Christoph Böhmer

Regulation of Channels by the Serum and Glucocorticoid-Inducible Kinase - Implications for Transport, Excitability and Cell Proliferation

The serum and glucocorticoid-inducible kinase SGK1 stimulates the Na+ channels ENaC and SCN5A, the K+ channels ROMK1, Kv1.3, and KCNE1/KCNQ1, the cation conductance induced by 4F2/LAT1 and the chloride conductance induced by CFTR. The isoforms SGK2 and SGK3 have similarly been shown to regulate ENaC, SCN5A, Kv1.3 and KCNE1/KCNQ1. The kinases regulate channel abundance in the plasma membrane in part by inhibition of the ubiquitin ligase Nedd4-2 and in part by interaction with trafficking molecules such as the Na+/H+ exchanger regulating factor NHERF2. An in vivo role of SGK1 mediated ENaC channel regulation in renal salt excretion and blood pressure control is documented by the impaired ability of SGK1 knockout mice to adequately reduce renal Na+ output and maintain blood pressure during dietary salt restriction and by enhanced blood pressure in individuals carrying certain polymorphisms in the SGK1 gene. The in vivo physiological significance of SGK dependent regulation of the other channels remains to be shown even though circumstantial evidence points to involvement in the regulation of epithelial transport, cell volume, cell proliferation, cardiac action potential and neuroexcitability. There is little doubt that further channels will be identified which are modulated by the SGKs and that further in vivo physiological functions will be defined where channel regulation by the SGKs plays a critical role.

Acid sphingomyelinase–ceramide system mediates effects of antidepressant drugs

Major depression is a highly prevalent severe mood disorder that is treated with antidepressants. The molecular targets of antidepressants require definition. We investigated the role of the acid sphingomyelinase (Asm)-ceramide system as a target for antidepressants. Therapeutic concentrations of the antidepressants amitriptyline and fluoxetine reduced Asm activity and ceramide concentrations in the hippocampus, increased neuronal proliferation, maturation and survival and improved behavior in mouse models of stress-induced depression. Genetic Asm deficiency abrogated these effects. Mice overexpressing Asm, heterozygous for acid ceramidase, treated with blockers of ceramide metabolism or directly injected with C16 ceramide in the hippocampus had higher ceramide concentrations and lower rates of neuronal proliferation, maturation and survival compared with controls and showed depression-like behavior even in the absence of stress. The decrease of ceramide abundance achieved by antidepressant-mediated inhibition of Asm normalized these effects. Lowering ceramide abundance may thus be a central goal for the future development of antidepressants.

The Shrinkage-activated Na+ Conductance of Rat Hepatocytes and its Possible Correlation to rENaC

At moderate cell shrinkage, activation of Na+ channels is the most prominent mechanism of regulatory cell volume increase in rat hepatocytes. The amiloride sensitivity of these channels suggests a relation to the family of epithelial Na+ channels (ENaCs). The present study was performed to determine the pharmacological profile of shrinkage-activated Na+ channels and to test for ENaC expression in primary cultures of rat hepatocytes; in addition, the influence of the cell volume regulated serine/threonine kinase hSGK on activity and pharmacological profile of rENaC was examined in Xenopus oocytes. Conventional electrophysiology in hepatocytes reveals that the shrinkage-activated Na+ channel is inhibited by amiloride and EIPA with IC50 values of 6.0 and 0.12 μmol/l, respectively. Western blots and RT-PCR demonstrate that rat hepatocytes do express all three subunits (α, β, γ) of ENaC. Coexpression of hSGK with rENaC in Xenopus oocytes reveals that the kinase stimulates ENaC by a factor of 4. Moreover, hSGK decreases the affinity to amiloride (increase of IC50 from 0.12 to 0.26 μmol/l) and increases the affinity to EIPA (decrease of IC50 from 250 to 50 μmol/l). In conclusion, rat hepatocytes express ENaC, which is activated by the cell volume-sensitive kinase hSGK. ENaC may contribute to the Na+ channels activated by osmotic cell shrinkage in hepatocytes, whereby the relatively low amiloride and high EIPA sensitivity of the channel could at least be partially due to modification by SGK, which decreases the amiloride and increases the EIPA sensitivity of ENaC.

