Almilaji A et al. (2013), Klotho sensitivity of the neuronal excitatory a...

XB-ART-48001

PLoS One 2013 Jan 01;87:e70988. doi: 10.1371/journal.pone.0070988.

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Klotho sensitivity of the neuronal excitatory amino acid transporters EAAT3 and EAAT4.

Almilaji A , Munoz C , Pakladok T , Alesutan I , Feger M , Föller M , Lang UE , Shumilina E , Lang F .

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Klotho, a transmembrane protein, which can be cleaved off as β-glucuronidase and hormone, is released in both, kidney and choroid plexus and encountered in blood and cerebrospinal fluid. Klotho deficiency leads to early appearance of age-related disorders and premature death. Klotho may modify transport by inhibiting 1,25(OH)2D3 formation or by directly affecting channel and carrier proteins. The present study explored whether Klotho influences the activity of the Na(+)-coupled excitatory amino acid transporters EAAT3 and EAAT4, which are expressed in kidney (EAAT3), intestine (EAAT3) and brain (EAAT3 and EAAT4). To this end, cRNA encoding EAAT3 or EAAT4 was injected into Xenopus oocytes with and without additional injection of cRNA encoding Klotho. EAAT expressing Xenopus oocytes were further treated with recombinant human β-Klotho protein with or without β-glucuronidase inhibitor D-saccharic acid 1,4-lactone monohydrate (DSAL). Electrogenic excitatory amino acid transport was determined as L-glutamate-induced current (Iglu) in two electrode voltage clamp experiments. EAAT3 and EAAT4 protein abundance in the Xenopus oocyte cell membrane was visualized by confocal microscopy and quantified utilizing chemiluminescence. As a result, coexpression of Klotho cRNA significantly increased Iglu in both, EAAT3 or EAAT4-expressing Xenopus oocytes. Klotho cRNA coexpression significantly increased the maximal current and cell membrane protein abundance of both EAAT3 and EAAT4. The effect of Klotho coexpression on EAAT3 and EAAT4 activity was mimicked by treating EAAT3 or EAAT4-expressing Xenopus oocytes with recombinant human β-Klotho protein. The effects of Klotho coexpression and of treatment with recombinant human β-Klotho protein were both abrogated in the presence of DSAL (10 µM). In conclusion, Klotho is a novel, powerful regulator of the excitatory amino acid transporters EAAT3 and EAAT4.

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Species referenced: Xenopus laevis
Genes referenced: slc1a1 slc1a6

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	Figure 2. Glutamate-induced currents as a function of glutamate concentration in EAAT3/EAAT4-expressing Xenopus oocytes wihout or with Klotho coexpression.A, C: Means ± SEM of glutamate-induced current (Iglu) as a function of glutamate concentration in Xenopus oocytes injected with cRNA encoding EAAT3 (A, n = 9) or EAAT4 (C, n = 6) without (open circles) or with (closed circles) additional coexpression of Klotho.,*(p<0.05, p<0.01) indicate statistically significant difference from Xenopus oocytes injected with cRNA encoding EAAT3 (A) or EAAT4 (C) alone (two-tailed unpaired t-test). B, D: Means ± SEM of glutamate induced current (Iglu) normalized to Iglu at 5 mM glutamate as a function of glutamate concentration in Xenopus oocytes injected with cRNA encoding EAAT3 (B, n = 9) or EAAT4 (D, n = 6) without (open circles) and with (closed circles) additional coexpression of Klotho. The values were fitted to a hyperbola function.
