Descripción: Dimethyl sulfoxide (DMSO) has been known to enhance cell membrane permeability of drugs or DNA. Molecular dynamics (MD) simulations with single-component lipid bilayers predicted the existence of three regimes of action of DMSO: membrane loosening, pore formation and bilayer collapse. We show here that these modes of action are also reproduced in the presence of cholesterol in the bilayer, and we provide a description at the atomic detail of the DMSO-mediated process of pore formation in cholesterol-containing lipid membranes. We also successfully explore the applicability of DMSO to promote plasma membrane permeability to water, calcium ions (Ca2+) and Yo-Pro-1 iodide (Yo-Pro-1) in living cell membranes. The experimental results on cells in culture can be easily explained according to the three expected regimes: in the presence of low doses of DMSO, the membrane of the cells exhibits undulations but no permeability increase can be detected, while at intermediate DMSO concentrations cells are permeabilized to water and calcium but not to larger molecules as Yo-Pro-1. These two behaviors can be associated to the MD-predicted consequences of the effects of the DMSO at low and intermediate DMSO concentrations. At larger DMSO concentrations, permeabilization is larger, as even Yo-Pro-1 can enter the cells as predicted by the DMSO-induced membrane-destructuring effects described in the MD simulations. © 2012 de Ménorval et al.

Descripción: Osmolarity not only plays a key role in celluar homeostasis but also challenges cell survival. The molecular understanding of osmosis has not yet been completely achieved, and the discovery of aquaporins as molecular entities involved in water transport has caused osmosis to again become a focus of research. The main questions that need to be answered are the mechanism underlying the osmotic permeability coefficients and the extent to which aquaporins change our understanding of osmosis. Here, attempts to answer these questions are discussed. Critical aspects of the state of the state of knowledge on osmosis, a topic that has been studied since 19th century, are reviewed and integrated with the available information provided by in vivo, in vitro and in silico approaches. © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Descripción: The plant aquaporin plasma membrane intrinsic proteins (PIP) subfamily represents one of the main gateways for water exchange at the plasma membrane (PM). A fraction of this subfamily, known as PIP1, does not reach the PM unless they are coexpressed with a PIP2 aquaporin. Although ubiquitous and abundantly expressed, the role and properties of PIP1 aquaporins have therefore remained masked. Here, we unravel how FaPIP1;1, a fruit-specific PIP1 aquaporin from Fragaria x ananassa, contributes to the modulation of membrane water permeability (Pf) and pH aquaporin regulation. Our approach was to combine an experimental and mathematical model design to test its activity without affecting its trafficking dynamics. We demonstrate that FaPIP1;1 has a high water channel activity when coexpressed as well as how PIP1-PIP2 affects gating sensitivity in terms of cytosolic acidification. PIP1-PIP2 random heterotetramerization not only allows FaPIP1;1 to arrive at the PMbut also produces an enhancement of FaPIP2;1 activity. In this context, we propose that FaPIP1;1 is a key participant in the regulation of water movement across the membranes of cells expressing both aquaporins.

Descripción: The purpose of this review is to examine and discuss the ways in which water is organized at the interface of a biological membrane. The relevance of this structure to the surface properties and to the adsorption of proteins in membranes is also analized. The approach is based on the idea that cell functions are confined to a restricted water media, the cell interior, in which the proximity of the membrane may be key to regulating the enzyme activity and the cell membrane permeability. As the lipid bilayer is the structural base of cell membranes, the distribution of water in the surface sites of a phospholipid membrane is analyzed by means of Fourier Transform spectrometry. The polarization of water at the surface was looked into through the measure of surface potentials and the dynamics of the surface hydration by cyclic voltammetry. Modification of these properties by the replacement of water by polyol molecules such as trehalose and phloretin and by the insertion of aqueous soluble enzymes, has also been investigated.

