Regulation of the glutamate transporter EAAT1 by the ubiquitin ligase Nedd4-2 and the serum and glucocorticoid-inducible kinase isoforms SGK1/3 and protein kinase B

Surface expression of the glial glutamate transporter EAAT1 is stimulated by insulin-like growth factor 1 through activation of phosphatidylinositol-3-kinase. Downstream targets include serum and glucocorticoid-sensitive kinase isoforms SGK1, SGK2 and SGK3, and protein kinase B. SGK1 regulates Nedd4-2, a ubiquitin ligase that prepares cell membrane proteins for degradation. To test whether Nedd4-2, SGK1, SGK3 and protein kinase B regulate EAAT1, cRNA encoding EAAT1 was injected into Xenopus oocytes with or without additional injection of wild-type Nedd4-2, constitutively active S422DSGK1, inactive K127NSGK1, wild-type SGK3 and/or constitutively active T308D,S473DPKB. Glutamate induces a current in Xenopus oocytes expressing EAAT1, but not in water-injected oocytes, which is decreased by co-expression of Nedd4-2, an effect reversed by additional co-expression of S422DSGK1, SGK3 and T308D,S473DPKB, but not K127NSGK1. Site-directed mutagenesis of the SGK1 phosphorylation sites in the Nedd4-2 protein (S382A,S468ANedd4-2) and in the EAAT1 protein (T482AEAAT1, T482DEAAT1) significantly blunts the effect of S422DSGK1. Moreover, the current is significantly larger in T482DEAAT1- than in T482AEAAT1-expressing oocytes, indicating that a negative charge mimicking phosphorylation at T482 increases transport. The experiments reveal a powerful novel mechanism that regulates the activity of EAAT1. This mechanism might participate in the regulation of neuronal excitability and glutamate transport in other tissues.     

Down-Regulation of Excitatory Amino Acid Transporters EAAT1 and EAAT2 by the Kinases SPAK and OSR1

SPAK (SPS1-related proline/alanine-rich kinase) and OSR1 (oxidative stress-responsive kinase 1) are cell volume-sensitive kinases regulated by WNK (with-no-K[Lys]) kinases. SPAK/OSR1 regulate several channels and carriers. SPAK/OSR1 sensitive functions include neuronal excitability. Orchestration of neuronal excitation involves the excitatory glutamate transporters EAAT1 and EAAT2. Sensitivity of those carriers to SPAK/OSR1 has never been shown. The present study thus explored whether SPAK and/or OSR1 contribute to the regulation of EAAT1 and/or EAAT2. To this end, cRNA encoding EAAT1 or EAAT2 was injected into Xenopus oocytes without or with additional injection of cRNA encoding wild-type SPAK or wild-type OSR1, constitutively active ^T233ESPAK, WNK insensitive ^T233ASPAK, catalytically inactive ^D212ASPAK, constitutively active ^T185EOSR1, WNK insensitive ^T185AOSR1 or catalytically inactive ^D164AOSR1. The glutamate (2 mM)-induced inward current (I _Glu) was taken as a measure of glutamate transport. As a result, I _Glu was observed in EAAT1- and in EAAT2-expressing oocytes but not in water-injected oocytes, and was significantly decreased by coexpression of SPAK and OSR1. As shown for EAAT2, SPAK, and OSR1 decreased significantly the maximal transport rate but significantly enhanced the affinity of the carrier. The effect of wild-type SPAK/OSR1 on EAAT1 and EAAT2 was mimicked by ^T233ESPAK and ^T185EOSR1, but not by ^T233ASPAK, ^D212ASPAK, ^T185AOSR1, or ^D164AOSR1. Coexpression of either SPAK or OSR1 decreased the EAAT2 protein abundance in the cell membrane of EAAT2-expressing oocytes. In conclusion, SPAK and OSR1 are powerful negative regulators of the excitatory glutamate transporters EAAT1 and EAAT2.     

