Molecular Determinants of Agonist Selectivity in Glutamate-Gated Chloride Channels Which Likely Explain the Agonist Selectivity of the Vertebrate Glycine and GABAA-ρ Receptors

Orthologous Cys-loop glutamate-gated chloride channels (GluClR’s) have been cloned and described electrophysiologically and pharmacologically in arthropods and nematodes (both members of the invertebrate ecdysozoan superphylum). Recently, GluClR’s from Aplysia californica (a mollusc from the lophotrochozoan superphylum) have been cloned and similarly studied. In spite of sharing a common function, the ecdysozoan and lophotrochozoan receptors have been shown by phylogenetic analyses to have evolved independently. The recent crystallization of the GluClR from C. elegans revealed the binding pocket of the nematode receptor. An alignment of the protein sequences of the nematode and molluscan GluClRs showed that the Aplysia receptor does not contain all of the residues defining the binding mode of the ecdysozoan receptor. That the two receptors have slightly different binding modes is not surprising since earlier electrophysiological and pharmacological experiments had suggested that they were differentially responsive to certain agonists. Knowledge of the structure of the C. elegans GluClR has permitted us to generate a homology model of the binding pocket of the Aplysia receptor. We have analyzed the differences between the two binding modes and evaluated the relative significance of their non-common residues. We have compared the GluClRs electrophysiologically and pharmacologically and we have used site-directed mutagenesis on both receptor types to test predictions made from the model. Finally, we propose an explanation derived from the model for why the nematode receptors are gated only by glutamate, whereas the molluscan receptors can also be activated by β-alanine, GABA and taurine. Like the Aplysia receptor, the vertebrate glycine and GABA_A-ρ receptors also respond to these other agonists. An alignment of the sequences of the molluscan and vertebrate receptors shows that the reasons we have given for the ability of the other agonists to activate the Aplysia receptor also explain the agonist profile seen in the glycine and GABA_A-ρ receptors.     

Changes in Cationic Selectivity of the Nicotinic Channel at the Rat Ganglionic Synapse: A Role for Chloride Ions?

The permeability of the nicotinic channel (nAChR) at the ganglionic synapse has been examined, in the intact rat superior cervical ganglion in vitro, by fitting the Goldman current equation to the synaptic current (EPSC) I-V relationship. Subsynaptic nAChRs, activated by neurally-released acetylcholine (ACh), were thus analyzed in an intact environment as natively expressed by the mature sympathetic neuron. Postsynaptic neuron hyperpolarization (from -40 to -90 mV) resulted in a change of the synaptic potassium/sodium permeability ratio (P(K)/P(Na)) from 1.40 to 0.92, corresponding to a reversible shift of the apparent acetylcholine equilibrium potential, E(ACh), by about +10 mV. The effect was accompanied by a decrease of the peak synaptic conductance (g(syn)) and of the EPSC decay time constant. Reduction of [Cl(-)](o) to 18 mM resulted in a change of P(K)/P(Na) from 1.57 (control) to 2.26, associated with a reversible shift of E(ACh) by about -10 mV. Application of 200 nM αBgTx evoked P(K)/P(Na) and g(syn) modifications similar to those observed in reduced [Cl(-)](o). The two treatments were overlapping and complementary, as if the same site/mechanism were involved. The difference current before and after chloride reduction or toxin application exhibited a strongly positive equilibrium potential, which could not be explained by the block of a calcium component of the EPSC. Observations under current-clamp conditions suggest that the driving force modification of the EPSC due to P(K)/P(Na) changes represent an additional powerful integrative mechanism of neuron behavior. A possible role for chloride ions is suggested: the nAChR selectivity was actually reduced by increased chloride gradient (membrane hyperpolarization), while it was increased, moving towards a channel preferentially permeable for potassium, when the chloride gradient was reduced.     