Descripción: In strawberry, the putative participation of aquaporins should be considered during fruit ripening. Furthermore, the availability of different firmness cultivars in this non-climacteric fruit is a very useful tool to determine their involvement in softening. In a previous work, the cloning of a strawberry fruit-specific aquaporin, FaPIP1;1, which showed an expression profile associated with fruit ripening was reported. Here, FaPIP2;1, an aquaporin subtype of PIP2 was cloned and its functional characterization in Xenopus oocytes determined. The FaPIP2;1 gene encodes a water channel with high water permeability (Pf) that is regulated by cytosolic pH. Interestingly, the co-expression of both FaPIP subtypes resulted in an enhancement of water permeability, showing Pf values that exceeds their individual contribution. The expression pattern of both aquaporin subtypes in two cultivars with contrasting fruit firmness showed that the firmer cultivar (Camarosa) has a higher accumulation of FaPIP1 and FaPIP2 mRNAs during fruit ripening when compared with the softer cultivar (Toyonoka). In conclusion, not only FaPIP aquaporins showed an expression pattern associated with fruit firmness but it was also shown that the enhancement of water transfer through the plasma membrane is coupled to the presence/absence of the co-expression of both subtypes. © 2010 The Author(s).

Descripción: The α9 and α10 cholinergic nicotinic receptor subunits assemble to form the receptor that mediates efferent inhibition of hair cell function within the auditory sensory organ, a mechanism thought to modulate the dynamic range of hearing. In contrast to all nicotinic receptors, which serve excitatory neurotransmission, the activation of α9α10 produces hyperpolarization of hair cells. An evolutionary analysis has shown that the α10 subunit exhibits signatures of positive selection only along the mammalian lineage, strongly suggesting the acquisition of a unique function. To establish whether mammalian α9α10 receptors have acquired distinct functional properties as a consequence of this evolutionary pressure, we compared the properties of rat and chicken recombinant and native α9α10 receptors. Our main finding in the present work is that, in contrast to the high (pCa 2+/pMonovalents ∼10) Ca 2+ permeability reported for rat α9α10 receptors, recombinant and native chicken α9α10 receptors have a much lower permeability (∼2) to this cation, comparable to that of neuronal α4β2 receptors. Moreover, we show that, in contrast to α10, α7 as well as α4 and β2 nicotinic subunits are under purifying selection in vertebrates, consistent with the conserved Ca 2+ permeability reported across species. These results have important consequences for the activation of signaling cascades that lead to hyperpolarization of hair cells after α9α10 gating at the cholinergic-hair cell synapse. In addition, they suggest that high Ca 2+ permeability of the α9α10 cholinergic nicotinic receptor might have evolved together with other features that have given the mammalian ear an expanded high-frequency sensitivity.

Descripción: Research done in the last years strongly support the hypothesis that PIP aquaporin can form heterooligomeric assemblies, specially combining PIP2 monomers with PIP1 monomers. Nevertheless, the structural elements involved in the ruling of homo versus heterooligomeric organization are not completely elucidated. In this work we unveil some features of monomer-monomer interaction in Beta vulgaris PIP aquaporins. Our results show that while BvPIP2;2 is able to interact with BvPIP1;1, BvPIP2;1 shows no functional interaction. The lack of functional interaction between BvPIP2;1 and BvPIP1;1 was further corroborated by dose-response curves of water permeability due to aquaporin activity exposed to different acidic conditions. We also found that BvPIP2;1 is unable to translocate BvPIP1;1-ECFP from an intracellular position to the plasma membrane when co-expressed, as BvPIP2;2 does. Moreover we postulate that the first extracellular loop (loop A) of BvPIP2;1, could be relevant for the functional interaction with BvPIP1;1. Thus, we investigate BvPIP2;1 loop A at an atomic level by Molecular Dynamics Simulation (MDS) and by direct mutagenesis. We found that, within the tetramer, each loop A presents a dissimilar behavior. Besides, BvPIP2;1 loop A mutants restore functional interaction with BvPIP1;1. This work is a contribution to unravel how PIP2 and PIP1 interact to form functional heterooligomeric assemblies. We postulate that BvPIP2;1 loop A is relevant for the lack of functional interaction with BvPIP1;1 and that the monomer composition of PIP assemblies determines their functional properties. © 2013 Jozefkowicz et al.

