The effects of membrane compartmentalization of csk on TCR signaling

The TCR signal transduction is initiated by the activation of Src-family kinases (SFK) which phosphorylate Immunoreceptor tyrosine-based activation motifs (ITAM) present in the intracellular parts of the T-cell receptor (TCR) signaling subunits. Numerous data suggest that after stimulation TCR interacts with membrane rafts and thus it gains access to SFK and other important molecules involved in signal transduction. However, the precise mechanism of this process is unclear. One of the key questions is how SFK access TCR and what is the importance of non-raft and membrane raft-associated SFK for the initiation and maintenance of the TCR signaling. To answer this question we targeted a negative regulator of SFK, C-terminal Src kinase (Csk) to membrane rafts, recently described “heavy rafts” or non-raft membrane. Our data show that only Csk targeted into “classical” raft but not to “heavy raft” or non-raft membrane effectively inhibits TCR signaling, demonstrating the critical role of membrane raft-associated SFK in this process.     

Sugar reception in the blowfly: a possible Ca(++) involvement

The present study investigates the effects of W-7 (a calmodulin antagonist involved in the Ca⁺⁺ cascade) on the response of the ‘sugar’ and ‘water’ cells of labellar chemosensilla in the blowfly Protophormia terraenovae to stimulation with sucrose or fructose. In order to ascertain whether Ca⁺⁺ conductance is involved, the effects of EGTA, one of the most used Ca⁺⁺ chelating agent, and of SK&F-96365, an inhibitor of receptor mediated calcium influx, were also studied. Our electrophysiological data indicate that W-7 addition strongly depresses the ‘sugar’ chemoreceptor response to both sugars and in the case of sucrose stimulation also influences adaptation rate. The Ca⁺⁺ chelator has no significant effects on the response of the ‘sugar’ cell following stimulation with sucrose, but lowers fructose stimulating effectiveness. In the presence of SK&F-96365 both sucrose and fructose responses are inhibited. A possible transduction mechanism for sugar reception is discussed.     

Expression and function of aquaporins in human skin: Is aquaporin-3 just a glycerol transporter?

The aquaporins (AQPs) are a family of transmembrane proteins forming water channels. In mammals, water transport through AQPs is important in kidney and other tissues involved in water transport. Some AQPs (aquaglyceroporins) also exhibit glycerol and urea permeability. Skin is the limiting tissue of the body and within skin, the stratum corneum (SC) of the epidermis is the limiting barrier to water loss by evaporation. The aquaglyceroporin AQP3 is abundantly expressed in keratinocytes of mammalian skin epidermis. Mice lacking AQP3 have dry skin and reduced SC hydration. Interestingly, however, results suggested that impaired glycerol, rather than water transport was responsible for this phenotype. In the present work, we examined the overall expression of AQPs in cells from human skin and we reviewed data on the functional role of AQPs in skin, particularly in the epidermis. By RT-PCR on primary cell cultures, we found that up to 6 different AQPs (AQP1, 3, 5, 7, 9 and 10) may be selectively expressed in various cells from human skin. AQP1, 5 are strictly water channels. But in keratinocytes, the major cell type of the epidermis, only the aquaglyceroporins AQP3, 10 were found. To understand the role of aquaglyceroporins in skin, we examined the relevance to human skin of the conclusion, from studies on mice, that skin AQP3 is only important for glycerol transport. In particular, we find a correlation between the absence of AQP3 and intercellular edema in the epidermis in two different experimental models: eczema and hyperplastic epidermis. In conclusion, we suggest that in addition to glycerol, AQP3 may be important for water transport and hydration in human skin epidermis.     

Expression and function of aquaporins in human skin: Is aquaporin-3 just a glycerol transporter?

The aquaporins (AQPs) are a family of transmembrane proteins forming water channels. In mammals, water transport through AQPs is important in kidney and other tissues involved in water transport. Some AQPs (aquaglyceroporins) also exhibit glycerol and urea permeability. Skin is the limiting tissue of the body and within skin, the stratum corneum (SC) of the epidermis is the limiting barrier to water loss by evaporation. The aquaglyceroporin AQP3 is abundantly expressed in keratinocytes of mammalian skin epidermis. Mice lacking AQP3 have dry skin and reduced SC hydration. Interestingly, however, results suggested that impaired glycerol, rather than water transport was responsible for this phenotype. In the present work, we examined the overall expression of AQPs in cells from human skin and we reviewed data on the functional role of AQPs in skin, particularly in the epidermis. By RT-PCR on primary cell cultures, we found that up to 6 different AQPs (AQP1, 3, 5, 7, 9 and 10) may be selectively expressed in various cells from human skin. AQP1, 5 are strictly water channels. But in keratinocytes, the major cell type of the epidermis, only the aquaglyceroporins AQP3, 10 were found. To understand the role of aquaglyceroporins in skin, we examined the relevance to human skin of the conclusion, from studies on mice, that skin AQP3 is only important for glycerol transport. In particular, we find a correlation between the absence of AQP3 and intercellular edema in the epidermis in two different experimental models: eczema and hyperplastic epidermis. In conclusion, we suggest that in addition to glycerol, AQP3 may be important for water transport and hydration in human skin epidermis.     

