Pascal Mäser

The Potassium Transporter AtHAK5 Functions in K+ Deprivation-Induced High-Affinity K+ Uptake and AKT1 K+ Channel Contribution to K+ Uptake Kinetics in Arabidopsis Roots

Potassium is an important macronutrient and the most abundant cation in plants. Because soil mineral conditions can vary, plants must be able to adjust to different nutrient availabilities. Here, we used Affymetrix Genechip microarrays to identify genes responsive to potassium (K+) deprivation in roots of mature Arabidopsis (Arabidopsis thaliana) plants. Unexpectedly, only a few genes were changed in their expression level after 6, 48, and 96 h of K+ starvation even though root K+ content was reduced by approximately 60%. AtHAK5, a potassium transporter gene from the KUP/HAK/KT family, was most consistently and strongly up-regulated in its expression level across 48-h, 96-h, and 7-d K+ deprivation experiments. AtHAK5 promoter-β-glucuronidase and -green fluorescent protein fusions showed AtHAK5 promoter activity in the epidermis and vasculature of K+ deprived roots. Rb+ uptake kinetics in roots of athak5 T-DNA insertion mutants and wild-type plants demonstrated the absence of a major part of an inducible high-affinity Rb+/K+ (Km approximately 15–24 μm) transport system in athak5 plants. In comparative analyses, uptake kinetics of the K+ channel mutant akt1-1 showed that akt1-1 roots are mainly impaired in a major transport mechanism, with an apparent affinity of approximately 0.9 mm K+(Rb+). Data show adaptation of apparent K+ affinities of Arabidopsis roots when individual K+ transporter genes are disrupted. In addition, the limited transcriptome-wide response to K+ starvation indicates that posttranscriptional mechanisms may play important roles in root adaptation to K+ availability in Arabidopsis. The results demonstrate an in vivo function for AtHAK5 in the inducible high-affinity K+ uptake system in Arabidopsis roots.

Altered shoot/root Na+ distribution and bifurcating salt sensitivity in Arabidopsis by genetic disruption of the Na+ transporter AtHKT1

Sodium (Na+) is toxic to most plants, but the molecular mechanisms of plant Na+ uptake and distribution remain largely unknown. Here we analyze Arabidopsis lines disrupted in the Na+ transporter AtHKT1. AtHKT1 is expressed in the root stele and leaf vasculature. athkt1 null plants exhibit lower root Na+ levels and are more salt resistant than wild‐type in short‐term root growth assays. In shoot tissues, however, athkt1 disruption produces higher Na+ levels, and athkt1 and athkt1/sos3 shoots are Na+‐hypersensitive in long‐term growth assays. Thus wild‐type AtHKT1 controls root/shoot Na+ distribution and counteracts salt stress in leaves by reducing leaf Na+ accumulation.

Plant Ion Channels: Gene Families, Physiology, and Functional Genomics Analyses

Distinct potassium, anion, and calcium channels in the plasma membrane and vacuolar membrane of plant cells have been identified and characterized by patch clamping. Primarily owing to advances in Arabidopsis genetics and genomics, and yeast functional complementation, many of the corresponding genes have been identified. Recent advances in our understanding of ion channel genes that mediate signal transduction and ion transport are discussed here. Some plant ion channels, for example, ALMT and SLAC anion channel subunits, are unique. The majority of plant ion channel families exhibit homology to animal genes; such families include both hyperpolarization- and depolarization-activated Shaker-type potassium channels, CLC chloride transporters/channels, cyclic nucleotide-gated channels, and ionotropic glutamate receptor homologs. These plant ion channels offer unique opportunities to analyze the structural mechanisms and functions of ion channels. Here we review gene families of selected plant ion channel classes and discuss unique structure-function aspects and their physiological roles in plant cell signaling and transport.

Potassium transporters in plants--involvement in K+ acquisition, redistribution and homeostasis

Potassium is a major plant nutrient which has to be accumulated in great quantity by roots and distributed throughout the plant and within plant cells. Membrane transport of potassium can be mediated by potassium channels and secondary potassium transporters. Plant potassium transporters are present in three families of membrane proteins: the K+ uptake permeases (KT/HAK/KUP), the K+ transporter (Trk/HKT) family and the cation proton antiporters (CPA). This review will discuss the contribution of members of each family to potassium acquisition, redistribution and homeostasis.