α-AP-2 Directs Myosin VI-dependent Endocytosis of Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channels in the Intestine

The actin motor myosin VI regulates endocytosis of cystic fibrosis transmembrane conductance regulator (CFTR) in the intestine, but the endocytic adaptor linking CFTR to myosin VI is unknown. Dab2 (Disabled 2) is the binding partner for myosin VI, clathrin, and α-AP-2 and directs endocytosis of low density lipoprotein receptor family members by recognizing a phosphotyrosine-binding domain. However, CFTR does not possess a phosphotyrosine-binding domain. We examined whether α-AP-2 and/or Dab2 were binding partners for CFTR and the role of myosin VI in localizing endocytic adaptors in the intestine. CFTR co-localized with α-AP-2, Dab2, and myosin VI and was identified in a complex with all three endocytic proteins in the intestine. Apical CFTR was increased in the intestines of Dab-2 KO mice, suggesting its involvement in regulating surface CFTR. Glutathione S-transferase pulldown assays revealed binding of CFTR to α-AP-2 (but not Dab2) in the intestine, whereas Dab-2 interacted with α-AP-2. siRNA silencing of α-AP-2 in cells significantly reduced CFTR endocytosis, further supporting α-AP-2 as the direct binding partner for CFTR. α-AP-2 and Dab2 localized to the terminal web regions of enterocytes, but Dab2 accumulated in this location in Snell''s Waltzer myosin VI^(sv/sv) intestine. Ultrastructural examination revealed that the accumulation of Dab2 correlated with prominent involution and the loss of normal positioning of the intermicrovillar membranes that resulted in expansion of the terminal web region in myosin VI^(sv/sv) enterocytes. The findings support α-AP-2 in directing myosin VI-dependent endocytosis of CFTR and a requirement for myosin VI in membrane invagination and coated pit formation in enterocytes.     

Phase Behavior of the ι-Carrageenan/Maltodextrin/Water System at Different Potassium Chloride Concentrations and Temperatures

Equilibrium phase diagrams of the ι-carrageenan/maltodextrin/water system have been established at potassium chloride (KCl) concentrations of 0.1, 0.2, and 0.3 M and 80, 85 and 90°C. All pseudo-binary phase diagrams of ι-carrageenan/maltodextrin mixtures suggested classic segregative phase separation. The binodal was heavily skewed toward the maltodextrin axis. The high asymmetry of the ι-carrageenan/maltodextrin/water phase diagram determined by the phase-volume-ratio method was consistent with the compositional analysis of phase-separated ι-carrageenan/maltodextrin samples and can be explained in terms of the Flory–Huggins interaction parameter, reflecting a higher water-binding ability of the charged ι-carrageenan than neutral maltodextrin. Increasing the concentration of ι-carrageenan-gel-promoting KCl from 0.1 to 0.3 M at 80°C enlarged the two-phase domain, whereas increasing temperature from 80 to 90°C at 0.3 M KCl enhanced biopolymer compatibility. The effects of salt concentration and temperature have been related to the differences in the Flory–Huggins interaction parameters of the two biopolymers with water as well as the helix formation of ι-carrageenan in the presence of KCl through the changes in the slopes of tie lines of phase-separated samples.

Intracellular GABA-Activated In→Out Permeation of Chloride Across the Deiters' Neuron Membrane: Modulation by Phosphorylating Activities

The modulation of intracellular GABA activated ³⁶Cl^– inout permeation across single Deiters' neuron membranes has been studied in a microchamber system. Addition of Mg²⁺/ATP on the membrane cytoplasmic side reduces strongly the GABA effect as does ATP alone. However, the greatest inhibition of the GABA effect is given by the addition of Mg²⁺ to the intracellular side buffer: a complete block of the stimulation by GABA of ³⁶Cl^– inout permeation. This is interpreted as due to the presence in this case of a constant concentration of exogenous Mg²⁺ acting together with endogenous ATP in the small cytoplasmic layer on the membrane inner side. The addition of ADP to Mg²⁺/ATP increases the inhibitory effect of the latter. This is presumably due to an extra increase of ATP, locally under the membrane, due to phosphorylation of ADP by endogenous phosphocreatine. Overall, the data confirm that phosphorylating conditions impair the intracellular GABA action on ³⁶Cl^– inout permeation.     