On the mechanism of conductance control of the arthropod visual cell membrane

It is assumed that dark channels determine a permanent dark conductance of the arthropod visual cell membrane. The light stimulus causes a transient opening of light channels. The ion selectivity of dark channels and light channels is roughly described. Factors influencing the activation of light channels, as membrane energy metabolism, membrane potential and adjusted calcium ion concentration are specified. The mechanism of the action of calcium ions on the conductance of the visual cell membrane is discussed.These considerations are mainly concerned with photoreceptor cells of Limulus, Astacus and Balanus Presented at the EMBO-Workshop on Transduction Mechanism of Photoreceptors, Jülich, Germany, October 4–8, 1976     

Hog1 mitogen-activated protein kinase phosphorylation targets the yeast Fps1 aquaglyceroporin for endocytosis, thereby rendering cells resistant to acetic acid

Aquaporins and aquaglyceroporins form the membrane channels that mediate fluxes of water and small solute molecules into and out of cells. Eukaryotes often use mitogen-activated protein kinase (MAPK) cascades for the intracellular signaling of stress. This study reveals an aquaglyceroporin being destabilized by direct MAPK phosphorylation and also a stress resistance being acquired through this channel loss. Hog1 MAPK is transiently activated in yeast exposed to high, toxic levels of acetic acid. This Hog1 then phosphorylates the plasma membrane aquaglyceroporin, Fps1, a phosphorylation that results in Fps1 becoming ubiquitinated and endocytosed and then degraded in the vacuole. As Fps1 is the membrane channel that facilitates passive diffusional flux of undissociated acetic acid into the cell, this loss downregulates such influx in low-pH cultures, where acetic acid (pKa, 4.75) is substantially undissociated. Consistent with this downregulation of the acid entry generating resistance, sensitivity to acetic acid is seen with diverse mutational defects that abolish endocytic removal of Fps1 from the plasma membrane (loss of Hog1, loss of the soluble domains of Fps1, a T231A S537A double mutation of Fps1 that prevents its in vivo phosphorylation, or mutations generating a general loss of endocytosis of cell surface proteins [doa4Delta and end3Delta]). Remarkably, targetting of Fps1 for degradation may be the major requirement for an active Hog1 in acetic acid resistance, since Hog1 is largely dispensable for such resistance when the cells lack Fps1. Evidence is presented that in unstressed cells, Hog1 exists in physical association with the N-terminal cytosolic domain of Fps1.     

1,3‐propanediol binds deep inside the channel to inhibit water permeation through aquaporins

Aquaporins and aquaglyceroporins (AQPs) are membrane channel proteins responsible for transport of water and for transport of glycerol in addition to water across the cell membrane, respectively. They are expressed throughout the human body and also in other forms of life. Inhibitors of human AQPs have been sought for therapeutic treatment for various medical conditions including hypertension, refractory edema, neurotoxic brain edema, and so forth. Conducting all‐atom molecular dynamics simulations, we computed the binding affinity of acetazolamide to human AQP4 that agrees closely with in vitro experiments. Using this validated computational method, we found that 1,3‐propanediol (PDO) binds deep inside the AQP4 channel to inhibit that particular aquaporin efficaciously. Furthermore, we used the same method to compute the affinities of PDO binding to four other AQPs and one aquaglyceroporin whose atomic coordinates are available from the protein data bank (PDB). For bovine AQP1, human AQP2, AQP4, AQP5, and Plasmodium falciparum PfAQP whose structures were resolved with high resolution, we obtained definitive predictions on the PDO dissociation constant. For human AQP1 whose PDB coordinates are less accurate, we estimated the dissociation constant with a rather large error bar. Taking into account the fact that PDO is generally recognized as safe by the US FDA, we predict that PDO can be an effective diuretic which directly modulates water flow through the protein channels. It should be free from the serious side effects associated with other diuretics that change the hydro‐homeostasis indirectly by altering the osmotic gradients.     

