Publications by authors named "Arne Franzen"

9 Publications

  • Page 1 of 1

Establishing a sensitive fluorescence-based quantification method for cyclic nucleotides.

BMC Biotechnol 2020 08 27;20(1):47. Epub 2020 Aug 27.

Institute of Biological Information Processing (Molecular and Cellular Physiology, IBI-1), Forschungszentrum Jülich, 52428, Jülich, Germany.

Background: Approximately 40% of prescribed drugs exert their activity via GTP-binding protein-coupled receptors (GPCRs). Once activated, these receptors cause transient changes in the concentration of second messengers, e.g., cyclic adenosine 3',5'-monophosphate (cAMP). Specific and efficacious genetically encoded biosensors have been developed to monitor cAMP fluctuations with high spatial and temporal resolution in living cells or tissue. A well characterized biosensor for cAMP is the Förster resonance energy transfer (FRET)-based Epac1-camps protein. Pharmacological characterization of newly developed ligands acting at GPCRs often includes numerical quantification of the second messenger amount that was produced.

Results: To quantify cellular cAMP concentrations, we bacterially over-expressed and purified Epac1-camps and applied the purified protein in a cell-free detection assay for cAMP in a multi-well format. We found that the biosensor can detect as little as 0.15 pmol of cAMP, and that the sensitivity is not impaired by non-physiological salt concentrations or pH values. Notably, the assay tolerated desiccation and storage of the protein without affecting Epac1-camps cyclic nucleotide sensitivity.

Conclusions: We found that determination cAMP in lysates obtained from cell assays or tissue samples by purified Epac1-camps is a robust, fast, and sensitive assay suitable for routine and high throughput analyses.
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http://dx.doi.org/10.1186/s12896-020-00633-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7450941PMC
August 2020

An amino-terminal point mutation increases EAAT2 anion currents without affecting glutamate transport rates.

J Biol Chem 2020 10 20;295(44):14936-14947. Epub 2020 Aug 20.

Molekular- und Zellphysiologie (IBI-1), Institute of Biological Information Processing, Forschungszentrum Jülich, Jülich, Germany. Electronic address:

Excitatory amino acid transporters (EAATs) are prototypical dual function proteins that function as coupled glutamate/Na/H/K transporters and as anion-selective channels. Both transport functions are intimately intertwined at the structural level: Secondary active glutamate transport is based on elevator-like movements of the mobile transport domain across the membrane, and the lateral movement of this domain results in anion channel opening. This particular anion channel gating mechanism predicts the existence of mutant transporters with changed anion channel properties, but without alteration in glutamate transport. We here report that the L46P mutation in the human EAAT2 transporter fulfills this prediction. L46 is a pore-forming residue of the EAAT2 anion channels at the cytoplasmic entrance into the ion conduction pathway. In whole-cell patch clamp recordings, we observed larger macroscopic anion current amplitudes for L46P than for WT EAAT2. Rapid l-glutamate application under forward transport conditions demonstrated that L46P does not reduce the transport rate of individual transporters. In contrast, changes in selectivity made gluconate permeant in L46P EAAT2, and nonstationary noise analysis revealed slightly increased unitary current amplitudes in mutant EAAT2 anion channels. We used unitary current amplitudes and individual transport rates to quantify absolute open probabilities of EAAT2 anion channels from ratios of anion currents by glutamate uptake currents. This analysis revealed up to 7-fold increased absolute open probability of L46P EAAT2 anion channels. Our results reveal an important determinant of the diameter of EAAT2 anion pore and demonstrate the existence of anion channel gating processes outside the EAAT uptake cycle.
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http://dx.doi.org/10.1074/jbc.RA120.013704DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7606670PMC
October 2020

Zinc modulation of proton currents in a new voltage-gated proton channel suggests a mechanism of inhibition.

FEBS J 2020 11 6;287(22):4996-5018. Epub 2020 Apr 6.