Descripción: The scope of the present review focuses on the interfacial properties of cell membranes that may establish a link between the membrane and the cytosolic components. We present evidences that the current view of the membrane as a barrier of permeability that contains an aqueous solution of macromolecules may be replaced by one in which the membrane plays a structural and functional role. Although this idea has been previously suggested, the present is the first systematic work that puts into relevance the relation water-membrane in terms of thermodynamic and structural properties of the interphases that cannot be ignored in the understanding of cell function. To pursue this aim, we introduce a new definition of interphase, in which the water is organized in different levels on the surface with different binding energies. Altogether determines the surface free energy necessary for the structural response to changes in the surrounding media. The physical chemical properties of this region are interpreted in terms of hydration water and confined water, which explain the interaction with proteins and could affect the modulation of enzyme activity. Information provided by several methodologies indicates that the organization of the hydration states is not restricted to the membrane plane albeit to a region extending into the cytoplasm, in which polar head groups play a relevant role. In addition, dynamic properties studied by cyclic voltammetry allow one to deduce the energetics of the conformational changes of the lipid head group in relation to the head-head interactions due to the presence of carbonyls and phosphates at the interphase. These groups are, apparently, surrounded by more than one layer of water molecules: a tightly bound shell, that mostly contributes to the dipole potential, and a second one that may be displaced by proteins and osmotic stress. Hydration water around carbonyl and phosphate groups may change by the presence of polyhydroxylated compounds or by changing the chemical groups esterified to the phosphates, mainly choline, ethanolamine or glycerol. Thus, surface membrane properties, such as the dipole potential and the surface pressure, are modulated by the water at the interphase region by changing the structure of the membrane components. An understanding of the properties of the structural water located at the hydration sites and the functional water confined around the polar head groups modulated by the hydrocarbon chains is helpful to interpret and analyze the consequences of water loss at the membranes of dehydrated cells. In this regard, a correlation between the effects of water activity on cell growth and the lipid composition is discussed in terms of the recovery of the cell volume and their viability. Critical analyses of the properties of water at the interface of lipid membranes merging from these results and others from the literature suggest that the interface links the membrane with the aqueous soluble proteins in a functional unit in which the cell may be considered as a complex structure stabilized by water rather than a water solution of macromolecules surrounded by a semi permeable barrier. © 2008 Elsevier B.V. All rights reserved.

Descripción: Glucocorticoid receptor (GR) is cytoplasmic in the absence of ligand and localizes to the nucleus after steroid binding. Previous evidence demonstrated that the hsp90-based heterocomplex bound to GR is required for the efficient retrotransport of the receptor to the nuclear compartment. We examined the putative association of GR and its associated chaperone heterocomplex with structures of the nuclear pore. We found that importin β and the integral nuclear pore glycoprotein Nup62 interact with hsp90, hsp70, p23, and the TPR domain proteins FKBP52 and PP5. Nup62 and GR were able to interact in a more efficient manner when chaperoned by the hsp90-based heterocomplex. Interestingly, the binding of hsp70 and p23 to Nup62 does not require the presence of hsp90, whereas the association of FKBP52 and PP5 is hsp90 dependent, as indicated by the results of experiments where the hsp90 function was disrupted with radicicol. The ability of both FKBP52 and PP5 to interact with Nup62 was abrogated in cells overexpressing the TPR peptide. Importantly, GR cross-linked to the hsp90 heterocomplex was able to translocate to the nucleus in digitonin-permeabilized cells treated with steroid, suggesting that GR could pass through the pore in its untransformed state. Copyright © 2009, American Society for Microbiology. All Rights Reserved.