	Figure 3. Effect of Klotho coexpression on protein abundance of both EAAT3 and EAAT4 in the Xenopus oocyte cell membrane.A, C: Confocal images of EAAT3 (A) and EAAT4 (C) protein abundance in the plasma membrane of Xenopus oocytes injected with water (1st panel), injected with cRNA encoding EAAT3 (A) or EAAT4 (C) without (2nd panel) or with additional coexpression of Klotho (3rd panel). B, D: Means ± SEM of EAAT3 (B, n = 75–80) and EAAT4 (D, n = 82–87) protein abundance as determined by chemiluminescence in the plasma membrane of Xenopus oocytes injected with cRNA encoding EAAT3 (B) or EAAT4 (D) without (white bars) or with (black bars) coexpression of Klotho. For comparison, water injected oocytes (grey bars).,*(p<0.01, p<0.001) indicate statistically significant difference from Xenopus oocytes injected with cRNA encoding EAAT3/EAAT4 alone (ANOVA).
	Figure 4. Effect of recombinant human β-Klotho protein on electrogenic glutamate transport in EAAT3 -expressing Xenopus oocytes.A: Means ± SEM (n = 7–16) of glutamate (2 mM)-induced current (Iglu) in Xenopus oocytes injected with water (grey bar) or injected with cRNA encoding EAAT3 and pretreated prior to measurements for 24 hours without (white bar) or with 10, 30 and 50 ng/ml recombinant human β-Klotho protein (1st, 2nd and 3rd black bar respectively).,(p<0.05, p<0.01) indicate statistically significant difference from untreated Xenopus oocytes (ANOVA). B: Means ± SEM (n = 12–17) of glutamate (2 mM)-induced current (Iglu) in Xenopus oocytes injected with water (grey bar) or injected with cRNA encoding EAAT3 pretreated prior to measurements with 30 ng/ml recombinant human β-Klotho protein for 0 hr (white bar) or 1, 6, 12 or 24hr (black bars respectively).*(p<0.01) indicates statistically significant difference from untreated Xenopus oocytes (ANOVA).
	Figure 5. Reversal of the effect of Klotho on electrogenic glutamate transport in EAAT3 or EAAT4 expressing Xenopus oocytes by β-glucuronidase inhibitor DSAL.A: Means ± SEM (n = 9–21) of glutamate (2 mM)-induced current (Iglu) in Xenopus oocytes injected with water (grey bar) or injected with cRNA encoding EAAT3 alone (white bar) or both EAAT3 and Klotho (black bars). Where indicated, the oocytes were treated with β-glucuronidase inhibitor DSAL (10 µM).(p<0.01) indicates statistically significant difference from oocytes injected with cRNA encoding EAAT3 alone (ANOVA). ### (p<0.001) indicates statistically significant difference from oocytes injected with cRNA encoding both EAAT3 and Klotho (ANOVA).B: Means ± SEM (n = 11–23) of glutamate (2 mM)-induced current (Iglu) in Xenopus oocytes injected with water (grey bar) or injected with cRNA encoding EAAT3 alone (white bar) or pretreated for 24 hours with 30ng/ ml recombinant human β-Klotho protein without (first black bar) or with the presence of β-glucuronidase inhibitor DSAL (10µM ) (second black bar).(p<0.001) indicates statistically significant difference from non-treated oocytes injected with cRNA encoding EAAT3 alone (ANOVA). ##(p<0.01). indicates statistically significant difference from oocytes injected with cRNA encoding EAAT3 and treated for 24 hours with 30ng/ ml recombinant human β-Klotho protein (ANOVA).C: Means ± SEM (n = 5–23) of glutamate (2 mM)-induced current (Iglu) in Xenopus oocytes injected with water (grey bar) or injected with cRNA encoding EAAT4 alone (white bar) or both EAAT4 and Klotho (black bars). Where indicated, the oocytes were treated with β-glucuronidase inhibitor DSAL (10 µM).(p<0.001) indicates statistically significant difference from oocytes injected with cRNA encoding EAAT4 alone (ANOVA). ###(p<0.001) indicates statistically significant difference from oocytes injected with cRNA encoding both EAAT4 and Klotho (ANOVA).D: Means ± SEM (n = 9–21) of glutamate (2 mM)-induced current (Iglu) in Xenopus oocytes injected with water (grey bar) or injected with cRNA encoding EAAT4 alone (white bar) and pretreated for 24 hours with 30 ng/ ml recombinant human β-Klotho protein without (first black bar) or with the presence of β-glucuronidase inhibitor DSAL (10µM, second black bar).*(p<0.001) indicates statistically significant difference from non-treated oocytes injected with cRNA encoding EAAT4 alone (ANOVA). ##(p<0.01). indicates statistically significant difference from oocytes injected with cRNA encoding EAAT4 and treated for 24 hours with 30 ng/ml recombinant human Klotho protein (ANOVA).
