Christoph Boehmer

The Serum and Glucocorticoid-Inducible Kinase SGK1 and the Na+/H+ Exchange Regulating Factor NHERF2 Synergize to Stimulate the Renal Outer Medullary K+ Channel ROMK1

Mineralocorticoids stimulate Na(+) reabsorption and K(+) secretion in principal cells of connecting tubule and collecting duct. The involved ion channels are ENaC and ROMK1, respectively. In Xenopus oocytes, the serum and glucocorticoid-sensitive kinase SGK1 has been shown to increase ENaC activity by enhancing its abundance in the plasma membrane. With the same method, ROMK1 appeared to be insensitive to regulation by SGK1. On the other hand, ROMK1 has been shown to colocalize with NHERF2, a protein mediating targeting and trafficking of transport proteins into the cell membrane. The present study has been performed to test whether NHERF2 is required for regulation of ROMK1 by SGK1. Coexpression of neither NHERF2 nor SGK1 with ROMK1 increases ROMK1 activity. However, coexpression of NHERF2 and SGK1 together with ROMK1 markedly increases K(+) channel activity. The combined effect of SGK1 and NHERF2 does not significantly alter the I/V relation of the channel but increases the abundance of the channel in the membrane and decreases the decay of channel activity after inhibition of vesicle insertion with brefeldin. Coexpression of NHERF2 and SGK1 does not modify cytosolic pH but leads to a slight shift of pK(a) of ROMK1 to more acidic values. In conclusion, NHERF2 and SGK1 interact to enhance ROMK1 activity in large part by enhancing the abundance of channel protein within the cell membrane. This interaction allows the integration of genomic regulation and activation of SGK1 and NHERF2 in the control of ROMK1 activity and renal K(+) excretion.

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.

Activating Mutation of the Renal Epithelial Chloride Channel ClC-Kb Predisposing to Hypertension

The chloride channel ClC-Kb is expressed in the basolateral cell membrane of the distal nephron and participates in renal NaCl reabsorption. Loss-of-function mutations of ClC-Kb lead to classic Bartter syndrome, a rare salt-wasting disorder. Recently, we identified the ClC-KbT481S polymorphism, which confers a strong gain-of-function effect on the ClC-Kb chloride channel. The present study has been performed to explore the prevalence of the mutation and its functional significance in renal salt handling and blood pressure regulation. As evident from electrophysiological analysis with the 2-electrode voltage-clamp technique, heterologous expression of ClC-KbT481S in Xenopus oocytes gave rise to a current that was 7-fold larger than the current produced by wild-type ClC-Kb. The prevalence of the mutant allele was significantly higher in an African population from Ghana (22%) than in whites (12%). As tested in 1 white population, carriers of ClC-KbT481S were associated with significantly higher systolic (by ≈6.0 mm Hg) and diastolic (by ≈4.2 mm Hg) blood pressures and significantly higher prevalence (45% versus 25%) of hypertensive (≥140/90 mm Hg) blood pressure levels. Individuals carrying ClC-KbT481S had significantly higher plasma Na+ concentrations and significantly decreased glomerular filtration rate. In conclusion, the mutation ClC-KbT481S of the renal epithelial Cl− channel ClC-Kb strongly activates ClC-Kb chloride channel function in vitro and may predispose to the development of essential hypertension in vivo.

Regulation of the voltage gated K+ channel Kv1.3 by the ubiquitin ligase Nedd4-2 and the serum and glucocorticoid inducible kinase SGK1

The stimulation of cell proliferation by insulin like growth factor IGF‐1 has previously been shown to depend on activation of voltage gated K+ channels. The signaling involved in activation of voltage gated K+ channel Kv1.3 includes the phosphatidylinositol‐3 (PI3) protein kinase, 3‐phosphoinositide dependent protein kinase PDK1 and the serum and glucocorticoid inducible kinase SGK1. However, nothing is known about mechanisms mediating the stimulation of Kv1.3 by SGK1. Most recently, SGK1 has been shown to phosphorylate and thus inactivate the ubiquitin ligase Nedd4‐2. The present study has been performed to explore whether the regulation of Kv1.3 involves Nedd4‐2. To this end Kv1.3 has been expressed in Xenopus oocytes with or without coexpression of Nedd4‐2 and/or constitutively active S422DSGK1. In oocytes expressing Kv1.3 but not in water injected oocytes, depolarization from a holding potential of −80 mV to +20 mV triggers rapidly inactivating currents typical for Kv1.3. Coexpression of Nedd4‐2 decreases, coexpression of S422DSGK1 enhances the currents significantly. The effects of either Nedd4‐2 or of SGK1 are abrogated by destruction of the respective catalytic subunits (C938SNedd4‐2 or K127NSGK1). Further experiments revealed that wild type SGK1 and SGK3 and to a lesser extent SGK2 are similarly effective in stimulating Kv1.3 in both, presence and absence of Nedd4‐2. It is concluded that Kv1.3 is downregulated by Nedd4‐2 and stimulates by SGK1, SGK2, and SGK3. The data thus disclose a novel mechanism of Kv1.3 channel regulation. J. Cell. Physiol. 199: 194–199, 2004© 2003 Wiley‐Liss, Inc.