Regulation of the excitatory amino acid transporter EAAT5 by the serum and glucocorticoid dependent kinases SGK1 and SGK3

In the mammalian retina, glutamate re-uptake is mediated by the sodium dependent cotransport systems EAAT1-5 thus terminating neuronal excitation and preventing neuroexcitotoxicity. In retinal amacrine and ganglion cells, EAAT5 is colocalized with the serum and glucocorticoid inducible kinase SGK1, a serine/threonine kinase known to regulate transport. The study explored the possible regulation of EAAT5 by SGK1, its isoform SGK3, and the closely related protein kinase B. EAAT5 was coexpressed in Xenopus laevis oocytes with or without the respective kinases. Transport activity was quantified by electrophysiology and cell surface expression was determined by chemiluminescence. Both EAAT5 mediated currents and EAAT5 protein abundance at the cell surface were increased by a factor of 1.5-2 upon coexpression of SGK1 or SGK3 but not following coexpression of PKB. In conclusion, the kinases SGK1 and SGK3 increase EAAT5 activity by increasing cell surface abundance of the carrier.     

Up-regulation of the betaine/GABA transporter BGT1 by JAK2

Janus-activated kinase-2 JAK2 is activated by hyperosmotic shock and modifies the activity of several Na(+) coupled transporters. Carriers up-regulated by osmotic shock include the Na(+) coupled osmolyte transporter BGT1 (betaine/GABA transporter 1), which accomplishes the concentrative cellular uptake of γ-amino-butyric acid (GABA). The present study thus explored whether JAK2 participates in the regulation of BGT1 activity. To this end, cRNA encoding BGT1 was injected into Xenopus oocytes with or without cRNA encoding wild type JAK2, constitutively active (V617F)JAK2 or inactive (K882E)JAK2, and electrogenic GABA transport determined by dual electrode voltage clamp. In oocytes injected with cRNA encoding BGT1 but not in oocytes injected with water or with cRNA encoding JAK2 alone, the addition of 1mM GABA to the extracellular fluid generated an inward current (I(BGT)). In BGT1 expressing oocytes I(BGT) was significantly increased by coexpression of JAK2 or (V617F)JAK2, but not by coexpression of (K882E)JAK2. According to kinetic analysis coexpression of JAK2 increased the maximal I(BGT) without significantly modifying the concentration required for halfmaximal I(BGT) (K(M)). In oocytes expressing BGT1 and (V617F)JAK2 I(BGT) was gradually decreased by JAK2 inhibitor AG490 (40 μM). The decline of I(BGT) following disruption of carrier insertion with brefeldin A (5 μM) was similar in the absence and presence of the JAK2 inhibitor AG490 (40 μM). In conclusion, JAK2 is a novel regulator of the GABA transporter BGT1. The kinase up-regulates the carrier presumably by enhancing the insertion of carrier protein into the cell membrane.     

Stimulation of the creatine transporter SLC6A8 by the protein kinase mTOR

Cellular accumulation of creatine is accomplished by the Na(+), Cl(-), and creatine transporter CreaT (SLC6A8). The mammalian target of rapamycin (mTOR) is a kinase stimulating cellular nutrient uptake. The present experiments explored whether SLC6A8 is regulated by mTOR. In Xenopus oocytes expressing SLC6A8 but not in water injected oocytes, creatine-induced a current which was significantly enhanced by coexpression of mTOR. Kinetic analysis revealed that mTOR enhanced maximal current without significantly altering affinity. Preincubation of the oocytes for 32 h with rapamycin (50 nM) decreased the creatine-induced current and abrogated its stimulation by mTOR. The effect of mTOR on CreaT was blunted by additional coexpression of the inactive mutant of the serum and glucocorticoid-inducible kinase (K119N)SGK1 and mimicked by coexpression of wild type SGK1. In conclusion, mTOR stimulates the creatine transporter SLC6A8 through mechanisms at least partially shared by the serum and glucocorticoid-inducible kinase SGK1.     