Institut für Physiologie und Pathophysiologie, Paracelsus Universität Salzburg Standort Nürnberg, Nuremberg, Germany.

The H 1 voltage-gated proton (H 1) channel is a key component of the cellular proton extrusion machinery and is pivotal for charge compensation during the respiratory burst of phagocytes. The best-described physiological inhibitor of H 1 is Zn . Externally applied ZnCl drastically reduces proton currents reportedly recorded in Homo sapiens, Rattus norvegicus, Mus musculus, Oryctolagus cuniculus, Rana esculenta, Helix aspersa, Ciona intestinalis, Coccolithus pelagicus, Emiliania huxleyi, Danio rerio, Helisoma trivolvis, and Lingulodinium polyedrum, but with considerable species variability. Here, we report the effects of Zn and Cd on H 1 from Nicoletia phytophila, NpH 1. We introduced mutations at potential Zn coordination sites and measured Zn inhibition in different extracellular pH, with Zn concentrations up to 1000 μm. Zn inhibition in NpH 1 was quantified by the slowing of the activation time constant and a positive shift of the conductance-voltage curve. Replacing aspartate in the S3-S4 loop with histidine (D145H) enhanced both the slowing of activation kinetics and the shift in the voltage-conductance curve, such that Zn inhibition closely resembled that of the human channel. Histidine is much more effective than aspartate in coordinating Zn in the S3-S4 linker. A simple Hodgkin Huxley model of NpH 1 suggests a decrease in the opening rate if it is inhibited by zinc or cadmium. Limiting slope measurements and high-resolution clear native gel electrophoresis (hrCNE) confirmed that NpH 1 functions as a dimer. The data support the hypothesis that zinc is coordinated in between the dimer instead of the monomer. Zinc coordination sites may be potential targets for drug development.
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http://dx.doi.org/10.1111/febs.15291DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7754295PMC
November 2020

Allosteric gate modulation confers K coupling in glutamate transporters.

EMBO J 2019 10 10;38(19):e101468. Epub 2019 Sep 10.

Institute of Complex Systems, Zelluläre Biophysik (ICS-4) and JARA-HPC, Forschungszentrum Jülich, Jülich, Germany.

Excitatory amino acid transporters (EAATs) mediate glial and neuronal glutamate uptake to terminate synaptic transmission and to ensure low resting glutamate concentrations. Effective glutamate uptake is achieved by cotransport with 3 Na and 1 H , in exchange with 1 K . The underlying principles of this complex transport stoichiometry remain poorly understood. We use molecular dynamics simulations and electrophysiological experiments to elucidate how mammalian EAATs harness K gradients, unlike their K -independent prokaryotic homologues. Glutamate transport is achieved via elevator-like translocation of the transport domain. In EAATs, glutamate-free re-translocation is prevented by an external gate remaining open until K  binding closes and locks the gate. Prokaryotic Glt contains the same K -binding site, but the gate can close without K . Our study provides a comprehensive description of K -dependent glutamate transport and reveals a hitherto unknown allosteric coupling mechanism that permits adaptions of the transport stoichiometry without affecting ion or substrate binding.
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http://dx.doi.org/10.15252/embj.2019101468DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6769379PMC
October 2019

Preferential association with ClC-3 permits sorting of ClC-4 into endosomal compartments.

J Biol Chem 2017 11 26;292(46):19055-19065. Epub 2017 Sep 26.

