The Domain Interface of the Human Glutamate Transporter EAAT1 Mediates Chloride Permeation

AbstractThe concentration of glutamate within the glutamatergic synapse is tightly regulated by the excitatory amino-acid transporters (EAATs). In addition to their primary role of clearing extracellular glutamate, the EAATs also possess a thermodynamically uncoupled Cl− conductance. Several crystal structures of an archaeal EAAT homolog, GltPh, at different stages of the transport cycle have been solved. In a recent structure, an aqueous cavity located at the interface of the transport and trimerization domains has been identified. This cavity is lined by polar residues, several of which have been implicated in Cl− permeation. We hypothesize that this cavity opens during the transport cycle to form the Cl− channel. Residues lining this cavity in EAAT1, including Ser-366, Leu-369, Phe-373, Arg-388, Pro-392, and Thr-396, were mutated to small hydrophobic residues. Wild-type and mutant transporters were expressed in Xenopus laevis oocytes and two-electrode voltage-clamp electrophysiology, and radiolabeled substrate uptake was used to investigate function. Significant alterations in substrate-activated Cl− conductance were observed for several mutant transporters. These alterations support the hypothesis that this aqueous cavity at the interface of the transport and trimerization domains is a partially formed Cl− channel, which opens to form a pore through which Cl− ions pass. This study enhances our understanding as to how glutamate transporters function as both amino-acid transporters and Cl− channels

Constitutive Ion Fluxes and Substrate Binding Domains of Human Glutamate Transporters

Application of L-glutamate activates ionic currents in voltage-clamped Xenopus oocytes expressing cloned human excitatory amino acid transporters (EAATs). However, even in the absence of L-glutamate, the membrane conductance of oocytes expressing EAAT1 was significantly increased relative to oocytes expressing EAAT2 or control oocytes. Whereas transport mediated by EAAT2 is blocked by the non-transported competitive glutamate analog kainate (K = 14 μM), EAAT1 is relatively insensitive (K \u3e 3 mM). Substitution of a block of 76 residues from EAAT2 into EAAT1, in which 18 residues varied from EAAT1, conferred high affinity kainate binding to EAAT1, and application of kainate to oocytes expressing the chimeric transporter blocked a pre-existing monovalent cation conductance that displayed a permeability sequence K \u3e Na \u3e Li choline. The results identify a structural domain of glutamate transporters that influences kainate binding and demonstrate the presence of a constitutive ion-selective pore in the transporter

Glycine transport inhibitors for the treatment of pain.

Opioids, local anesthetics, anticonvulsant drugs, antidepressants, and non-steroidal anti-inflammatory drugs (NSAIDs) are used to provide pain relief but they do not provide adequate pain relief in a large proportion of chronic pain patients and are often associated with unacceptable side effects. Inhibitory glycinergic neurotransmission is impaired in chronic pain states, and this provides a novel target for drug development. Inhibitors of the glycine transporter 2 (GlyT2) enhance inhibitory neurotransmission and show particular promise for the treatment of neuropathic pain. N-arachidonyl-glycine (NAGly) is an endogenous lipid that inhibits glycine transport by GlyT2 and also shows potential as an analgesic, which may be further exploited in drug development. In this review we discuss the role of glycine neurotransmission in chronic pain and future prospects for the use of glycine transport inhibitors in the treatment of pain.NHMRC Grant: 104596

Directed Mutagenesis in Structure Activity Studies of Neurotransmitter Transporters

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Identification of a 3rd Na+ binding site of the glycine transporter, GlyT2

The Na+/Cl- dependent glycine transporters GlyT1 and GlyT2 regulate synaptic glycine concentrations. Glycine transport by GlyT2 is coupled to the co-transport of three Na+ ions, whereas transport by GlyT1 is coupled to the co-transport of only two Na+ ions. These differences in ion-flux coupling determine their respective concentrating capacities and have a direct bearing on their functional roles in synaptic transmission. The crystal structures of the closely related bacterial Na+-dependent leucine transporter, LeuTAa, and the Drosophila dopamine transporter, dDAT, have allowed prediction of two Na+ binding sites in GlyT2, but the physical location of the third Na+ site in GlyT2 is unknown. A bacterial betaine transporter, BetP, has also been crystallized and shows structural similarity to LeuTAa. Although betaine transport by BetP is coupled to the co-transport of two Na+ ions, the first Na+ site is not conserved between BetP and LeuTAa, the so called Na1' site. We hypothesized that the third Na+ binding site (Na3 site) of GlyT2 corresponds to the BetP Na1' binding site. To identify the Na3 binding site of GlyT2, we performed molecular dynamics (MD) simulations. Surprisingly, a Na+ placed at the location consistent with the Na1' site of BetP spontaneously dissociated from its initial location and bound instead to a novel Na3 site. Using a combination of MD simulations of a comparative model of GlyT2 together with an analysis of the functional properties of wild type and mutant GlyTs we have identified an electrostatically favorable novel third Na+ binding site in GlyT2 formed by Trp263 and Met276 in TM3, Ala481 in TM6 and Glu648 in TM10