Negligible Effect of Sodium Chloride on the Development and Function of TGF-β-Induced CD4+ Foxp3+ Regulatory T Cells

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Chloride in airway smooth muscle: the ignored anion no longer?

This Perspectives accompanies an Editorial Focus that summarizes new developments concerning the role of chloride in airway smooth muscle physiology. We provide several observations and mechanistic insights to reconcile recent experimental evidence with existing paradigms concerning chloride channel-mediated effects on airway smooth muscle tone. In addition, we highlight the potentially complex and dynamic nature that chloride currents and membrane potential have on calcium handling and airway smooth muscle contractility.     

Rescue of Dysfunctional ΔF508-CFTR Chloride Channel Activity by IBMX

Nucleotide-dependent gating of ΔF508-CFTR was evaluated in membrane patches excised from HEK 293 and mouse L-cells and compared to observations on wt-CFTR channels recorded in the same expression systems. ΔF508-CFTR exhibited PKA activated, ATP-dependent channel gating. When compared to wt-CFTR, the K _m for ATP was increased by ninefold (260 μm vs. 28 μm) and maximal open probability (P _o ) was reduced by 49% (0.21 ± 0.06 vs. 0.41 ± 0.02). Additionally, in the absence of PKA, ΔF508-CFTR inactivated over a 1 to 5 min period whereas wt-CFTR remained active. Time-dependent inactivation could be mimicked in wt-CFTR by the intermittent absence of ATP in the cytosolic solution. The effects of 3-isobutyl-1-methyl xanthine (IBMX), a compound reported to stimulate ΔF508-CFTR, were evaluated on wt- and ΔF508-CFTR channels. At concentrations up to 5 mm, IBMX caused a concentration dependent reduction in the observed single channel amplitude (i) of wt-CFTR (maximal observed reduction 35 ± 3%). However, IBMX failed to significantly alter total patch current because of a concomitant 30% increase in P _o . The effects of IBMX on ΔF508-CFTR were similar to effects on wt-CFTR in that i was reduced and P _o was increased by similar magnitudes. Additionally, ΔF508-CFTR channel inactivation was dramatically slowed by IBMX. These results suggest that IBMX interacts with the ATP-bound open state of CFTR to introduce a short-lived nonconducting state which prolongs burst duration and reduces apparent single channel amplitude. A secondary effect observed in ΔF508-CFTR, which may result from this interaction, is a prolongation of the activated state. In light of previously proposed linear kinetic models of CFTR gating, these results suggest that IBMX traps CFTR in an ATP-bound state which may preclude inactivation of ΔF508-CFTR. Received: 5 February 1999/Revised: 25 March 1999     

Effects of Cadmium Chloride and Sodium Selenite Alone or in Combination on the Liver of Male Sprague–Dawley Rats Assessed by Different Assays

This study assessed the impact of either cadmium chloride (Cd) or sodium selenite (Se) alone or in combination on male Sprague–Dawley rats. For this purpose, body and liver weights, comet and TUNEL assays, histological analysis and levels of lipid peroxidation and antioxidants in liver were determined in four groups of male Sprague–Dawley rats. The rats were given subcutaneous doses of 1 mg/kg body weight (BW) of either normal saline (control = Ct) or Cd or Se or Cd plus Se (Cd + Se) on alternate days for 4 weeks. The Cd group showed increased DNA damage, apoptosis and hepatic levels of lipid peroxidation and altered histology. Conversely, the antioxidant levels in this group were decreased as compared with the control group. The Se group also showed DNA damage, apoptosis and altered histology and reduced catalase activity, but it was less severe than the Cd group. In the Cd + Se group, ameliorating effects of Se on Cd-induced changes were observed. While the Se was able to curtail the toxic effect of Cd, the Cd or Se alone were genotoxic and cytotoxic for rats receiving a high pharmacological but non-fatal dose of 1 mg/kg BW.     