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.

Properties and regulation of glutamine transporter SN1 by protein kinases SGK and PKB.

The amino acid transporter SN1 with substrate specificity identical to the amino acid transport system N is expressed mainly in astrocytes and hepatocytes where it accomplishes Na(+)-coupled glutamine uptake and efflux. To characterize properties and regulation of SN1, substrate-induced currents and/or radioactive glutamine uptake were determined in Xenopus oocytes injected with cRNA encoding SN1, the ubiquitin ligase Nedd4-2, and/or the constitutively active serum and glucocorticoid inducible kinase S422DSGK1, its isoform SGK3, and the constitutively active protein kinase B T308D,S473DPKB. The substrate-induced currents were enhanced by increasing glutamine and/or Na(+) concentrations, hyperpolarization, and alkalinization (pH 8.0). They were inhibited by acidification (pH 6.0). Coexpression of Nedd4-2 downregulated SN1-mediated transport, an effect reversed by coexpression of S422DSGK1, SGK3, and T308D,S473DPKB. It is concluded that SN1 is a target for the ubiquitin ligase Nedd4-2, which is inactivated by the serum and glucocorticoid inducible kinase SGK1, its isoform SGK3, and protein kinase B.

Serum and glucocorticoid inducible kinases in the regulation of the cardiac sodium channel SCN5A

The serum and glucocorticoid inducible kinase SGK1 and its isoform SGK3 are both expressed in cardiac tissue. One of the functions of SGK1 is the phosphorylation and inactivation of the ubiquitin ligase Nedd4-2, which in turn could be shown to downregulate the voltage-gated Na+ channel SCN5A (hH1). The present study has been performed to test for a role of SGK1 and SGK3 in the regulation of SCN5A. To this end cRNA encoding the human Na+ channel SCN5A was injected into Xenopus laevis oocytes with or without cRNA encoding the wild-type kinases SGK1, the constitutively active kinase (S422D)SGK1, the inactive form K127NSGK1 or the wild-type SGK3. SCN5A currents were activated by coexpression of either wild-type SGK1 or SGK3 or the constitutively active S422DSGK1. In contrast, the inactive mutant K127NSGK1 significantly decreased the currents. Moreover, coexpression of SGK3 significantly altered SCN5A gating, i.e. it hyperpolarized the activation threshold and depolarized the prepotential required for 50% availability of the channel. Opposite shifts of gating properties were elicited by mutation of serine to alanine (S483ASCN5A and S663ASCN5A) in the SGK consensus sequences of SCN5A. The present observations disclose a role of the kinases SGK1 and SGK3 in the regulation of cardiac Na+ channels. As SGK1 is upregulated by glucocorticoids, mineralocorticoids and a variety of inflammatory mediators and both kinases are activated by insulin and IGF1, the kinases could mediate effects of those hormones and mediators on cardiac function.