	Figure 1. Effect of Klotho coexpression on electrogenic glutamate transport in EAAT3 or EAAT4 expressing Xenopus oocytes. A: Representative original tracings of glutamate (2 mM)-induced current (Iglu) at −60 mV in Xenopus oocytes injected with water (i), or with cRNA encoding Klotho alone (ii), EAAT3 alone (iii) or both, EAAT3 and Klotho (iv). B: Means ± SEM (n = 7–36) of glutamate (2 mM)-induced current (Iglu) in Xenopus oocytes injected without (left bars) or with (right bars) cRNA encoding EAAT3 and injected without (white bars) or with (black bars) cRNA encoding Klotho.*(p<0.001) indicates statistically significant difference from Xenopus oocytes injected with cRNA encoding EAAT3 alone (ANOVA). C: Representative original tracings of glutamate (2 mM)-induced current (Iglu) measured at a holding potential of −60 mV in Xenopus oocytes injected with water (i), or with cRNA encoding Klotho alone (ii), EAAT4 alone (iii) or both EAAT4 and Klotho (iv). D: Means ± SEM (n = 5–8) of glutamate (2 mM)-induced current (Iglu) in Xenopus oocytes injected without (left bars) or with (right bars) cRNA encoding EAAT4, and injected without (white bars) or with (black bars) cRNA encoding Klotho.(p<0.01) indicate statistically significant difference from Xenopus oocytes injected with cRNA encoding EAAT3 or EAAT4 alone (ANOVA).

References [+] :

Abraham, Small-molecule Klotho enhancers as novel treatment of neurodegeneration. 2012, Pubmed

Abraham, Small-molecule Klotho enhancers as novel treatment of neurodegeneration. 2012, Pubmed
Alesutan, Regulation of the glutamate transporter EAAT4 by PIKfyve. 2010, Pubmed , Xenbase
Alesutan, Upregulation of Na-coupled glucose transporter SGLT1 by Tau tubulin kinase 2. 2012, Pubmed , Xenbase
Amara, Excitatory amino acid transporters: keeping up with glutamate. 2002, Pubmed
Bailey, Loss-of-function mutations in the glutamate transporter SLC1A1 cause human dicarboxylic aminoaciduria. 2011, Pubmed , Xenbase
Bauer, Abnormal glycosylation of EAAT1 and EAAT2 in prefrontal cortex of elderly patients with schizophrenia. 2010, Pubmed
Bogatikov, Up-regulation of amino acid transporter SLC6A19 activity and surface protein abundance by PKB/Akt and PIKfyve. 2012, Pubmed , Xenbase
Boros, Active Ca(2+) reabsorption in the connecting tubule. 2009, Pubmed
Böhmer, Stimulation of the EAAT4 glutamate transporter by SGK protein kinase isoforms and PKB. 2004, Pubmed , Xenbase
Cha, Regulation of renal outer medullary potassium channel and renal K(+) excretion by Klotho. 2009, Pubmed
Chan, Effects of ammonia on high affinity glutamate uptake and glutamate transporter EAAT3 expression in cultured rat cerebellar granule cells. 2003, Pubmed
Chen, The antiaging protein Klotho enhances oligodendrocyte maturation and myelination of the CNS. 2013, Pubmed
Collin, Plasma membrane and vesicular glutamate transporter mRNAs/proteins in hypothalamic neurons that regulate body weight. 2003, Pubmed
Crino, Increased expression of the neuronal glutamate transporter (EAAT3/EAAC1) in hippocampal and neocortical epilepsy. 2002, Pubmed
Deng, Association study of polymorphisms in the glutamate transporter genes SLC1A1, SLC1A3, and SLC1A6 with schizophrenia. 2007, Pubmed
Dowd, Comparison of Na+-dependent glutamate transport activity in synaptosomes, C6 glioma, and Xenopus oocytes expressing excitatory amino acid carrier 1 (EAAC1). 1996, Pubmed , Xenbase