Identification of GPI anchor attachment signals by a Kohonen self-organizing map

MOTIVATION Anchoring of proteins to the extracytosolic leaflet of membranes via C-terminal attachment of glycosylphosphatidylinositol (GPI) is ubiquitous and essential in eukaryotes. The signal for GPI-anchoring is confined to the C-terminus of the target protein. In order to identify anchoring signals in silico, we have trained neural networks on known GPI-anchored proteins, systematically optimizing input parameters. RESULTS A Kohonen self-organizing map, GPI-SOM, was developed that predicts GPI-anchored proteins with high accuracy. In combination with SignalP, GPI-SOM was used in genome-wide surveys for GPI-anchored proteins in diverse eukaryotes. Apart from specialized parasites, a general trend towards higher percentages of GPI-anchored proteins in larger proteomes was observed. AVAILABILITY GPI-SOM is accessible on-line at http://gpi.unibe.ch. The source code (written in C) is available on the same website. SUPPLEMENTARY INFORMATION Positive training set, performance test sets and lists of predicted GPI-anchored proteins from different eukaryotes in fasta format.

Glycine residues in potassium channel-like selectivity filters determine potassium selectivity in four-loop-per-subunit HKT transporters from plants

Plant HKT proteins comprise a family of cation transporters together with prokaryotic KtrB, TrkH, and KdpA transporter subunits and fungal Trk proteins. These transporters contain four loop domains in one polypeptide with a proposed distant homology to K+ channel selectivity filters. Functional expression in yeast and Xenopus oocytes revealed that wheat HKT1 mediates Na+-coupled K+ transport. Arabidopsis AtHKT1, however, transports only Na+ in eukaryotic expression systems. To understand the molecular basis of this difference we constructed a series of AtHKT1/HKT1 chimeras and introduced point mutations to AtHKT1 and wheat HKT1 at positions predicted to be critical for K+ selectivity. A single-point mutation, Ser-68 to glycine, was sufficient to restore K+ permeability to AtHKT1. The reverse mutation in HKT1, Gly-91 to serine, abrogated K+ permeability. This glycine in P-loop A of AtHKT1 and HKT1 can be modeled as the first glycine of the K+ channel selectivity filter GYG motif. The importance of such filter glycines for K+ selectivity was confirmed by interconversion of Ser-88 and Gly-88 in the rice paralogues OsHKT1 and OsHKT2. Surprisingly, all HKT homologues known from dicots have a serine at the filter position in P-loop A, suggesting that these proteins function mainly as Na+ transporters in plants and that Na+/K+ symport in HKT proteins is associated with a glycine in the filter residue. These data provide experimental evidence that the glycine residues in selectivity filters of HKT proteins are structurally related to those of K+ channels.

Mechanisms of arsenical and diamidine uptake and resistance in Trypanosoma brucei.

ABSTRACT Sleeping sickness, caused by Trypanosoma brucei spp., has become resurgent in sub-Saharan Africa. Moreover, there is an alarming increase in treatment failures with melarsoprol, the principal agent used against late-stage sleeping sickness. In T. brucei, the uptake of melarsoprol as well as diamidines is thought to be mediated by the P2 aminopurine transporter, and loss of P2 function has been implicated in resistance to these agents. The trypanosomal gene TbAT1 has been found to encode a P2-type transporter when expressed in yeast. Here we investigate the role of TbAT1 in drug uptake and drug resistance in T. brucei by genetic knockout of TbAT1. Tbat1-null trypanosomes were deficient in P2-type adenosine transport and lacked adenosine-sensitive transport of pentamidine and melaminophenyl arsenicals. However, the null mutants were only slightly resistant to melaminophenyl arsenicals and pentamidine, while resistance to other diamidines such as diminazene was more pronounced. Nevertheless, the reduction in drug sensitivity might be of clinical significance, since mice infected with tbat1-null trypanosomes could not be cured with 2 mg of melarsoprol/kg of body weight for four consecutive days, whereas mice infected with the parental line were all cured by using this protocol. Two additional pentamidine transporters, HAPT1 and LAPT1, were still present in the null mutant, and evidence is presented that HAPT1 may be responsible for the residual uptake of melaminophenyl arsenicals. High-level arsenical resistance therefore appears to involve the loss of more than one transporter.