Galaxy Zoo: Disentangling the Environmental Dependence of Morphology and Colour

We analyze the environmental dependence of galaxy morphology and colour with two-point clustering statistics, using data from the Galaxy Zoo, the largest sample of visually classified morphologies yet compiled, extracted from the Sloan Digital Sky Survey. We present two-point correlation functions of spiral and early-type galaxies, and we quantify the correlation between morphology and environment with marked correlation functions. These yield clear and precise environmental trends across a wide range of scales, analogous to similar measurements with galaxy colours, indicating that the Galaxy Zoo classifications themselves are very precise. We measure morphology marked correlation functions at fixed colour and find that they are relatively weak, with the only residual correlation being that of red galaxies at small scales, indicating a morphology gradient within haloes for red galaxies. At fixed morphology, we find that the environmental dependence of colour remains strong, and these correlations remain for fixed morphology \textit{and} luminosity. An implication of this is that much of the morphology--density relation is due to the relation between colour and density. Our results also have implications for galaxy evolution: the morphological transformation of galaxies is usually accompanied by a colour transformation, but not necessarily vice versa. A spiral galaxy may move onto the red sequence of the colour-magnitude diagram without quickly becoming an early-type. We analyze the significant population of red spiral galaxies, and present evidence that they tend to be located in moderately dense environments and are often satellite galaxies in the outskirts of haloes. Finally, we combine our results to argue that central and satellite galaxies tend to follow different evolutionary paths.Comment: 19 pages, 18 figures. Accepted for publication in MNRA

Genetic Variation in the Base Excision Repair Pathway, Environmental Risk Factors, and Colorectal Adenoma Risk

Cigarette smoking, high alcohol intake, and low dietary folate levels are risk factors for colorectal adenomas. Oxidative damage caused by these three factors can be repaired through the base excision repair pathway (BER). We hypothesized that genetic variation in BER might modify colorectal adenoma risk. In a sigmoidoscopy-based study, we examined associations between 182 haplotype tagging SNPs in 14 BER genes, and colorectal adenoma risk, and examined their potential role as modifiers of the effect cigarette smoking, alcohol intake, and dietary folate levels. Among all individuals, no statistically significant associations between BER SNPs and adenoma risk persisted after correction for multiple comparisons. However, among Asian-Pacific Islanders we observed two SNPs in FEN1 and one in NTHL1, and among African-Americans one SNP in APEX1 that were associated with colorectal adenoma risk. Significant associations were also observed between SNPs in the NEIL2 gene and rectal adenoma risk. Three SNPS modified the effect of smoking (MUTYH interaction p = 0.002; OGG1 interaction p = 0.013); FEN1 interaction p = 0.013)), one SNP in LIG3 modified the effect of alcohol consumption (interaction p = 0.024) and two SNPs in LIG3 modified the effect of dietary folate (interaction p = 0.001 and p = 0.08) on colorectal adenoma risk. These findings support a role for genetic variants in the BER pathway as potential modifiers of colorectal adenoma risk. Our findings strengthen the role of oxidative damage induced by key lifestyle and dietary risk factors in colorectal adenoma formation

A Star Catalog for the Open Cluster NGC188

We present new BVRI broad-band photometry for the old open cluster NGC188 based upon analysis of 299 CCD images either obtained by us, donated by colleagues, or retrieved from public archives. We compare our results on a star-by-star basis with data from eleven previous photometric investigations of the cluster. We homogenize and merge the data from all the photometric studies, and also merge membership probabilities from four previous proper-motion studies of the cluster field. Fiducial cluster sequences in the BV (Johnson) RI (Cousins) photometric system of Landolt (1992, AJ, 104, 340) represent the principal result of this paper. These have been compared to reference samples defined by (a) Landolt's standard stars, (b) the old open clusters M67 and NGC6791, and (c) stars within 25 pc having modern photometry and precise Hipparcos parallaxes. In a companion paper we show that our derived cluster results agree well with the predictions of modern stellar-interior and -evolution theory, given reasonable estimates of the cluster chemical abundances and foreground reddening. The individual and combined datasets for NGC188 have been made available through our web site.Comment: Accepted for PAS

Metabolism disrupting chemicals and metabolic disorders

The recent epidemics of metabolic diseases, obesity, type 2 diabetes(T2D), liver lipid disorders and metabolic syndrome have largely been attributed to genetic background and changes in diet, exercise and aging. However, there is now considerable evidence that other environmental factors may contribute to the rapid increase in the incidence of these metabolic diseases. This review will examine changes to the incidence of obesity, T2D and non-alcoholic fatty liver disease (NAFLD), the contribution of genetics to these disorders and describe the role of the endocrine system in these metabolic disorders. It will then specifically focus on the role of endocrine disrupting chemicals (EDCs) in the etiology of obesity, T2D and NAFLD while finally integrating the information on EDCs on multiple metabolic disorders that could lead to metabolic syndrome. We will specifically examine evidence linking EDC exposures during critical periods of development with metabolic diseases that manifest later in life and across generations