Dowd, Rapid stimulation of EAAC1-mediated Na+-dependent L-glutamate transport activity in C6 glioma cells by phorbol ester. 1996, Pubmed
Dërmaku-Sopjani, Downregulation of NaPi-IIa and NaPi-IIb Na-coupled phosphate transporters by coexpression of Klotho. 2011, Pubmed , Xenbase
Furuta, Glutamate transporter protein subtypes are expressed differentially during rat CNS development. 1997, Pubmed
Furuta, Expression of glutamate transporter subtypes during normal human corticogenesis and type II lissencephaly. 2005, Pubmed
Gourley, Action control is mediated by prefrontal BDNF and glucocorticoid receptor binding. 2012, Pubmed
Grewer, Glutamate forward and reverse transport: from molecular mechanism to transporter-mediated release after ischemia. 2008, Pubmed
Henrion, Overlapping cardiac phenotype associated with a familial mutation in the voltage sensor of the KCNQ1 channel. 2012, Pubmed , Xenbase
Hertz, Bioenergetics of cerebral ischemia: a cellular perspective. 2008, Pubmed
Hosseinzadeh, Downregulation of ClC-2 by JAK2. 2012, Pubmed , Xenbase
Hu, Klotho: a novel phosphaturic substance acting as an autocrine enzyme in the renal proximal tubule. 2010, Pubmed
Huang, Climbing fiber activation of EAAT4 transporters and kainate receptors in cerebellar Purkinje cells. 2004, Pubmed
Huang, Astrocyte glutamate transporters regulate metabotropic glutamate receptor-mediated excitation of hippocampal interneurons. 2004, Pubmed
Huerta, Expression of excitatory amino acid transporter interacting protein transcripts in the thalamus in schizophrenia. 2006, Pubmed
Imura, Secreted Klotho protein in sera and CSF: implication for post-translational cleavage in release of Klotho protein from cell membrane. 2004, Pubmed
Imura, alpha-Klotho as a regulator of calcium homeostasis. 2007, Pubmed
Kavalali, Synaptic mechanisms underlying rapid antidepressant action of ketamine. 2012, Pubmed
Kim, Effects of chronic exposure to ethanol on glutamate transporter EAAT3 expressed in Xenopus oocytes: evidence for protein kinase C involvement. 2005, Pubmed , Xenbase
Klaus, PIKfyve-dependent regulation of the Cl- channel ClC-2. 2009, Pubmed , Xenbase
Kosakai, Degeneration of mesencephalic dopaminergic neurons in klotho mouse related to vitamin D exposure. 2011, Pubmed
Kuro-o, Mutation of the mouse klotho gene leads to a syndrome resembling ageing. 1997, Pubmed
Kuro-o, Klotho as a regulator of fibroblast growth factor signaling and phosphate/calcium metabolism. 2006, Pubmed
Kuro-o, Klotho. 2010, Pubmed
Lang, Potassium channels in renal epithelial transport regulation. 1992, Pubmed
Lang, Molecular mechanisms of schizophrenia. 2007, Pubmed
Maddock, MR spectroscopic studies of the brain in psychiatric disorders. 2012, Pubmed
Maragakis, Glutamate transporter expression and function in human glial progenitors. 2004, Pubmed
Mathern, Hippocampal GABA and glutamate transporter immunoreactivity in patients with temporal lobe epilepsy. 1999, Pubmed
McCullumsmith, Striatal excitatory amino acid transporter transcript expression in schizophrenia, bipolar disorder, and major depressive disorder. 2002, Pubmed
Mia, Downregulation of Kv1.5 K channels by the AMP-activated protein kinase. 2012, Pubmed , Xenbase
Miralles, Na+ dependent glutamate transporters (EAAT1, EAAT2, and EAAT3) in primary astrocyte cultures: effect of oxidative stress. 2001, Pubmed
