Jost Ludwig

A sequence motif responsible for ER export and surface expression of Kir2.0 inward rectifier K(+) channels.

Integral membrane proteins are sorted via the secretory pathway. It was proposed that this pathway is non‐selective provided that the cargo protein is properly assembled and lacks an endoplasmic reticulum (ER) retention signal. However, recent experimental evidence suggests that efficient export of proteins from the ER to the Golgi complex is not simply a default pathway. Here we demonstrate a novel sequence motif (FxYENEV) in the cytoplasmic C‐terminus of mammalian inward rectifier potassium (Kir) channels which determines ER export. This motif is found to be both necessary and sufficient for efficient export from the ER that eventually leads to efficient surface expression of Kir2.1 channels.

Characterization of potassium transport in wild‐type and isogenic yeast strains carrying all combinations of trk1, trk2 and tok1 null mutations

Saccharomyces cerevisiae cells express three defined potassium‐specific transport systems en‐coded by TRK1 , TRK2 and TOK1 . To gain a more complete understanding of the physiological function of these transport proteins, we have constructed a set of isogenic yeast strains carrying all combinations of trk1 Δ, trk2 Δ and tok1 Δ null mutations. The in vivo K + transport characteristics of each strain have been documented using growth‐based assays, and the in vitro biochemical and electrophysiological properties associated with K + transport have been determined. As has been reported previously, Trk1p and Trk2p facilitate high‐affinity potassium uptake and appear to be functionally redundant under a wide range of environmental conditions. In the absence of TRK1 and TRK2 , strains lack the ability specifically to take up K + , and trk1 Δ trk2 Δ double mutant cells depend upon poorly understood non‐specific cation uptake mechanisms for growth. Under conditions that impair the activity of the non‐specific uptake system, termed NSC1, we have found that the presence of functional Tok1p renders cells sensitive to Cs + . Based on this finding, we have established a growth‐based assay that monitors the in vivo activity of Tok1p.

AMINO TERMINAL-DEPENDENT GATING OF THE POTASSIUM CHANNEL RAT EAG IS COMPENSATED BY A MUTATION IN THE S4 SEGMENT

1 Rat eag potassium channels (r‐eag) were expressed in Xenopus oocytes. They gave rise to delayed rectifying K+ currents with a strong Cole‐Moore effect. 2 Deletions in the N‐terminal structure of r‐eag either shifted the activation threshold to more negative potentials and slowed the activation kinetics (Δ2–190, Δ2–12 and Δ7–12) or resulted in a shift to more positive potentials and faster activation kinetics (Δ150–162). 3 The impact of the deletion Δ7–12 was investigated in more detail: it almost abolished the Cole‐Moore effect and markedly slowed down channel deactivation. 4 Unlike wild‐type channels, the deletion mutants Δ7–12 exhibited a rapid inactivation which, in combination with the slow deactivation, resulted in current characteristics which were similar to those of the related potassium channel HERG. 5 Both the slowing of deactivation and the inactivation induced by the deletion Δ7–12 were compensated by a single histidine‐to‐arginine change in the S4 segment, while this mutation (H343R) only had minor effects on the gating kinetics of the full‐length r‐eag channel. 6 These results demonstrate a functional role of the N‐terminus in the voltage‐dependent gating of potassium channels which is presumably mediated by an interaction of the N‐terminal protein structure with the S4 motif during the gating process.

Cloning and functional expression of rat ether-à-go-go-like K+ channel genes

1 Screening of rat cortex cDNA resulted in cloning of two complete and one partial orthologue of the Drosophilaether‐à‐go‐go‐like K+ channel (elk). 2 Northern blot and reverse transcriptase‐polymerase chain reaction (RT‐PCR) analysis revealed predominant expression of rat elk mRNAs in brain. Each rat elk mRNA showed a distinct, but overlapping expression pattern in different rat brain areas. 3 Transient transfection of Chinese hamster ovary (CHO) cells with rat elk1 or rat elk2 cDNA gave rise to voltage‐activated K+ channels with novel properties. 4 RELK1 channels mediated slowly activating sustained potassium currents. The threshold for activation was at −90 mV. Currents were insensitive to tetraethylammonium (TEA) and 4‐aminopyridine (4‐AP), but were blocked by micromolar concentrations of Ba2+. RELK1 activation kinetics were not dependent on prepulse potential like REAG‐mediated currents. 5 RELK2 channels produced currents with a fast inactivation component and HERG‐like tail currents. RELK2 currents were not sensitive to the HERG channel blocker E4031.

A yeast-based method for the detection of cyto and genotoxicity

A miniaturized short-term in vivo genotoxicity screening assay based on genetically modified yeast (Saccharomyces cerevisiae) cells was performed to explore the capacity of this eukaryotic organism to detect the presence of genotoxic compounds. An increased general sensitivity of yeast cells to toxic compounds was obtained by using a strain being deleted in the prominent pleiotropic drug resistance mediating efflux transporters PDR5, SNQ2 and YOR1. In order to detect genotoxic effects, a yeast optimized version of the green fluorescent protein (GFP) was fused to the RAD54 promoter that is activated upon DNA damage. Various model substances including the oxygenated fuel additive methyl tertiary-butyl ether (MTBE) and the direct acting genotoxins methyl-N-nitro-N-nitrosoguanidine (MNNG) and 4-nitroquinoline-1-oxide (4-NQO) were tested. All model substances were in parallel examined for chronic cytotoxicity. The results point out the sufficiency of both the sensitivity of the yeast cells to detect chronic cytotoxicity and the intensity of the fluorescence signal for the assessment of genotoxic effects. Thus, the test enables simultaneous detection of cytotoxic and genotoxic effects. By partial automation and implementation of the test in the microtitre scale this bioassay allows parallel sensitive pre-screening of numerous samples.

