unique lipid exerts on membrane components. The aim of this review is to describe how cholesterol interacts with membrane lipids and proteins at the molecular/atomic scale, with special emphasis on transmembrane domains of proteins containing either the consensus cholesterol-binding motifs CRAC and CARC or a tilted peptide. Despite their broad structural diversity, all these domains bind cholesterol through common molecular mechanisms, leading to the identification of a subset of amino acid residues that are overrepresented in both linear and three-dimensional membrane cholesterol-binding sites.Fil: Fantini, J.. Aix-Marseille Université. Interactions Moléculaires et Systèmes Membranaires; FranciaFil: Barrantes, Francisco Jose. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Facultad de Ciencias Médicas. Instituto de Investigaciones Biomédicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Nanoscale Sub-Compartmentalization of the Dendritic Spine Compartment

Compartmentalization of the membrane is essential for cells to perform highly specific tasks and spatially constrained biochemical functions in topographically defined areas. These membrane lateral heterogeneities range from nanoscopic dimensions, often involving only a few molecular constituents, to micron-sized mesoscopic domains resulting from the coalescence of nanodomains. Short-lived domains lasting for a few milliseconds coexist with more stable platforms lasting from minutes to days. This panoply of lateral domains subserves the great variety of demands of cell physiology, particularly high for those implicated in signaling. The dendritic spine, a subcellular structure of neurons at the receiving (postsynaptic) end of central nervous system excitatory synapses, exploits this compartmentalization principle. In its most frequent adult morphology, the mushroom-shaped spine harbors neurotransmitter receptors, enzymes, and scaffolding proteins tightly packed in a volume of a few femtoliters. In addition to constituting a mesoscopic lateral heterogeneity of the dendritic arborization, the dendritic spine postsynaptic membrane is further compartmentalized into spatially delimited nanodomains that execute separate functions in the synapse. This review discusses the functional relevance of compartmentalization and nanodomain organization in synaptic transmission and plasticity and exemplifies the importance of this parcelization in various neurotransmitter signaling systems operating at dendritic spines, using two fast ligand-gated ionotropic receptors, the nicotinic acetylcholine receptor and the glutamatergic receptor, and a second-messenger G-protein coupled receptor, the cannabinoid receptor, as paradigmatic examples.Fil: Valles, Ana Sofia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; ArgentinaFil: Barrantes, Francisco Jose. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; Argentin

Dysregulation of Neuronal Nicotinic Acetylcholine Receptor-Cholesterol Crosstalk in Autism Spectrum Disorder

Autism spectrum disorder (ASD) is a set of complex neurodevelopmental diseases that include impaired social interaction, delayed and disordered language, repetitive or stereotypic behavior, restricted range of interests, and altered sensory processing. The underlying causes of the core symptoms remain unclear, as are the factors that trigger their onset. Given the complexity and heterogeneity of the clinical phenotypes, a constellation of genetic, epigenetic, environmental, and immunological factors may be involved. The lack of appropriate biomarkers for the evaluation of neurodevelopmental disorders makes it difficult to assess the contribution of early alterations in neurochemical processes and neuroanatomical and neurodevelopmental factors to ASD. Abnormalities in the cholinergic system in various regions of the brain and cerebellum are observed in ASD, and recently altered cholesterol metabolism has been implicated at the initial stages of the disease. Given the multiple effects of the neutral lipid cholesterol on the paradigm rapid ligand-gated ion channel, the nicotinic acetylcholine receptor, we explore in this review the possibility that the dysregulation of nicotinic receptor-cholesterol crosstalk plays a role in some of the neurological alterations observed in ASD.Fil: Valles, Ana Sofia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; ArgentinaFil: Barrantes, Francisco Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Biomédicas; Argentin

Chaperoning a7 neuronal nicotinic acetylcholine receptors

The α7 subtype of nicotinic acetylcholine receptors (AChRs) is one of the most abundant members of the Cysloop family of receptors present in the central nervous system. It participates in various physiological processes and has received much attention as a potential therapeutic target for a variety of pathologies. The importance of understanding the mechanisms controlling AChR assembly and cell-surface delivery lies in the fact that these two processes are key to determining the functional pool of receptors actively engaged in synaptic transmission. Here we review recent studies showing that RIC-3, a protein originally identified in the worm Caenorhabditis elegans, modulates the expression of α7 AChRs in a subtype-specific manner. Potentiation of AChR expression by post-transcriptional events is also critically assessed.Fil: Valles, Ana Sofia. Consejo Nacional de Investigaciones Cientificas y Técnicas. Centro Científico Tecnológico Bahia Blanca. Instituto de Investigaciones Bioquímicas Bahia Blanca (i); ArgentinaFil: Barrantes, Francisco Jose. Consejo Nacional de Investigaciones Cientificas y Técnicas. Centro Científico Tecnológico Bahia Blanca. Instituto de Investigaciones Bioquímicas Bahia Blanca (i); Argentina. Pontificia Universidad Católica "Santa María de Los Buenos Aires"; Argentin