Klotho Sensitivity of the Neuronal Excitatory Amino Acid Transporters EAAT3 and EAAT4

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 (I_glu) 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 I_glu 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.     

EAAT4 phosphorylation at the SGK1 consensus site is required for transport modulation by the kinase

EAAT4 (SLC1A6) is a Purkinje-Cell-specific post-synaptic excitatory amino acid transporter that plays a major role in clearing synaptic glutamate. EAAT4 abundance and function is known to be modulated by the serum and glucocorticoid inducible kinase (SGK) 1 but the precise mechanism of kinase action has not been defined yet. The present work aims to identify the molecular mechanism of EAAT4 modulation by the kinase. The EAAT4 sequence bears two putative SGK1 consensus sites (at Thr40 and Thr504) at the amino and carboxy terminus that are conserved among species. Expression studies in Xenopus oocytes demonstrated that EAAT4-mediated [(3)H] glutamate uptake and cell surface abundance are enhanced by co-expression of SGK1. Disruption of the SGK1 phosphorylation site at threonine 40 ((T40A)EAAT4) or of both phosphorylation sites ((T40AT504A)EAAT4) abrogated the effect of SGK1 on transporter function and expression. SGK1 modulates several transport proteins via inhibition of the ubiquitin ligase Nedd4-2. Co-expression of Nedd4-2 inhibited wild-type EAAT4 but not the (T40AT504A)EAAT4 mutant. Besides, RNA interference-mediated reduction of endogenous Nedd4-2 (xNedd4-2) expression increased the activity of the transporter. In conclusion, maximal glutamate transport modulation by SGK1 is accomplished by direct EAAT4 stimulation and to a lesser extent by inhibition of intrinsic Nedd4-2.     

Stimulation of the EAAT4 glutamate transporter by SGK protein kinase isoforms and PKB

The serum and glucocorticoid inducible kinase (SGK) 1 is expressed in brain tissue and upregulated by ischemia, neuronal excitation, and dehydration. The present study has been performed to elucidate the expression of SGK1 in cerebellar Purkinje cells and to explore whether it influences the colocalized glutamate transporter EAAT4. Intense SGK1 staining was observed in Purkinje cells following 48h of water deprivation. The kinase activates glutamate induced current (I(GLU)) in Xenopus oocytes heterologously expressing EAAT4, an effect mimicked by its isoforms SGK2, 3 and PKB. I(GLU) was decreased by the ubiquitin ligase Nedd4-2, an effect partially but not completely reversed by additional coexpression of the SGK kinase isoforms or PKB. According to immunohistochemistry EAAT4 protein abundance in the cell membrane was enhanced by SGK1 and decreased by Nedd4-2. In conclusion, SGK1 expression is upregulated by ischemia, excitation, and dehydration in cerebellar Purkinje cells. The upregulation of SGK1 may serve to stimulate EAAT4 and thus to reduce neuroexcitotoxicity.     

Post-translational regulation of EAAT2 function by co-expressed ubiquitin ligase Nedd4-2 is impacted by SGK kinases

The human excitatory amino acid transporter (EAAT)2 is the major glutamate carrier in the mammalian CNS. Defective expression of the transporter results in neuroexcitotoxicity that may contribute to neuronal disorders such as amyotrophic lateral sclerosis (ALS). The serum and glucocorticoid inducible kinase (SGK) 1 is expressed in the brain and is known to interact with the ubiquitin ligase Nedd4-2 to modulate membrane transporters and ion channels. The present study aimed to investigate whether SGK isoforms and the related kinase, protein kinase B (PKB), regulate EAAT2. Expression studies in Xenopus oocytes demonstrated that glutamate-induced inward current (IGLU) was stimulated by co-expression of SGK1, SGK2, SGK3 or PKB. IGLU is virtually abolished by Nedd4-2, an effect abrogated by additional co-expression of either kinase. The kinases diminish the effect through Nedd4-2 phosphorylation without altering Nedd4-2 protein abundance. SGKs increase the transporter maximal velocity without significantly affecting substrate affinity. Similar to glutamate-induced currents, [3H] glutamate uptake and cell surface abundance of the transporter were increased by the SGK isoforms and down-regulated by the ubiquitin ligase Nedd4-2. In conclusion, all three SGK isoforms and PKB increase EAAT2 activity and plasma membrane expression and thus, may participate in the regulation of neuroexcitability.     