Mucolipins: Intracellular TRPML1-3 channels

The mucolipin family of Transient Receptor Potential (TRPML) proteins is predicted to encode ion channels expressed in intracellular endosomes and lysosomes. Loss-of-function mutations of human TRPML1 cause type IV mucolipidosis (ML4), a childhood neurodegenerative disease. Meanwhile, gain-of-function mutations in the mouse TRPML3 result in the varitint-waddler (Va) phenotype with hearing and pigmentation defects. The broad spectrum phenotypes of ML4 and Va appear to result from certain aspects of endosomal/lysosomal dysfunction. Lysosomes, traditionally believed to be the terminal “recycling center” for biological “garbage”, are now known to play indispensable roles in intracellular signal transduction and membrane trafficking. Studies employing animal models and cell lines in which TRPML genes have been genetically disrupted or depleted have uncovered roles of TRPMLs in multiple cellular functions including membrane trafficking, signal transduction, and organellar ion homeostasis. Physiological assays of mammalian cell lines in which TRPMLs are heterologously overexpressed have revealed the channel properties of TRPMLs in mediating cation (Ca²⁺/Fe²⁺) efflux from endosomes and lysosomes in response to unidentified cellular cues. This review aims to summarize these recent advances in the TRPML field and to correlate the channel properties of endolysosomal TRPMLs with their biological functions. We will also discuss the potential cellular mechanisms by which TRPML deficiency leads to neurodegeneration.     

Fungal aquaporins: cellular functions and ecophysiological perspectives

Three aspects have to be taken into consideration when discussing cellular water and solute permeability of fungal cells: cell wall properties, membrane permeability, and transport through proteinaceous pores (the main focus of this review). Yet, characterized major intrinsic proteins (MIPs) can be grouped into three functional categories: (mainly) water transporting aquaporins, aquaglyceroporins that confer preferentially solute permeability (e.g., glycerol and ammonia), and bifunctional aquaglyceroporins that can facilitate efficient water and solute transfer. Two ancestor proteins, a water (orthodox aquaporin) and a solute facilitator (aquaglyceroporin), are supposed to give rise to today’s MIPs. Based on primary sequences of fungal MIPs, orthodox aquaporins/X-intrinsic proteins (XIPs) and FPS1-like/Yfl054-like/other aquaglyceroporins are supposed to be respective sister groups. However, at least within the fungal kingdom, no easy functional conclusion can be drawn from the phylogenetic position of a given protein within the MIP pedigree. In consequence, ecophysiological prediction of MIP relevance is not feasible without detailed functional analysis of the respective protein and expression studies. To illuminate the diverse MIP implications in fungal lifestyle, our current knowledge about protein function in two organisms, baker’s yeast and the Basidiomycotic Laccaria bicolor, an ectomycorrhizal model fungus, was exemplarily summarized in this review. MIP function has been investigated in such a depth in Saccharomyces cerevisiae that a system-wide view is possible. Yeast lifestyle, however, is special in many circumstances. Therefore, L. bicolor as filamentous Basidiomycete was added and allows insight into a very different way of life. Special emphasis was laid in this review onto ecophysiological interpretation of MIP function.     

Pseudoapoptosis induced by brief activation of ATP-gated P2X7 receptors

P2X7 receptors are ATP-gated ion channels primarily expressed on antigen-presenting immune cells where they play a role in the acute inflammatory response. These ion channels couple not only to influx of cations, including calcium, but also to rapid alterations in cell morphology (membrane blebbing, phosphatidylserine exposure, microvesicle shedding). These features resemble the extranuclear events associated with end stages of apoptosis but cell death does not occur if receptor activation is brief. Here we delineate two signaling pathways underlying these apoptotic-like processes. Loss of membrane asymmetry occurs within seconds, which directly triggers cytoskeletal disruption and zeiotic membrane blebbing; this is readily reversible and requires both calcium influx through P2X7 channels and mitochondrial calcium increase but is not associated with cytochrome c release. A slower, calcium-independent, ROCK-1-dependent cascade that does not involve rapid loss of membrane asymmetry but is associated with cytochrome c release is secondarily activated. The ROCK-1 pathway appears largely responsible for cell death, which occurs after prolonged stimulation of P2X7 receptors. We suggest that the former mechanism underlies the reversible pseudoapoptotic events induced by brief activation of P2X7 receptors.     