Nieoullon, The neuronal excitatory amino acid transporter EAAC1/EAAT3: does it represent a major actor at the brain excitatory synapse? 2006, Pubmed
Nudmamud-Thanoi, Deficits of neuronal glutamatergic markers in the caudate nucleus in schizophrenia. 2007, Pubmed
O'Kane, Na(+)-dependent glutamate transporters (EAAT1, EAAT2, and EAAT3) of the blood-brain barrier. A mechanism for glutamate removal. 1999, Pubmed
Ohnishi, Reversal of mineral ion homeostasis and soft-tissue calcification of klotho knockout mice by deletion of vitamin D 1alpha-hydroxylase. 2009, Pubmed
Pathare, OSR1-sensitive renal tubular phosphate reabsorption. 2012, Pubmed , Xenbase
Peghini, Glutamate transporter EAAC-1-deficient mice develop dicarboxylic aminoaciduria and behavioral abnormalities but no neurodegeneration. 1997, Pubmed
Proper, Distribution of glutamate transporters in the hippocampus of patients with pharmaco-resistant temporal lobe epilepsy. 2002, Pubmed
Rajamanickam, EAAT4 phosphorylation at the SGK1 consensus site is required for transport modulation by the kinase. 2007, Pubmed , Xenbase
Rakhade, Focal reduction of neuronal glutamate transporters in human neocortical epilepsy. 2008, Pubmed
Ramasamy, Recent advances in physiological calcium homeostasis. 2006, Pubmed
Rao, RETRACTED: Dysregulated glutamate and dopamine transporters in postmortem frontal cortex from bipolar and schizophrenic patients. 2012, Pubmed
Razzaque, Premature aging-like phenotype in fibroblast growth factor 23 null mice is a vitamin D-mediated process. 2006, Pubmed
Sanacora, Preliminary evidence of riluzole efficacy in antidepressant-treated patients with residual depressive symptoms. 2007, Pubmed
Schmitt, Decreased gene expression of glial and neuronal glutamate transporters after chronic antipsychotic treatment in rat brain. 2003, Pubmed
Schniepp, Retinal colocalization and in vitro interaction of the glutamate transporter EAAT3 and the serum- and glucocorticoid-inducible kinase SGK1 [correction]. 2004, Pubmed , Xenbase
Segawa, Correlation between hyperphosphatemia and type II Na-Pi cotransporter activity in klotho mice. 2007, Pubmed
Shan, Abnormal expression of glutamate transporters in temporal lobe areas in elderly patients with schizophrenia. 2013, Pubmed
Shashidharan, Immunohistochemical localization of the neuron-specific glutamate transporter EAAC1 (EAAT3) in rat brain and spinal cord revealed by a novel monoclonal antibody. 1997, Pubmed
Simantov, Changes in expression of neuronal and glial glutamate transporters in rat hippocampus following kainate-induced seizure activity. 1999, Pubmed
Smith, Expression of excitatory amino acid transporter transcripts in the thalamus of subjects with schizophrenia. 2001, Pubmed
Sopjani, Regulation of the Na+/K+ ATPase by Klotho. 2011, Pubmed , Xenbase
Strutz-Seebohm, Structural basis of slow activation gating in the cardiac I Ks channel complex. 2011, Pubmed , Xenbase
Takeshita, Sinoatrial node dysfunction and early unexpected death of mice with a defect of klotho gene expression. 2004, Pubmed
Teocchi, Hippocampal gene expression dysregulation of Klotho, nuclear factor kappa B and tumor necrosis factor in temporal lobe epilepsy patients. 2013, Pubmed
Tsujikawa, Klotho, a gene related to a syndrome resembling human premature aging, functions in a negative regulatory circuit of vitamin D endocrine system. 2003, Pubmed