Effects of the level of mRNA expression on biophysical properties, sensitivity to neurotoxins, and regulation of the brain delayed-rectifier K+ channels Kv1.2.

Injection of 0.2 ng of cRNA encoding the brain Kv1.2 channel into Xenopus oocytes leads to the expression of a very slowly inactivating K+ current. Inactivation is absent in oocytes injected with 20 ng of cRNA although activation remains unchanged. Low cRNA concentrations generate a channel which is sensitive to dendrotoxin I (IC50 = 2 nM at 0.2 ng of cRNA/oocyte) and to less potent analogs of this toxin from Dendroaspis polylepis venom. A good correlation is found between blockade of the K+ current and binding of the different toxins to rat brain membranes. High cRNA concentrations generate another form of the K+ channel which is largely insensitive to dendrotoxin I (IC50 = 200 nM at 20 ng of cRNA per oocyte). At low cRNA concentrations, the expressed Kv1.2 channel is also blocked by other polypeptide toxins such as MCD peptide (IC50 = 20 nM), charybdotoxin (IC50 = 50 nM), and beta-bungarotoxin (IC50 = 50 nM), which bind to distinct and allosterically related sites on the channel protein. The pharmacologically distinct type of K+ channel expressed at high cRNA concentrations (20 ng of cRNA/oocyte) is nearly totally resistant to 100 nM MCD peptide and hardly altered by charybdotoxin and beta-bungarotoxin at concentrations as high as 1 microM. Both at low and at high cRNA concentrations, the expressed Kv1.2 channel is blocked by an increase in intracellular Ca2+ from the inositol trisphosphate sensitive pools and by the phorbol ester PMA that activates protein kinase C.     

Elevated ammonium levels: differential acute effects on three glutamate transporter isoforms

Increased ammonium (NH(4)(+)/NH(3)) in the brain is a significant factor in the pathophysiology of hepatic encephalopathy, which involves altered glutamatergic neurotransmission. In glial cell cultures and brain slices, glutamate uptake either decreases or increases following acute ammonium exposure but the factors responsible for the opposing effects are unknown. Excitatory amino acid transporter isoforms EAAT1, EAAT2, and EAAT3 were expressed in Xenopus oocytes to study effects of ammonium exposure on their individual function. Ammonium increased EAAT1- and EAAT3-mediated [(3)H]glutamate uptake and glutamate transport currents but had no effect on EAAT2. The maximal EAAT3-mediated glutamate transport current was increased but the apparent affinities for glutamate and Na(+) were unaltered. Ammonium did not affect EAAT3-mediated transient currents, indicating that EAAT3 surface expression was not enhanced. The ammonium-induced stimulation of EAAT3 increased with increasing extracellular pH, suggesting that the gaseous form NH(3) mediates the effect. An ammonium-induced intracellular alkalinization was excluded as the cause of the enhanced EAAT3 activity because 1) ammonium acidified the oocyte cytoplasm, 2) intracellular pH buffering with MOPS did not reduce the stimulation, and 3) ammonium enhanced pH-independent cysteine transport. Our data suggest that the ammonium-elicited uptake stimulation is not caused by intracellular alkalinization or changes in the concentrations of cotransported ions but may be due to a direct effect on EAAT1/EAAT3. We predict that EAAT isoform-specific effects of ammonium combined with cell-specific differences in EAAT isoform expression may explain the conflicting reports on ammonium-induced changes in glial glutamate uptake.     