From the Institute of Complex Systems, Zelluläre Biophysik (ICS-4), Forschungszentrum Jülich, 52425 Jülich, Germany

ClC-4 is an intracellular Cl/H exchanger that is highly expressed in the brain and whose dysfunction has been linked to intellectual disability and epilepsy. Here we studied the subcellular localization of human ClC-4 in heterologous expression systems. ClC-4 is retained in the endoplasmic reticulum (ER) upon overexpression in HEK293T cells. Co-expression with distinct ClC-3 splice variants targets ClC-4 to late endosome/lysosomes (ClC-3a and ClC-3b) or recycling endosome (ClC-3c). When expressed in cultured astrocytes, ClC-4 sorted to endocytic compartments in WT cells but was retained in the ER in cells. To understand the virtual absence of ER-localized ClC-4 in WT astrocytes, we performed association studies by high-resolution clear native gel electrophoresis. Although other CLC channels and transporters form stable dimers, ClC-4 was mostly observed as monomer, with ClC-3-ClC-4 heterodimers being more stable than ClC-4 homodimers. We conclude that unique oligomerization properties of ClC-4 permit regulated targeting of ClC-4 to various endosomal compartment systems via expression of different ClC-3 splice variants.
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http://dx.doi.org/10.1074/jbc.M117.801951DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5704486PMC
November 2017

Fusogenic Liposomes as Nanocarriers for the Delivery of Intracellular Proteins.

Langmuir 2017 01 17;33(4):1051-1059. Epub 2017 Jan 17.

Biophysical Chemistry, Friedrich-Wilhelms-University Bonn , D-53012 Bonn, Germany.

Direct delivery of proteins and peptides into living mammalian cells has been accomplished using phospholipid liposomes as carrier particles. Such liposomes are usually taken up via endocytosis where the main part of their cargo is degraded in lysosomes before reaching its destination. Here, fusogenic liposomes, a newly developed molecular carrier system, were used for protein delivery. When such liposomes were loaded with water-soluble proteins and brought into contact with mammalian cells, the liposomal membrane efficiently fused with the cellular plasma membrane delivering the liposomal content to the cytoplasm without degradation. To explore the key factors of proteofection processes, the complex formation of fusogenic liposomes and proteins of interest and the size and zeta potential of the formed fusogenic proteoliposoms were monitored. Intracellular protein delivery was analyzed using fluorescence microscopy and flow cytometry. Proteins such as EGFP, Dendra2, and R-phycoerythrin or peptides such as LifeAct-FITC and NTF2-AlexaFluor488 were successfully incorporated into mammalian cells with high efficiency. Moreover, correct functionality and faithful transport to binding sites were also proven for the imported proteins.
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http://dx.doi.org/10.1021/acs.langmuir.6b04304DOI Listing
January 2017

Identification of an HV 1 voltage-gated proton channel in insects.

FEBS J 2016 Apr 6;283(8):1453-64. Epub 2016 Mar 6.

Institute of Complex Systems, Zelluläre Biophysik (ICS-4) Forschungszentrum Jülich, Germany.

Unlabelled: The voltage-gated proton channel 1 (HV 1) is an important component of the cellular proton extrusion machinery and is essential for charge compensation during the respiratory burst of phagocytes. HV 1 has been identified in a wide range of eukaryotes throughout the animal kingdom, with the exception of insects. Therefore, it has been proposed that insects do not possess an HV 1 channel. In the present study, we report the existence of an HV 1-type proton channel in insects. We searched insect transcriptome shotgun assembly (TSA) sequence databases and found putative HV 1 orthologues in various polyneopteran insects. To confirm that these putative HV 1 orthologues were functional channels, we studied the HV 1 channel of Nicoletia phytophila (NpHV 1), an insect of the Zygentoma order, in more detail. NpHV 1 comprises 239 amino acids and is 33% identical to the human voltage-gated proton channel 1. Patch clamp measurements in a heterologous expression system showed proton selectivity, as well as pH- and voltage-dependent gating. Interestingly, NpHV 1 shows slightly enhanced pH-dependent gating compared to the human channel. Mutations in the first transmembrane segment at position 66 (Asp66), the presumed selectivity filter, lead to a loss of proton-selective conduction, confirming the importance of this aspartate residue in voltage-gated proton channels.