Probiotics as a Complementary Therapy in the Model of Cadmium Chloride Toxicity: Crosstalk of β-Catenin,BDNF, and StAR Signaling Pathways

Cadmium chloride (CdCl₂) is a ubiquitous environmental toxicant that causes a variety of disturbances in biological systems, including brain dysfunction and testicular tissue degeneration. On the other hand, it is supposed that beneficial properties of probiotic bacteria (Lactobacillus and Acidobacillus) are related to their capacity to adhere or bind different targets, thus leading to improved intestinal microbial balance and other benefits to the host. Bearing aforementioned in mind, the present study was undertaken to investigate the protective effect of probiotic supplementation against cadmium chloride-induced brain and testis toxicity in mice model. Animals received Lactobacillus and Acidobacillus either alone or added to folic acid for 1 week before CdCl2 intoxication in a dose of 20 mg/kg BW followed by probiotics (5?×?10⁹⁾ and/or folic acid (12 mg/kg) treatment for 3 weeks. The levels of malondialdehyde (MDA), butyrl choline esterase (BCHE), reduced glutathione (GSH), and total superoxide dismutase (SOD) activities were investigated. Finally, cadmium neurotoxicity was determined by estimating the gene expression of β-catenin and brain-derived neurotrophic factor (BDNF), as well as estimating the alterations in testicular function by determining acid phosphatase level in addition to steroidogenic acute regulatory protein (StAR) and 17-hydroxy steroid dehydrogenase (17-β HSD) gene expression. Based on our results, we can conclude that exposure of mice to cadmium chloride resulted in a significant elevation in MDA, BCHE levels accompanied with a significant reduction in GSH and SOD activities compared to the control value. CdCl₂ also downregulated the gene expression of β-catenin and BDNF, as well as acid phosphatase level, in addition to StAR and 17-β HSD gene expression. These deviated parameters were significantly modulated in the co-treated animals with probiotics compared with the cadmium-treated group. In conclusion, Lactobacillus and folic acid in a mixture with cadmium acted beneficially to an organism, increasing the cadmium excretion in feces, and consequently increasing β-catenin and BDNF in brain tissue and StAR and 17-β HSD in testis and improving their functions. Histoarchitecture analysis confirmed these results.     

Effect of cortisone on the developmental pattern of the neutral and the acid β-galactosidases of the small intestine of the rat

1. The developmental pattern and effect of cortisone on acid beta-galactosidase and neutral beta-galactosidase were studied in postnatal rats by a recently proposed method for their independent determination. 2. After birth the acid beta-galactosidase activity increases in the ileum, whereas it decreases slightly in the jejunum. On day 16 after birth the activity in the ileum decreases and in 20-day-old rats activity in both parts of the intestine decreases to adult values. In suckling animals the activity in the ileum exceeds the jejunal activity severalfold and in adult animals the activity in the jejunum is slightly higher than that in the ileum. 3. Neutral beta-galactosidase activity is high after birth and decreases in both jejunum and ileum after day 20 after birth. In 12-20-day-old rats activity in both parts is essentially the same, but in adult animals jejunal activity exceeds ileal activity four-to five-fold. 4. Cortisone (0.5, 2.0 or 5.0mg/100g body wt. daily for 4 days) does not influence the activity of either enzyme in 60-day-old rats. Acid beta-galactosidase activity is decreased after cortisone treatment in 8-, 12-, 16-and 18-day-old rats, with sensitivity to cortisone increasing with the approach of weaning. No effect of cortisone on acid beta-galactosidase is seen in 8-day-old rats. Neutral beta-galactosidase activity is increased in the ileum of 8-, 12-, 16- and 18-day old rats, but only in the jejunum of 8-and 12-day-old rats.     

The TMEM16 Protein Family: A New Class of Chloride Channels?