K+-dependent gating of Kir1.1 channels is linked to pH gating through a conformational change in the pore

1 We have used giant patch‐clamp recording to investigate the interaction between pH gating and K+‐dependent gating in rat Kir1.1 (ROMK) channels heterologously expressed in Xenopus oocytes. 2 Gating by intracellular protons (pH gating) and extracellular K+ ions (K+‐dependent gating) is a hallmark of Kir1.1 channels that mediate K+ secretion and control NaCl reabsorption in the kidney. pH gating is driven by protonation of an intracellular lysine residue (K80 in Kir1.1). K+‐dependent gating occurs upon withdrawal of K+ ions from the extracellular side of the channel. Both gating mechanisms are thought to interact allosterically. 3 K+‐dependent gating was shown to be strictly coupled to pH gating; it only occurred when channels were in the pH‐inactivated closed state, but not in the open state. Moreover, K+‐dependent gating was absent in the non‐pH‐gated mutant Kir1.1(K80M). 4 Channels inactivated by K+‐dependent gating were reactivated upon addition of permeant ions to the extracellular side of the membrane, while impermeant ions failed to induce channel reactivation. Moreover, mutagenesis identified two residues in the P‐helix (L136 and V140 in Kir1.1) that are crucial for K+‐dependent gating. Replacement of these residues with the ones present in the non‐K+‐gated Kir2.1 abolished K+‐dependent gating of Kir1.1 channels without affecting pH gating. 5 The results indicate that pH gating and K+‐dependent gating are coupled to each other via structural rearrangements in the inner pore involving the P‐helix.

Estrogenic effects of natural and synthetic compounds including tibolone assessed in Saccharomyces cerevisiae expressing the human estrogen α and β receptors

The human estrogen receptors (hER)α and hERβ, differentially expressed and localized in various tissues and cell types, mediate transcriptional activation of target genes. These encode a variety of physiological reproductive and nonreproductive functions involved in energy metabolism, salt balance, immune system, development, and differentiation. As a step toward developing a screening assay for the use in applications where significant numbers of compounds or complex matrices need to be tested for (anti) estrogenic bioactivity, hERα and hERβ were expressed in a genetically modified Saccharomyces cerevisiae strain, devoid of three endogenous xenobiotic transporters (PDR5, SNQ2, and FOR1). By using receptor‐mediated transcriptional activation of the green fluorescent protein optimized for expression in yeast (yEGFP) as reporter 17 natural, comprising estrogens and phytoestrogens or synthetic compounds among which tibolone with its metabolites, gestagens, and antiestrogens were investigated. The reporter assay deployed a simple and robust protocol for the rapid detection of estrogenic effects within a 96‐well microplate format. Results were expressed as effective concentrations (EC50) and correlated to other yeast based and cell line assays. Tibolone and its metabolites exerted clear estrogenic effects, though considerably less potent than all other natural and synthetic compounds. For the blood serum of two volunteers, considerable higher total estrogenic bioactivity than single estradiol concentrations as determined by immunoassay was found. Visualization of a hERα/GFP fusion protein in yeast revealed a sub cellular cytosolic localization. This study demonstrates the versatility of (anti) estrogenic bioactivity determination using sensitized S. cerevisiae cells to assess estrogenic exposure and effects.—Hasenbrink, G., Sievernich, A., Wildt, L., Ludwig, J., and Lichtenberg‐Fraté, H. Estrogenic effects of natural and synthetic compunds including tibolone assessed in Saccharomyces cerevisiae expressing the human estrogen α and β receptors. FASEB J. 20, E861–E870 (2006)

A physiological role for ether-à-go-go K+ channels?

The physiology of the ether-a-go-go K + channel resembles that of the mammalian M-current when expressed in vitro.

Analysis of the mKir2.1 channel activity in potassium influx defective Saccharomyces cerevisiae strains determined as changes in growth characteristics.

Potassium uptake defective Saccharomyces cerevisiae strains (Δtrk1,2 and Δtrk1,2 Δtok1) were used for the phenotypic analysis of the mouse inward rectifying Kir2.1 channel by growth analysis. Functional expression of both, multi‐copy plasmid and chromosomally expressed GFP‐mKir2.1 fusion constructs complemented the potassium uptake deficient phenotype in a pHout dependent manner. Upon application of Hygromycin B to chromosomally mKir2.1 expressing cells, significantly lower toxin sensitivity (EC50 15.4 μM) compared to Δtrk1,2 Δtok1 cells (EC50 2.6 μM) was observed. Growth determination of mKir2.1 expressing strains upon application of Ag+, Cs+ and Ba2+ as known blockers of mKir2.1 channels revealed significantly decreased channel function. Cells with mKir2.1 were about double sensitive to AgNO3, 350‐fold more sensitive to CsCl and 1500‐fold more sensitive to BaCl2 in comparison to the respective controls indicating functional expression and correct pharmacology.

Systems biology of monovalent cation homeostasis in yeast: the translucent contribution

Maintenance of monovalent cation homeostasis (mainly K(+) and Na(+)) is vital for cell survival, and cation toxicity is at the basis of a myriad of relevant phenomena, such as salt stress in crops and diverse human diseases. Full understanding of the importance of monovalent cations in the biology of the cell can only be achieved from a systemic perspective. Translucent is a multinational project developed within the context of the SysMO (System Biology of Microorganisms) initiative and focussed in the study of cation homeostasis using the well-known yeast Saccharomyces cerevisiae as a model. The present review summarize how the combination of biochemical, genetic, genomic and computational approaches has boosted our knowledge in this field, providing the basis for a more comprehensive and coherent vision of the role of monovalent cations in the biology of the cell.