Involvement of rBAT in Na(+)-dependent and -independent transport of the neurotransmitter candidate L-DOPA in Xenopus laevis oocytes injected with rabbit small intestinal epithelium poly A(+) RNA

Although L-3,4-dihydroxyphenylalanine (L-DOPA) is claimed to be a neurotransmitter in the central nervous system (CNS), receptor or transporter molecules for L-DOPA have not been determined. In an attempt to identify a transporter for L-DOPA, we examined whether or not an active and high affinity L-DOPA transport system is expressed in Xenopus laevis oocytes injected with poly A(+) RNA prepared from several tissues. Among the poly A(+) RNAs tested, rabbit intestinal epithelium poly A(+) RNA gave the highest transport activity for L-[(14)C]DOPA in the oocytes. The uptake was approximately five times higher than that of water-injected oocytes, and was partially Na(+)-dependent. L-Tyrosine, L-phenylalanine, L-leucine and L-lysine inhibited this transport activity, whereas D-DOPA, dopamine, glutamate and L-DOPA cyclohexylester, an L-DOPA antagonist did not affect this transport. Coinjection of an antisense cRNA, as well as oligonucleotide complementary to rabbit rBAT (NBAT) cDNA almost completely inhibited the uptake of L-[(14)C]DOPA in the oocytes. On the other hand, an antisense cRNA of rabbit 4F2hc barely affected this L-[(14)C]DOPA uptake activity. rBAT was thus responsible for the L-[(14)C]DOPA uptake activity expressed in X. laevis oocytes injected with poly A(+) RNA from rabbit intestinal epithelium. As rBAT is localized at the target regions of L-DOPA in the CNS, rBAT might be one of the components involved in L-DOPAergic neurotransmission.     

Stimulation of the intestinal phosphate transporter SLC34A2 by the protein kinase mTOR

Adequate phosphate homeostasis is of critical importance for a wide variety of functions including bone mineralization and energy metabolism. Phosphate balance is a function of intestinal absorption and renal elimination, which are both under tight hormonal control. Intestinal phosphate absorption is accomplished by the Na(+), phosphate cotransporter NaPi IIb (SLC34A2). Signaling mechanisms mediating hormonal regulation of SLC34A2 are incompletely understood. The mammalian target of rapamycin (mTOR) is a kinase regulating a variety of nutrient transporters. The present experiments explored whether mTOR regulates the activity of SLC34A2. In Xenopus oocytes expressing SLC34A2 but not in water injected oocytes phosphate (1 mM) induced a current (Ip) which was significantly enhanced by coexpression of mTOR. Preincubation of the oocytes for 24 h with rapamycin (50 nM) did not significantly affect Ip in the absence of mTOR but virtually abolished the increase of Ip following coexpression of mTOR. The wild type serum and glucocorticoid inducible kinase SGK1 and the constitutively active (S422D)SGK1 similarly stimulated Ip, an effect again reversed by rapamycin. Coexpression of the inactive mutant of the serum and glucocorticoid inducible kinase (K119N)SGK1 significantly decreased Ip and abrogated the stimulating effect of mTOR on Ip. In conclusion, mTOR and SGK1 cooperate in the stimulation of the intestinal phosphate transporter SLC34A2.     

Inhibition of voltage-gated K+ channels in dendritic cells by rapamycin

Rapamycin, an inhibitor of the serine/threonine kinase mammalian target of rapamycin (mTOR), is a widely used immunosuppressive drug. Rapamycin affects the function of dendritic cells (DCs), antigen-presenting cells participating in the initiation of primary immune responses and the establishment of immunological memory. Voltage-gated K(+) (Kv) channels are expressed in and impact on the function of DCs. The present study explored whether rapamycin influences Kv channels in DCs. To this end, DCs were isolated from murine bone marrow and ion channel activity was determined by whole cell patch clamp. To more directly analyze an effect of mTOR on Kv channel activity, Kv1.3 and Kv1.5 were expressed in Xenopus oocytes with or without the additional expression of mTOR and voltage-gated currents were determined by dual-electrode voltage clamp. As a result, preincubation with rapamycin (0-50 nM) led to a gradual decline of Kv currents in DCs, reaching statistical significance within 6 h and 50 nM of rapamycin. Rapamycin accelerated Kv channel inactivation. Coexpression of mTOR upregulated Kv1.3 and Kv1.5 currents in Xenopus oocytes. Furthermore, mTOR accelerated Kv1.3 channel activation and slowed down Kv1.3 channel inactivation. In conclusion, mTOR stimulates Kv channels, an effect contributing to the immunomodulating properties of rapamycin in DCs.     