Domains of calcium channels as dissipative structures in a simulated neuron

The lateral distribution of open calcium channels of the fluid-mosaic membrane were investigated in a phenomenological model of a cylinder-shaped nerve cell. The local density of the channels changed due to their lateral diffusion and voltage- and calcium-dependent conformation transitions between open and closed states. Domains with an increased steady density of the open calcium channels were created as a result of action of intracellular calcium on its own channels, increasing the probability of the open state of the latter. These spatially nonuniform distributions of the channels are considered dissipative structures emerged in the active nonlinear medium at the expense of energy of active transport.Neirofiziologiya/Neurophysiology, Vol. 26, No. 2, pp. 99–107, March–April, 1994.     

Glycerol facilitator GlpF and the associated aquaporin family of channels

The aqua (glycero) porins conduct water (and glycerol) across cell membranes. The structure of these channels reveals a tripathic channel that supports a hydrophobic surface and, opposite to this, a line of eight hydrogen-bond acceptors and four hydrogen-bond donors. The eight carbonyls act as acceptors for water (or glycerol OH) molecules. The central water molecule in the channel is oriented to polarize hydrogen atoms outward from the center. This arrangement suggests how the structure prevents the potentially lethal conduction of protons across the membrane. The structure also suggests the mechanism behind the selectivity of aquaglyceroporins for glycerol, the basis for enantioselectivity among alditols, and the basis for the prevention of any leakage of the electrochemical gradient.     

The salting-in hypothesis of post-hypertonic lysis

The phenomenon of slow cooling cryoinjury has remained one of the primary areas of research in cryobiology since the early 1950s when it was first investigated thoroughly. Lovelock demonstrated that cell death from freezing and thawing was mainly due to exposure to hypertonic solutions and the subsequent dilution back to isotonic conditions. He suggested that the cell became permeable to sodium in hypertonic conditions leading to a loading of sodium during the hypertonic exposure, which caused the cell to swell past its elastic limit during resuspension in isotonic media (post-hypertonic lysis). This idea was pursued by Zade-Oppen, Farrant, and others who were able to show that the membrane became leaky to cations in hypertonic media but they could not provide any mechanism that would cause the cell to load up with sodium (other than an exchange of extracellular sodium for intracellular potassium, leaving the cell with the same cation concentration that it started out with). In the absence of such a mechanism, predicting post-hypertonic lysis from osmotic simulations cannot be done.A simplified model is proposed in which the intracellular milieu is composed of both KCl and a proteinaceous component that normally forms many salt bridges between amino acids with fixed charges. When the intracellular salt concentration increases, the proteins are “salted in” to solution (salt bridges are replaced with ionic interactions) thereby decreasing the intracellular cation concentration. Cation channels in the plasma membrane are opened by exposure to a high salt concentration (either inside or outside the membrane) allowing extracellular sodium to take the place of the intracellular potassium that is interacting with anionic groups on the proteins. Dilution of the external medium (which also occurs during melting) causes water to move into the cells, diluting the cytoplasm. The proteins are then “salted out” of solution and release the salt back to free ions in solution. The cell has an excess of intracellular ions and may swell past its elastic limit due to water influx. A simulation engine is developed based on the model and compared to results in the literature for freeze–thaw injury in human red blood cells.     

Coupling of metabolic, second messenger pathways and insulin granule dynamics in pancreatic beta-cells: a computational analysis

Insulin secretory responses to nutrient stimuli and hormonal modulators in pancreatic beta-cells are controlled by a variety of secondary messengers. We have analyzed numerous mechanisms responsible for regulated exocytosis in these cells and present an integrated mathematical model of cytosolic Ca²⁺, cAMP and granule dynamics. The insulin-containing granules in the beta-cell were divided into four classes: a large “reserve” granule pool, a smaller pool of the morphologically docked granules that is chemically ‘primed’ for release or the “readily releasable pool”, and a pool of “restless newcomer granules” that undergoes preferential exocytosis. The model incorporates glucose and other aspects of metabolism, the cAMP amplifying pathway, insulin granule dynamics and the exocyst concept for granule binding. The values of most of the model parameters were inferred from available experimental data. The model can generate both the fast first phase and slow biphasic insulin secretion found experimentally in response to a step increase of membrane potential or of glucose. The numerical simulations have also reproduced a variety of experimental conditions, such as periodic stimulation by high K⁺ and the potentiation induced in islets by pre-incubation with cAMP pathway activators. The explicit incorporation of Ca²⁺ channels, Ca²⁺ and cAMP dynamics allows the model to be further connected to current models for calcium and metabolic dynamics and provides an interpretation of the roles of the triggering and amplifying pathways of glucose-stimulated insulin secretion. The model may be important in the identification of pharmacological targets for improving insulin secretion in type 2 diabetes.     