Database: Nucleotide sequence data have been deposited in the GenBank database under accession number KT780722.
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http://dx.doi.org/10.1111/febs.13680DOI Listing
April 2016

Carboxyl-terminal Truncations of ClC-Kb Abolish Channel Activation by Barttin Via Modified Common Gating and Trafficking.

J Biol Chem 2015 Dec 9;290(51):30406-16. Epub 2015 Oct 9.

From the Institute of Complex Systems 4 (ICS-4), Zelluläre Biophysik, Forschungszentrum Jülich, 52425 Jülich

ClC-K chloride channels are crucial for auditory transduction and urine concentration. Mutations in CLCNKB, the gene encoding the renal chloride channel hClC-Kb, cause Bartter syndrome type III, a human genetic condition characterized by polyuria, hypokalemia, and alkalosis. In recent years, several Bartter syndrome-associated mutations have been described that result in truncations of the intracellular carboxyl terminus of hClC-Kb. We here used a combination of whole-cell patch clamp, confocal imaging, co-immunoprecipitation, and surface biotinylation to study the functional consequences of a frequent CLCNKB mutation that creates a premature stop codon at Trp-610. We found that W610X leaves the association of hClC-Kb and the accessory subunit barttin unaffected, but impairs its regulation by barttin. W610X attenuates hClC-Kb surface membrane insertion. Moreover, W610X results in hClC-Kb channel opening in the absence of barttin and prevents further barttin-mediated activation. To describe how the carboxyl terminus modifies the regulation by barttin we used V166E rClC-K1. V166E rClC-K1 is active without barttin and exhibits prominent, barttin-regulated voltage-dependent gating. Electrophysiological characterization of truncated V166E rClC-K1 demonstrated that the distal carboxyl terminus is necessary for slow cooperative gating. Since barttin modifies this particular gating process, channels lacking the distal carboxyl-terminal domain are no longer regulated by the accessory subunit. Our results demonstrate that the carboxyl terminus of hClC-Kb is not part of the binding site for barttin, but functionally modifies the interplay with barttin. The loss-of-activation of truncated hClC-Kb channels in heterologous expression systems fully explains the reduced basolateral chloride conductance in affected kidneys and the clinical symptoms of Bartter syndrome patients.
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http://dx.doi.org/10.1074/jbc.M115.675827DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4683263PMC
December 2015

Neuronal ClC-3 Splice Variants Differ in Subcellular Localizations, but Mediate Identical Transport Functions.

J Biol Chem 2015 Oct 4;290(43):25851-62. Epub 2015 Sep 4.

From the Institute of Complex Systems, Zelluläre Biophysik (ICS-4), Forschungszentrum Jülich, 52425 Jülich, Germany

ClC-3 is a member of the CLC family of anion channels and transporters, for which multiple functional properties and subcellular localizations have been reported. Since alternative splicing often results in proteins with diverse properties, we investigated to what extent alternative splicing might influence subcellular targeting and function of ClC-3. We identified three alternatively spliced ClC-3 isoforms, ClC-3a, ClC-3b, and ClC-3c, in mouse brain, with ClC-3c being the predominant splice variant. Whereas ClC-3a and ClC-3b are present in late endosomes/lysosomes, ClC-3c is targeted to recycling endosomes via a novel N-terminal isoleucine-proline (IP) motif. Surface membrane insertion of a fraction of ClC-3c transporters permitted electrophysiological characterization of this splice variant through whole-cell patch clamping on transfected mammalian cells. In contrast, neutralization of the N-terminal dileucine-like motifs was required for functional analysis of ClC-3a and ClC-3b. Heterologous expression of ClC-3a or ClC-3b carrying mutations in N-terminal dileucine motifs as well as WTClC-3c in HEK293T cells resulted in outwardly rectifying Cl(-) currents with significant capacitive current components. We conclude that alternative splicing of Clcn3 results in proteins with different subcellular localizations, but leaves the transport function of the proteins unaffected.
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http://dx.doi.org/10.1074/jbc.M115.668186DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4646242PMC
October 2015
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