Stimulation of Xenopus oocyte Na+,K+ATPase by the serum and glucocorticoid-dependent kinase sgk1

Abstract. The serum and glucocorticoid-dependent kinase-1 (sgk1) is expressed in a wide variety of tissues including renal epithelial cells. As it is up-regulated by aldosterone, it is considered to participate in the regulation of renal Na+ reabsorption. Indeed, co-expression of sgk1 with the renal epithelial Na+ channel (ENaC) augments Na+ channel activity. The aim of the present study was to examine possible effects of sgk1 on Na+/K+-ATPase activity. To this end dual-electrode voltage-clamp experiments were performed in Xenopus oocytes expressing the active kinase S422Dsgk1 or the inactive mutant K127Nsgk1. Na+/K+-ATPase activity was estimated from the hyperpolarization (ΔVm) and the outwardly-directed current (IP) created by addition of extracellular K+ in the presence of K+ channel blocker Ba2+. Both ΔVm and IP were significantly larger in oocytes expressing S422Dsgk1 than in those expressing K127Nsgk1 or having been injected with water. IP was fully inhibited by ouabain. Ion-selective microelectrodes showed that the stimulation of pump current was not the result of altered cytosolic Na+ activity or pH. The present results thus point to an additional action of sgk1 that may participate in the regulation of renal tubular Na+ transport. Moreover, sgk1 may be involved in the regulation of Na+/K+-ATPase in extrarenal tissues.

Characterization of mouse amino acid transporter B0AT1 (slc6a19)

The mechanism of the mouse (m)B0AT1 (slc6a19) transporter was studied in detail using two electrode voltage-clamp techniques and tracer studies in the Xenopus oocyte expression system. All neutral amino acids induced inward currents at physiological potentials, but large neutral non-aromatic amino acids were the preferred substrates of mB0AT1. Substrates were transported with K0.5 values ranging from approx. 1 mM to approx. 10 mM. The transporter mediates Na+-amino acid co-transport with a stoichiometry of 1:1. No other ions were involved in the transport mechanism. An increase in the extracellular Na+ concentration reduced the K0.5 for leucine, and vice versa. Moreover, the K0.5 values and Vmax values of both substrates varied with the membrane potential. As a result, K0.5 and Vmax values are a complex function of the concentration of substrate and co-substrate and the membrane potential. A model is presented assuming random binding order and a positive charge associated with the ternary [Na+-substrate-transporter] complex, which is consistent with the experimental data.

Activation of Na+/K+-ATPase by the Serum and Glucocorticoid-Dependent Kinase Isoforms

Background/Aim: Expression of the constitutively active form of serum and glucocorticoid-dependent kinase (^S422DSGK1) in Xenopus oocytes has recently been shown to upregulate endogenous Na⁺/K⁺-ATPase activity, an effect presumably participating in the regulation of cellular K⁺ uptake and transepithelial Na⁺ transport. SGK1 and the two isoforms SGK2 and SGK3 are stimulated by insulin and insulin-like growth factor-1 (IGF-1), which have been shown to enhance Na⁺/K⁺-ATPase activity in a variety of cells. The present experiments have been performed to elucidate whether or not wild-type SGK1, SGK2 and SGK3 are similar to ^S422DSGK1 in being effective regulators of Na⁺/K⁺-ATPase. Methods: To this end, dual-electrode voltage clamp experiments were performed in Xenopus oocytes injected either with water or with mRNA of constitutively active ^S422DSGK1 and wild-type SGK1, SGK2 or SGK3. Na⁺/K⁺-ATPase activity was estimated from the outward-directed current created by readdition of extracellular K⁺ in the presence of K⁺ channel blocker Ba²⁺ following a 10-min exposure to K⁺-free extracellular fluid. Results: The outward-directed current was fully abolished by incubation with 1 mM ouabain and was significantly larger in oocytes expressing ^S422DSGK1, SGK1, SGK2 or SGK3, as compared to those injected with water. Conclusion: The stimulating effect of SGK1 on the Xenopus oocyte Na⁺/K⁺-ATPase is mimicked by the isoforms SGK2 and SGK3. Thus, all three kinases may participate in the regulation of Na⁺/K⁺-ATPase activity by hormones such as insulin and IGF-1.

Upregulation of HERG Channels by the Serum and Glucocorticoid Inducible Kinase Isoform SGK3

Human ether-a-go-go (HERG) channels participate in the repolarization of the cardiac action potential. Loss of function mutations of HERG lead to delayed cardiac repolarization reflected by prolonged QT interval. HERG channels are regulated through a signaling cascade involving phosphatidylinositol 3 (PI3) kinase. Downstream targets of PI3 kinase include the serum and glucocorticoid inducible kinase (SGK) and protein kinase B (PKB) isoforms. The present study has been performed to explore whether SGK1 and SGK3 participate in the regulation of HERG channel activity. HERG was expressed in Xenopus oocytes with or without additional expression of SGK1 or SGK3. Chemiluminescence was employed to determine HERG plasma membrane protein abundance. Coexpression of SGK3 but not of SGK1 in Xenopus oocytes resulted in an increase of steady state current (IHERG) and enhanced cell membrane protein abundance without affecting gating kinetics of the channel. Replacement of serine by alanine at the two SGK consensus sites decreased IHERG but neither mutation abolished the stimulating effect of SGK3. In conclusion, SGK3 participates in the regulation of HERG by increasing HERG protein abundance in the plasma membrane and may thus modify the duration of the cardiac action potential.