Aquaporins in yeasts and filamentous fungi

Recently, genome sequences from different fungi have become available. This information reveals that yeasts and filamentous fungi possess up to five aquaporins. Functional analyses have mainly been performed in budding yeast, Saccharomyces cerevisiae, which has two orthodox aquaporins and two aquaglyceroporins. Whereas Aqy1 is a spore-specific water channel, Aqy2 is only expressed in proliferating cells and controlled by osmotic signals. Fungal aquaglyceroporins often have long, poorly conserved terminal extensions and differ in the otherwise highly conserved NPA motifs, being NPX and NXA respectively. Three subgroups can be distinguished. Fps1-like proteins seem to be restricted to yeasts. Fps1, the osmogated glycerol export channel in S. cerevisiae, plays a central role in osmoregulation and determination of intracellular glycerol levels. Sequences important for gating have been identified within its termini. Another type of aquaglyceroporin, resembling S. cerevisiae Yfl054, has a long N-terminal extension and its physiological role is currently unknown. The third group of aquaglyceroporins, only found in filamentous fungi, have extensions of variable size. Taken together, yeasts and filamentous fungi are a fruitful resource to study the function, evolution, role and regulation of aquaporins, and the possibility to compare orthologous sequences from a large number of different organisms facilitates functional and structural studies.     

Biochemical and physiological evidence that calmodulin is involved in the taste response of the sugar receptor cells of the blowfly, Phormia regina

The gustatory system is essential for almost all animals. However, the signal transduction mechanisms have not yet been fully elucidated. We isolated labellar chemosensilla from blowfly, Phormia regina, and purified calcium binding proteins from the water soluble fraction. The most abundant calcium-binding protein was calmodulin. To investigate the role of calmodulin in taste transduction, electrophysiological responses were recorded with the calmodulin inhibitor, W-7. When we stimulated the labellar chemosensillum with sucrose plus W-7, a dose-dependent decrease of impulse frequency was observed when the concentration was <50 microM. In addition, when W-7 at 50 microM or higher concentration was added, an initial short-term impulse generation from the sugar receptor cell was observed, but this was followed by a silent period. When the sensillum was stimulated with W-7 plus a membrane-permeable cGMP analog, dibtyryl-cGMP or 8-bromo-cGMP, impulses of the sugar receptor cell were induced but the frequency was decreased. By the sidewall-recording method, we observed that the receptor potential induced by sucrose stimulation was decreased by W-7 in the sugar receptor cell, and corresponded with a disappearance of impulses. These data strongly suggest that the cGMP-gated channel generating receptor potential in the sugar receptor cell requires calmodulin for its gating.     

Receptor-Operated Calcium Influx Mediated by Protein Tyrosine Kinase Pathways

Abstract

Calcium influx from the extracellular space elicited by activation of heterotrimeric G protein-coupled and heptahelical receptors plays a critical role in transmembrane signal transduction in a wide variety of cell systems. In nonexcitable cells, the precise voltage-independent mechanism by which calcium enters the cell remains unknown. Multiple mechanisms appear to be operating in different cell types (1–3): 1. G protein-operated calcium influx, 2. second messenger-operated calcium influx, 3. capacitative calcium influx, and 4. phosphorylation of calcium channels. Receptor-operated calcium channels have a fundamental role in stimulus-secretion coupling in many different cells, but these channels remain to be purified and cloned. This review proposes that receptor-operated calcium influx is mediated by protein tyrosine kinase pathways. The function of protein tyrosine kinase pathways and their interactions with other receptor-operated calcium influx mechanisms are described.     

Single-Channel Current Through Nicotinic Receptor Produced by Closure of Binding Site C-Loop

We investigated the initial coupling of agonist binding to channel gating of the nicotinic acetylcholine receptor using targeted molecular-dynamics (TMD) simulation. After TMD simulation to accelerate closure of the C-loops at the agonist binding sites, the region of the pore that passes through the cell membrane expands. To determine whether the structural changes in the pore result in ion conduction, we used a coarse-grained ion conduction simulator, Biology Boltzmann transport Monte Carlo, and applied it to two structural frames taken before and after TMD simulation. The structural model before TMD simulation represents the channel in the proposed “resting” state, whereas the model after TMD simulation represents the channel in the proposed “active” state. Under external voltage biases, the channel in the “active” state was permeable to cations. Our simulated ion conductance approaches that obtained experimentally and recapitulates several functional properties characteristic of the nicotinic acetylcholine receptor. Thus, closure of the C-loop triggers a structural change in the channel sufficient to account for the open channel current. This approach of applying Biology Boltzmann transport Monte Carlo simulation can be used to further investigate the binding to gating transduction mechanism and the structural bases for ion selection and translocation.