Modulation of the Voltage-Gated Potassium Channel Kv1.5 by the SGK1 Protein Kinase Involves Inhibition of Channel Ubiquitination

The serum and glucocorticoid inducible kinase SGK1 is involved in dexamethasone-induced inhibition of insulin secretion by increasing voltage-gated potassium channel (Kv) activity. SGK1 upregulates the Kv1.5 channel but the precise mechanism underlying the SGK1 dependent regulation of Kv1.5 has not been defined yet. The present study explored the signal transduction processes involved. Expression studies in Xenopus oocytes revealed that SGK1 promotes channel activity by interfering with the Nedd4-2 ubiquitination pathway, irrespective of the presence of putative SGK1 phosphorylation sites on Kv1.5. Expression of the ubiquitin ligase Nedd4-2 declined Kv1.5 currents by ubiquitinating and thereby reducing Kv1.5 plasma membrane expression. Increasing concentrations of SGK1 gradually compensated the inhibiting effect of Nedd4-2 on Kv1.5. Enhanced Kv1.5 surface abundance by SGK1 reflects decreased channel internalization as indicated by Brefeldin A experiments. In conclusion, Kv1.5 upregulation by SGK1 involves inhibition of channel ubiquitination by Nedd4-2 that leads to Kv1.5 stabilization in the plasma membrane. Our results suggest that the kinase might participate in the regulation of insulin secretion in part by controlling Kv1.5 surface abundance.

Cloning, Heterologous Expression, and Characterization of Three Aquaglyceroporins from Trypanosoma brucei

Trypanosoma brucei, causative for African sleeping sickness, relies exclusively on glycolysis for ATP production. Under anaerobic conditions, glucose is converted to equimolar amounts of glycerol and pyruvate, which are both secreted from the parasite. As we have shown previously, glycerol transport in T. brucei occurs via specific membrane proteins (Wille, U., Schade, B., and Duszenko, M. (1998) Eur. J. Biochem. 256, 245–250). Here, we describe cloning and biochemical characterization of the three trypanosomal aquaglyceroporins (AQP; TbAQP1–3), which show a 40–45% identity to mammalian AQP3 and -9. AQPs belong to the major intrinsic protein family and represent channels for small non-ionic molecules. Both TbAQP1 and TbAQP3 contain two highly conserved NPA motifs within the pore-forming region, whereas TbAQP2 contains NSA and NPS motifs instead, which are only occasionally found in AQPs. For functional characterization, all three proteins were heterologously expressed in yeast and Xenopus oocytes. In the yeast fps1Δ mutant, TbAQPs suppressed hypoosmosensitivity and rendered cells to a hyper-osmosensitive phenotype, as expected for unregulated glycerol channels. Under iso- and hyperosmotic conditions, these cells constitutively released glycerol, consistent with a glycerol efflux function of TbAQP proteins. TbAQP expression in Xenopus oocytes increased permeability for water, glycerol and, interestingly, dihydroxyacetone. Except for urea, TbAQPs were virtually impermeable for other polyols; only TbAQP3 transported erythritol and ribitol. Thus, TbAQPs represent mainly water/glycerol/dihydroxyacetone channels involved in osmoregulation and glycerol metabolism in T. brucei. This function and especially the so far not investigated transport of dihydroxyacetone may be pivotal for the survival of the parasite survival under non-aerobic or osmotic stress conditions.

Regulation of the Glutamate Transporter EAAT4 by PIKfyve

The excitatory amino-acid transporter EAAT4 (SLC1A6), a Na+,glutamate cotransporter expressed mainly in Purkinje cells, serves to clear glutamate from the synaptic cleft. EAAT4 activity is stimulated by the serum and glucocorticoid inducible kinase SGK1. SGK1-dependent regulation of the Na+,glucose transporter SGLT1 (SLC5A1) and the creatine transporter CreaT (SLC6A8) has recently been shown to involve the mammalian phosphatidylinositol-3-phosphate-5-kinase PIKfyve (PIP5K3). The present experiments thus explored whether SGK1-dependent EAAT4-regulation similarly involves PIKfyve. In Xenopus oocytes expressing EAAT4, but not in water injected oocytes, glutamate induced a current which was significantly enhanced by coexpression of PIKfyve and SGK1. The glutamate induced current in Xenopus oocytes coexpressing EAAT4 and both, PIKfyve and SGK1, was significantly larger than the current in Xenopus oocytes expressing EAAT4 together with either kinase alone. Coexpression of the inactive SGK1 mutant K127NSGK1 did not significantly alter glutamate induced current in EAAT4-expressing Xenopus oocytes and abolished the stimulation of glutamate induced current by coexpression of PIKfyve. The stimulating effect of PIKfyve was abrogated by replacement of the serine with alanine in the SGK consensus sequence (S318APIKfyve). Furthermore, coexpression of S318APIKfyve significantly blunted the stimulating effect of SGK1 on EAAT4 activity. The observations disclose that PIKfyve indeed participates in the regulation of EAAT4.