The Hypertonicity-Induced Na+ Conductance of Rat Hepatocytes: Physiological Significance and Molecular Correlate

The initial event in the regulatory volume increase (RVI) of rat hepatocytes is an uptake of extracellular Na⁺ that is then exchanged for K⁺ via stimulation of Na⁺/K⁺-ATPase. While it was generally assumed that this Na⁺ uptake is mediated by the activation of Na⁺/H⁺ antiport and Na⁺-K⁺-2Cl^- symport it could be shown recently that, in addition to these transporters, hypertonic stress also stimulates conductive Na⁺ entry. In a quantitative study, it was found that the relative contribution of Na⁺ conductance, Na⁺/H⁺ antiport, and Na⁺-K⁺-2Cl^- symport to the initial Na⁺ import as well as to the RVI process (at 300 → 400 mosmol/l) is approximately 4 : 1 : 1. When the osmotic sensitivity of these Na⁺ importers was tested (at 300 mosmol/l → 327, 360, 400, 450 mosmol/l) it became clear that Na⁺ conductance is the prominent mechanism of RVI from 360 mosmol/l upwards whereas Na⁺/H⁺ antiport is the most sensitive transporter with 65 % of its maximal activation at 327 mosmol/l already. Concerning the intracellular regulation of the Na⁺ importers involved in RVI it was found that Na⁺ concuctance as well as Na⁺-K⁺-2Cl^- symport – but not Na⁺/H⁺ antiport – are activated via PKC. With respect to the molecular correlate of the volume activated Na⁺ conductance it could be shown that it exhibits a rather low affinity to amiloride (IC⁵⁰ = 6.0 μmol/l) and an overall sensitivity profile of EIPA > amiloride > benzamil = phenamil that, at first sight, would not speak in favor of a typical epithelial type of Na⁺ channel (ENaC). Western-blot analysis and RT-PCR techniques, however, revealed that α-, β-, as well as γ-ENaC are, in fact, expressed in rat hepatocytes. Moreover, by use of an antisense-DNA based approach it could be shown that at least α-ENaC is part of the hypertonicity induced Na⁺ conductance.

Negative charge at the consensus sequence for the serum- and glucocorticoid-inducible kinase, SGK1, determines pH sensitivity of the renal outer medullary K+ channel, ROMK1.

The renal outer medullary K(+)-channel ROMK1 is upregulated by the serum- and glucocorticoid-inducible kinase SGK1, an effect potentiated by Na(+)/H(+)-exchanger-regulating-factor NHERF2. SGK1 phosphorylates ROMK1 at serine44. To explore the role of SGK1 phosphorylation, serine44 was replaced by an alanine ([S44A]ROMK1) or an aspartate ([S44D]ROMK1). Wild type ROMK1, [S44A]ROMK1, and [S44D]ROMK1 were expressed in Xenopus oocytes with or without constitutively active [S422D]SGK1 and NHERF2, and K(+) current (I(KR)) determined. Cytosolic pH required for halfmaximal I(KR) (pK(a)) amounted to 7.05+/-0.01 for ROMK1, 7.07+/-0.02 for [S44A]ROMK1, and 6.83+/-0.05 for [S44D]ROMK1. Maximal I(KR) was [S44D]ROMK1>wild type ROMK1>[S44A]ROMK1. Coexpression of [S422D]SGK1 and NHERF2 enhanced the activity of ROMK1, [S44A]ROMK1 and [S44D]ROMK1, but led to a significant shift of pK(a) only in wild type ROMK1 (6.95+/-0.03). In conclusion, phosphorylation by SGK1 or introduction of a negative charge at serine44 shifts the pH sensitivity of the channel and contributes to the stimulation of maximal channel activity by the kinase.

Regulation of the epithelial calcium channel TRPV6 by the serum and glucocorticoid‐inducible kinase isoforms SGK1 and SGK3

Epithelial calcium (re)absorption is mediated by TRPV5 and TRPV6 channels. TRPV5 is modulated by the SGK1 kinase, a process requiring the PDZ‐domain containing scaffold protein NHERF2. The present study explored whether TRPV6 is similarly regulated by SGKs and the scaffold proteins NHERF1/2. In Xenopus oocytes, SGKs activate TRPV6 by increasing its plasma membrane abundance. Deletion of the putative PDZ binding motif on TRPV6 did not abolish channel activation by SGKs. Furthermore, coexpression of neither NHERF1 nor NHERF2 affected TRPV6 or potentiated the SGKs stimulating effect. The present observations disclose a novel TRPV6 regulatory mechanism which presumably participates in calcium homeostasis.