Voelkl, Spironolactone ameliorates PIT1-dependent vascular osteoinduction in klotho-hypomorphic mice. 2013, Pubmed
Wang, Klotho sensitizes human lung cancer cell line to cisplatin via PI3k/Akt pathway. 2013, Pubmed
Yoshida, Mediation of unusually high concentrations of 1,25-dihydroxyvitamin D in homozygous klotho mutant mice by increased expression of renal 1alpha-hydroxylase gene. 2002, Pubmed
van Landeghem, Expression of PACAP and glutamate transporter proteins in satellite oligodendrocytes of the human CNS. 2007, Pubmed

	Figure 2. Glutamate-induced currents as a function of glutamate concentration in EAAT3/EAAT4-expressing Xenopus oocytes wihout or with Klotho coexpression.A, C: Means ± SEM of glutamate-induced current (Iglu) as a function of glutamate concentration in Xenopus oocytes injected with cRNA encoding EAAT3 (A, n = 9) or EAAT4 (C, n = 6) without (open circles) or with (closed circles) additional coexpression of Klotho.,*(p<0.05, p<0.01) indicate statistically significant difference from Xenopus oocytes injected with cRNA encoding EAAT3 (A) or EAAT4 (C) alone (two-tailed unpaired t-test). B, D: Means ± SEM of glutamate induced current (Iglu) normalized to Iglu at 5 mM glutamate as a function of glutamate concentration in Xenopus oocytes injected with cRNA encoding EAAT3 (B, n = 9) or EAAT4 (D, n = 6) without (open circles) and with (closed circles) additional coexpression of Klotho. The values were fitted to a hyperbola function.
	Figure 3. Effect of Klotho coexpression on protein abundance of both EAAT3 and EAAT4 in the Xenopus oocyte cell membrane.A, C: Confocal images of EAAT3 (A) and EAAT4 (C) protein abundance in the plasma membrane of Xenopus oocytes injected with water (1st panel), injected with cRNA encoding EAAT3 (A) or EAAT4 (C) without (2nd panel) or with additional coexpression of Klotho (3rd panel). B, D: Means ± SEM of EAAT3 (B, n = 75–80) and EAAT4 (D, n = 82–87) protein abundance as determined by chemiluminescence in the plasma membrane of Xenopus oocytes injected with cRNA encoding EAAT3 (B) or EAAT4 (D) without (white bars) or with (black bars) coexpression of Klotho. For comparison, water injected oocytes (grey bars).,*(p<0.01, p<0.001) indicate statistically significant difference from Xenopus oocytes injected with cRNA encoding EAAT3/EAAT4 alone (ANOVA).
	Figure 4. Effect of recombinant human β-Klotho protein on electrogenic glutamate transport in EAAT3 -expressing Xenopus oocytes.A: Means ± SEM (n = 7–16) of glutamate (2 mM)-induced current (Iglu) in Xenopus oocytes injected with water (grey bar) or injected with cRNA encoding EAAT3 and pretreated prior to measurements for 24 hours without (white bar) or with 10, 30 and 50 ng/ml recombinant human β-Klotho protein (1st, 2nd and 3rd black bar respectively).,(p<0.05, p<0.01) indicate statistically significant difference from untreated Xenopus oocytes (ANOVA). B: Means ± SEM (n = 12–17) of glutamate (2 mM)-induced current (Iglu) in Xenopus oocytes injected with water (grey bar) or injected with cRNA encoding EAAT3 pretreated prior to measurements with 30 ng/ml recombinant human β-Klotho protein for 0 hr (white bar) or 1, 6, 12 or 24hr (black bars respectively).*(p<0.01) indicates statistically significant difference from untreated Xenopus oocytes (ANOVA).