Regulation of the Na+-coupled glutamate transporter EAAT3 by PIKfyve

The Na⁺,glutamate cotransporter EAAT3 is expressed in a wide variety of tissues. It accomplishes transepithelial transport and the cellular uptake of acidic amino acids. Regulation of EAAT3 activity involves a signaling cascade including the phosphatidylinositol-3 (PI3)-kinase, the phosphoinositide dependent kinase PDK1, and the serum and glucocorticoid inducible kinase SGK1. Targets of SGK1 include the mammalian phosphatidylinositol-3-phosphate-5-kinase PIKfyve (PIP5K3). The present experiments explored whether PIKfyve participates in the regulation of EAAT3 activity. To this end, EAAT3 was expressed in Xenopus oocytes with or without SGK1 and/or PIKfyve and glutamate-induced current (I_glu) determined by dual electrode voltage clamp. In Xenopus oocytes expressing EAAT3 but not in water injected oocytes glutamate induced an inwardly directed I_glu. Coexpression of either, SGK1 or PIKfyve, significantly enhanced I_glu in EAAT3 expressing oocytes. The increased I_glu was paralleled by increased EAAT3 protein abundance in the oocyte cell membrane. I_glu and EAAT3 protein abundance were significantly larger in oocytes coexpressing EAAT3, SGK1 and PIKfyve than in oocytes expressing EAAT3 and either, SGK1 or PIKfyve, alone. Coexpression of the inactive SGK1 mutant ^K127NSGK1 did not significantly alter I_glu in EAAT3 expressing oocytes and completely reversed the stimulating effect of PIKfyve coexpression on I_glu. The stimulating effect of PIKfyve on I_glu was abolished by replacement of the serine by alanine in the SGK consensus sequence (^S318APIKfyve). Moreover, additional coexpression of ^S318APIKfyve significantly blunted I_glu in Xenopus oocytes coexpressing SGK1 and EAAT3. The observations demonstrate that PIKfyve participates in EAAT3 regulation likely downstream of SGK1.     

Hetero-oligomerization of neuronal glutamate transporters

Excitatory amino acid transporters (EAATs) mediate the uptake of glutamate into neuronal and glial cells of the mammalian central nervous system. Two transporters expressed primarily in glia, EAAT1 and EAAT2, are crucial for glutamate homeostasis in the adult mammalian brain. Three neuronal transporters (EAAT3, EAAT4, and EAAT5) appear to have additional functions in regulating and processing cellular excitability. EAATs are assembled as trimers, and the existence of multiple isoforms raises the question of whether certain isoforms can form hetero-oligomers. Co-expression and pulldown experiments of various glutamate transporters showed that EAAT3 and EAAT4, but neither EAAT1 and EAAT2, nor EAAT2 and EAAT3 are capable of co-assembling into heterotrimers. To study the functional consequences of hetero-oligomerization, we co-expressed EAAT3 and the serine-dependent mutant R501C EAAT4 in HEK293 cells and Xenopus laevis oocytes and studied glutamate/serine transport and anion conduction using electrophysiological methods. Individual subunits transport glutamate independently of each other. Apparent substrate affinities are not affected by hetero-oligomerization. However, polarized localization in Madin-Darby canine kidney cells was different for homo- and hetero-oligomers. EAAT3 inserts exclusively into apical membranes of Madin-Darby canine kidney cells when expressed alone. Co-expression with EAAT4 results in additional appearance of basolateral EAAT3. Our results demonstrate the existence of heterotrimeric glutamate transporters and provide novel information about the physiological impact of EAAT oligomerization.     