Cl⁻ channels play important roles in many physiological processes, including transepithelial ion absorption and secretion, smooth and skeletal muscle contraction, neuronal excitability, sensory perception, and cell volume regulation. The molecular identity of many types of Cl⁻ channels is still unknown. Recently, three research groups have arrived independently at the identification of TMEM16A (also known as anoctamin-1) as a membrane protein strongly related to the activity of Ca²⁺-activated Cl⁻ channels (CaCCs). Site-specific mutagenesis of TMEM16A alters the properties of the channels, thus suggesting that TMEM16A forms, at least in part, the CaCC. TMEM16A is a member of a family that includes nine other membrane proteins. All TMEM16 proteins have a similar structure, with eight putative transmembrane domains and cytosolic amino- and carboxy-termini. TMEM16B expression also evokes the appearance of CaCCs, but with biophysical characteristics (voltage dependence, unitary conductance) different from those associated to TMEM16A. The roles of the other TMEM16 proteins are still unknown. The study of TMEM16 proteins may lead to identification of novel molecular mechanisms underlying ion transport and channel gating by voltage and Ca²⁺.     

Cytoprotective Effect of Estrogen on Ammonium Chloride–Treated C6-Glioma Cells

The potential cytoprotective effects of estrogen in the brain are of special interest in aging, neurodegenerative diseases, exposure to toxins, and trauma. Estrogen effects on neurons have been widely explored, but less is known about estrogen effects on glia. Glial cells are primary targets of ammonia toxicity, which arises from liver disease or failure (such as from cirrhosis in alcoholics), urea cycle disorders, or inborn errors of metabolism. We examined the ability of estrogen to protect glial cells from ammonium chloride toxicity using an in vitro model system. C6-glioma cells in later passage have many astrocytic characteristics and provided a convenient and well established model system for this work. When C6-glioma cells were exposed to 15 mM ammonium chloride, we observed major cell death (only 32% cell survival relative to control) within 72 h. Pretreatment with 17beta-estradiol (10 microM) significantly protected C6-glioma cells from ammonia toxicity (99% cell survival relative to control). In addition to enhancing the viability of C6-glioma cells against ammonia challenge, estrogen pretreatment was also found to protect mitochondrial function as assayed using the MTT reduction assay. Mitochondrial function was reduced to 39% of control levels in ammonia-challenged cultures and was mostly protected by estrogen (72% of control levels). The findings are potentially relevant for the development of therapeutic strategies to protect glial cells against ammonia toxicity resulting from hepatic failure or other causes.     

Association of the subunits of the calcium-independent receptor of α-latrotoxin

CIRL-1 also called latrophilin 1 or CL belongs to the family of adhesion G protein-coupled receptors (GPCRs). As all members of adhesion GPSR family CIRL-1 consists of two heterologous subunits, extracellular hydrophilic p120 and heptahelical membrane protein p85. Both CIRL-1 subunits are encoded by one gene but as a result of intracellular proteolysis of precursor, mature receptor has two-subunit structure. It was also shown that a minor portion of the CIRL-1 receptor complexes dissociates, producing the soluble receptor ectodomain, and this dissociation is due to the second cleavage at the site between the site of primary proteolysis and the first transmembrane domain. Recently model of independent localization p120 and p85 on the cell surface was proposed. In this article we evaluated the amount of p120-p85 complex still presented on the cellular membrane and confirmed that on cell surface major amount of mature CIRL-1 presented as a p120-p85 subunit complex.     