	Figure 5. Reversal of the effect of Klotho on electrogenic glutamate transport in EAAT3 or EAAT4 expressing Xenopus oocytes by β-glucuronidase inhibitor DSAL.A: Means ± SEM (n = 9–21) of glutamate (2 mM)-induced current (Iglu) in Xenopus oocytes injected with water (grey bar) or injected with cRNA encoding EAAT3 alone (white bar) or both EAAT3 and Klotho (black bars). Where indicated, the oocytes were treated with β-glucuronidase inhibitor DSAL (10 µM).(p<0.01) indicates statistically significant difference from oocytes injected with cRNA encoding EAAT3 alone (ANOVA). ### (p<0.001) indicates statistically significant difference from oocytes injected with cRNA encoding both EAAT3 and Klotho (ANOVA).B: Means ± SEM (n = 11–23) of glutamate (2 mM)-induced current (Iglu) in Xenopus oocytes injected with water (grey bar) or injected with cRNA encoding EAAT3 alone (white bar) or pretreated for 24 hours with 30ng/ ml recombinant human β-Klotho protein without (first black bar) or with the presence of β-glucuronidase inhibitor DSAL (10µM ) (second black bar).(p<0.001) indicates statistically significant difference from non-treated oocytes injected with cRNA encoding EAAT3 alone (ANOVA). ##(p<0.01). indicates statistically significant difference from oocytes injected with cRNA encoding EAAT3 and treated for 24 hours with 30ng/ ml recombinant human β-Klotho protein (ANOVA).C: Means ± SEM (n = 5–23) of glutamate (2 mM)-induced current (Iglu) in Xenopus oocytes injected with water (grey bar) or injected with cRNA encoding EAAT4 alone (white bar) or both EAAT4 and Klotho (black bars). Where indicated, the oocytes were treated with β-glucuronidase inhibitor DSAL (10 µM).(p<0.001) indicates statistically significant difference from oocytes injected with cRNA encoding EAAT4 alone (ANOVA). ###(p<0.001) indicates statistically significant difference from oocytes injected with cRNA encoding both EAAT4 and Klotho (ANOVA).D: Means ± SEM (n = 9–21) of glutamate (2 mM)-induced current (Iglu) in Xenopus oocytes injected with water (grey bar) or injected with cRNA encoding EAAT4 alone (white bar) and pretreated for 24 hours with 30 ng/ ml recombinant human β-Klotho protein without (first black bar) or with the presence of β-glucuronidase inhibitor DSAL (10µM, second black bar).*(p<0.001) indicates statistically significant difference from non-treated oocytes injected with cRNA encoding EAAT4 alone (ANOVA). ##(p<0.01). indicates statistically significant difference from oocytes injected with cRNA encoding EAAT4 and treated for 24 hours with 30 ng/ml recombinant human Klotho protein (ANOVA).
	Figure 1. Effect of Klotho coexpression on electrogenic glutamate transport in EAAT3 or EAAT4 expressing Xenopus oocytes. A: Representative original tracings of glutamate (2 mM)-induced current (Iglu) at −60 mV in Xenopus oocytes injected with water (i), or with cRNA encoding Klotho alone (ii), EAAT3 alone (iii) or both, EAAT3 and Klotho (iv). B: Means ± SEM (n = 7–36) of glutamate (2 mM)-induced current (Iglu) in Xenopus oocytes injected without (left bars) or with (right bars) cRNA encoding EAAT3 and injected without (white bars) or with (black bars) cRNA encoding Klotho.*(p<0.001) indicates statistically significant difference from Xenopus oocytes injected with cRNA encoding EAAT3 alone (ANOVA). C: Representative original tracings of glutamate (2 mM)-induced current (Iglu) measured at a holding potential of −60 mV in Xenopus oocytes injected with water (i), or with cRNA encoding Klotho alone (ii), EAAT4 alone (iii) or both EAAT4 and Klotho (iv). D: Means ± SEM (n = 5–8) of glutamate (2 mM)-induced current (Iglu) in Xenopus oocytes injected without (left bars) or with (right bars) cRNA encoding EAAT4, and injected without (white bars) or with (black bars) cRNA encoding Klotho.(p<0.01) indicate statistically significant difference from Xenopus oocytes injected with cRNA encoding EAAT3 or EAAT4 alone (ANOVA).