Stimulation of renal Na+ dicarboxylate cotransporter 1 by Na+/H+ exchanger regulating factor 2, serum and glucocorticoid inducible kinase isoforms, and protein kinase B

Renal tubular citrate transport is accomplished by electrogenic Na(+) coupled dicarboxylate transporter NaDC-1, a carrier subjected to regulation by acidosis. Trafficking of the Na(+)/H(+) exchanger NHE3 is controlled by NHE regulating factors NHERF-1 and NHERF-2 and the serum and glucocorticoid inducible kinase SGK1. To test for a possible involvement in NaDC-1 regulation, mRNA encoding NaDC-1 was injected into Xenopus oocytes with or without cRNA encoding NHERF-1, NHERF-2, SGK1, SGK2, SGK3, and/or the constitutively active form of the related protein kinase B ((T308,S473D)PKB). Succinate induced inward currents (I(succ)) were taken as a measure of transport rate. Coexpression of neither NHERF-1 nor NHERF-2 in NaDC-1 expressing oocytes significantly altered I(succ). On the other hand, coexpression of SGK1, SGK3, and (T308,S473D)PKB stimulated I(succ), an effect further stimulated by additional coexpression of NHERF-2 but not of NHERF-1. The action of the kinases and NHERF-2 may link urinary citrate excretion to proximal tubular H(+) secretion.     

Functional significance of N- and C-terminus of the amino acid transporters EAAC1 and ASCT1: characterization of chimeric transporters

To localize functionally significant domains in the amino acid transporters of mouse brain mEAAC1 and mASCT1, cRNA encoding for wild-type and chimeric transporters was injected into Xenopus oocytes. Activity of expressed transporters was investigated by measurements of uptake of 3H-labeled glutamate and serine and of glutamate- and serine-induced currents under voltage clamp. Though all transporters accept glutamate and serine as substrate, the central part of the protein (Ala94-Met418 of mEAAC1 and Ala119-Ile393 of mASCT1) determines substrate selectivity. The C-terminus rectifies the interaction with the respective substrate. A channel mode of the glutamate transporter can be activated by glutamate and serine, and the N- and C-termini of the mEAAC1 seem to be essential for the channel formation.     

Identification of monocarboxylate transporter 8 as a specific thyroid hormone transporter

Transport of thyroid hormone across the cell membrane is required for its action and metabolism. Recently, a T-type amino acid transporter was cloned which transports aromatic amino acids but not iodothyronines. This transporter belongs to the monocarboxylate transporter (MCT) family and is most homologous with MCT8 (SLC16A2). Therefore, we cloned rat MCT8 and tested it for thyroid hormone transport in Xenopus laevis oocytes. Oocytes were injected with rat MCT8 cRNA, and after 3 days immunofluorescence microscopy demonstrated expression of the protein at the plasma membrane. MCT8 cRNA induced an approximately 10-fold increase in uptake of 10 nM 125I-labeled thyroxine (T4), 3,3',5-triiodothyronine (T3), 3,3',5'-triiodothyronine (rT3) and 3,3'-diiodothyronine. Because of the rapid uptake of the ligands, transport was only linear with time for <4 min. MCT8 did not transport Leu, Phe, Trp, or Tyr. [125I]T4 transport was strongly inhibited by L-T4, D-T4, L-T3, D-T3, 3,3',5-triiodothyroacetic acid, N-bromoacetyl-T3, and bromosulfophthalein. T3 transport was less affected by these inhibitors. Iodothyronine uptake in uninjected oocytes was reduced by albumin, but the stimulation induced by MCT8 was markedly increased. Saturation analysis provided apparent Km values of 2-5 microM for T4, T3, and rT3. Immunohistochemistry showed high expression in liver, kidney, brain, and heart. In conclusion, we have identified MCT8 as a very active and specific thyroid hormone transporter.