Drug resistance in African trypanosomiasis: the melarsoprol and pentamidine story

Melarsoprol and pentamidine represent the two main classes of drugs, the arsenicals and diamidines, historically used to treat the diseases caused by African trypanosomes: sleeping sickness in humans and Nagana in livestock. Cross-resistance to these drugs was first observed over 60 years ago and remains the only example of cross-resistance among sleeping sickness therapies. A Trypanosoma brucei adenosine transporter is well known for its role in the uptake of both drugs. More recently, aquaglyceroporin 2 (AQP2) loss of function was linked to melarsoprol-pentamidine cross-resistance. AQP2, a channel that appears to facilitate drug accumulation, may also be linked to clinical cases of resistance. Here, we review these findings and consider some new questions as well as future prospects for tackling the devastating diseases caused by these parasites.

The genome of the heartworm, Dirofilaria immitis, reveals drug and vaccine targets

The heartworm Dirofilaria immitis is an important parasite of dogs. Transmitted by mosquitoes in warmer climatic zones, it is spreading across southern Europe and the Americas at an alarming pace. There is no vaccine, and chemotherapy is prone to complications. To learn more about this parasite, we have sequenced the genomes of D. immitis and its endosymbiont Wolbachia. We predict 10,179 protein coding genes in the 84.2 Mb of the nuclear genome, and 823 genes in the 0.9‐Mb Wolbachia genome. The D. immitis genome harbors neither DNA transposons nor active retrotransposons, and there is very little genetic variation between two sequenced isolates from Europe and the United States. The differential presence of anabolic pathways such as heme and nucleotide biosynthesis hints at the intricate metabolic interrelationship between the heartworm and Wolbachia. Comparing the proteome of D. immitis with other nematodes and with mammalian hosts, we identify families of potential drug targets, immune modulators, and vaccine candidates. This genome sequence will support the development of new tools against dirofilariasis and aid efforts to combat related human pathogens, the causative agents of lymphatic filariasis and river blindness.—Godel, C., Kumar, S., Koutsovoulos, G., Ludin, P., Nilsson, D., Comandatore, F., Wrobel, N., Thompson, M., Schmid, C. D., Goto, S., Bringaud, F., Wolstenholme, A., Bandi, C., Epe, C., Kaminsky, R., Blaxter, M., Mäser, P. The genome of the heartworm, Dirofilaria immitis, reveals drug and vaccine targets. FASEB J. 26, 4650–4661 (2012). www.fasebj.org

Loss of the High-Affinity Pentamidine Transporter Is Responsible for High Levels of Cross-Resistance between Arsenical and Diamidine Drugs in African Trypanosomes

Treatment of many infectious diseases is under threat from drug resistance. Understanding the mechanisms of resistance is as high a priority as the development of new drugs. We have investigated the basis for cross-resistance between the diamidine and melaminophenyl arsenical classes of drugs in African trypanosomes. We induced high levels of pentamidine resistance in a line without the tbat1 gene that encodes the P2 transporter previously implicated in drug uptake. We isolated independent clones that displayed very considerable cross-resistance with melarsen oxide but not phenylarsine oxide and reduced uptake of [3H]pentamidine. In particular, the high-affinity pentamidine transport (HAPT1) activity was absent in the pentamidine-adapted lines, whereas the low affinity pentamidine transport (LAPT1) activity was unchanged. The parental tbat1–/– line was sensitive to lysis by melarsen oxide, and this process was inhibited by low concentrations of pentamidine, indicating the involvement of HAPT1. This pentamidine-inhibitable lysis was absent in the adapted line KO-B48. Likewise, uptake of the fluorescent diamidine 4′,6-diamidino-2-phenylindole dihydrochloride was much delayed in live KO-B48 cells and insensitive to competition with up to 10 μM pentamidine. No overexpression of the Trypanosoma brucei brucei ATP-binding cassette transporter TbMRPA could be detected in KO-B48. We also show that a laboratory line of Trypanosoma brucei gambiense, adapted to high levels of resistance for the melaminophenyl arsenical drug melarsamine hydrochloride (Cymelarsan), had similarly lost TbAT1 and HAPT1 activity while retaining LAPT1 activity. It seems therefore that selection for resistance to either pentamidine or arsenical drugs can result in a similar phenotype of reduced drug accumulation, explaining the occurrence of cross-resistance.