Phylogeography of the harlequin beetle-riding pseudoscorpion and the rise of the Isthmus of Panamá

Molecular and geological evidence indicates that the emergence of the Isthmus of Panamá influenced the historical biogeography of the Neotropics in a complex, staggered manner dating back at least 9 Myr bp. To assess the influence of Isthmus formation on the biogeography of the harlequin beetle-riding pseudoscorpion, Cordylochernes scorpioides, we analysed mitochondrial COI sequence data from 71 individuals from 13 locations in Panamá and northern South America. Parsimony and likelihood-based phylogenies identified deep divergence between South American and Panamanian clades. In contrast to low haplotype diversity in South America, the Panamanian Cordylochernes clade is comprised of three highly divergent lineages: one clade consisting predominantly of individuals from central Panamá (PAN A), and two sister clades (PAN B1 and PAN B2) of western Panamanian pseudoscorpions. Breeding experiments demonstrated a strictly maternal mode of inheritance, indicating that our analyses were not confounded by nuclear-mitochondrial pseudogenes. Haplotype diversity is striking in western Atlantic Panamá, where all three Panamanian clades can occur in a single host tree. This sympatry points to the existence of a cryptic species hybrid zone in western Panamá, a conclusion supported by interclade crosses and coalescence-based migration rates. Molecular clock estimates yield a divergence time of approximately 3 Myr between the central and western Panamanian clades. Taken together, these results are consistent with a recent model in which a transitory proto-Isthmus enabled an early wave of colonization out of South America at the close of the Miocene, followed by sea level rise, inundation of the terrestrial corridor and then a second wave of colonization that occurred when the Isthmus was completed approximately 3 Myr bp.     

Did the evolution of the phytoplankton fuel the diversification of the marine biosphere?

We discuss the possible links between the fossil record of marine biodiversity, nutrient availability and primary productivity. The parallelism of the fossil records of marine phytoplankton and faunal biodiversity implicates the quantity (primary productivity) and quality (stoichiometry) of phytoplankton as being critical to the diversification of the marine biosphere through the Phanerozoic. The relatively subdued marine biodiversity of the Palaeozoic corresponds to a time of relatively low macronutrient availability and poor food quality of the phytoplankton as opposed to the diversification of the Modern Fauna through the Mesozoic–Cenozoic. Increasing nutrient runoff to the oceans through the Phanerozoic resulted from orogeny, the emplacement of Large Igneous Provinces (LIPs), the evolution of deep-rooting forests and the appearance of more easily decomposable terrestrial organic matter that enhanced weathering. Positive feedback by bioturbation of an expanding benthos played a critical role in evolving biogeochemical cycles by linking the oxidation of dead organic matter and the recycling of nutrients back to the water column where they could be re-utilized. We assess our conclusions against a recently published biogeochemical model for geological time-scales. Major peaks of marine diversity often occur near rising or peak fluxes of silica, phosphorus and dissolved reactive oceanic phosphorus; either major or minor ⁸⁷Sr/⁸⁶Sr peaks; and frequently in the vicinity of major (Circum-Atlantic Magmatic Province) and minor volcanic events, some of which are associated with Oceanic Anoxic Events. These processes appear to be scale-dependent in that they lie on a continuum between biodiversification on macroevolutionary scales of geological time and mass extinction.     

Imperfect Batesian mimicry—the effects of the frequency and the distastefulness of the model

Batesian mimicry is the resemblance between unpalatable models and palatable mimics. The widely accepted idea is that the frequency and the unprofitability of the model are crucial for the introduction of a Batesian mimic into the prey population. However, experimental evidence is limited and furthermore, previous studies have considered mainly perfect mimicry (automimicry). We investigated imperfect Batesian mimicry by varying the frequency of an aposematic model at two levels of distastefulness. The predator encountered prey in a random order, one prey item at a time. The prey were thus presented realistically in a sequential way. Great tits (Parus major) were used as predators. This experiment, with a novel signal, supports the idea that Batesian mimics gain most when the models outnumber them. The mortalities of the mimics as well as the models were significantly dependent on the frequency of the model. Both prey types survived better the fewer mimics there were confusing the predator. There were also indications that the degree of distastefulness of the model had an effect on the survival of the Batesian mimic: the models survived significantly better the more distasteful they were. The experiment supports the most classical predictions in the theories of the origin and maintenance of Batesian mimicry.