Publications by authors named "Robert Lukowski"

60 Publications

cGMP and mitochondrial K channels-Compartmentalized but closely connected in cardioprotection.

Br J Pharmacol 2021 May 15. Epub 2021 May 15.

Department of Pharmacology, Toxicology and Clinical Pharmacy, Institute of Pharmacy, University of Tuebingen, Tuebingen, Germany.

The 3',5'-cGMP pathway triggers cytoprotective responses and improves cardiomyocyte survival during myocardial ischaemia and reperfusion (I/R) injury. These beneficial effects were attributed to NO-sensitive GC induced cGMP production leading to activation of cGMP-dependent protein kinase I (cGKI). cGKI in turn phosphorylates many substrates, which eventually facilitate opening of mitochondrial ATP-sensitive potassium channels (mitoK ) and Ca -activated potassium channels of the BK type (mitoBK). Accordingly, agents activating mitoK or mitoBK provide protection against I/R-induced damages. Here, we provide an up-to-date summary of the infarct-limiting actions exhibited by the GC/cGMP axis and discuss how mitoK and mitoBK, which are present at the inner mitochondrial membrane, confer mito- and cytoprotective effects on cardiomyocytes exposed to I/R injury. In view of this, we believe that the functional connection between the cGMP cascade and mitoK channels should be exploited further as adjunct to reperfusion therapy in myocardial infarction.
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http://dx.doi.org/10.1111/bph.15536DOI Listing
May 2021

Potassium ions promote hexokinase-II dependent glycolysis.

iScience 2021 Apr 22;24(4):102346. Epub 2021 Mar 22.

Gottfried Schatz Research Center, Molecular Biology and Biochemistry, Medical University of Graz, Neue Stiftingtalstraße 6/6, 8010 Graz, Austria.

High expression levels of mitochondria-associated hexokinase-II (HKII) represent a hallmark of metabolically highly active cells such as fast proliferating cancer cells. Typically, the enzyme provides a crucial metabolic switch towards aerobic glycolysis. By imaging metabolic activities on the single-cell level with genetically encoded fluorescent biosensors, we here demonstrate that HKII activity requires intracellular K. The K dependency of glycolysis in cells expressing HKII was confirmed in cell populations using extracellular flux analysis and nuclear magnetic resonance-based metabolomics. Reductions of intracellular K by gramicidin acutely disrupted HKII-dependent glycolysis and triggered energy stress pathways, while K re-addition promptly restored glycolysis-dependent adenosine-5'-triphosphate generation. Moreover, expression and activation of K1.3, a voltage-gated K channel, lowered cellular K content and the glycolytic activity of HEK293 cells. Our findings unveil K as an essential cofactor of HKII and provide a mechanistic link between activities of distinct K channels and cell metabolism.
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http://dx.doi.org/10.1016/j.isci.2021.102346DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8047173PMC
April 2021

Potassium Channels in Cancer.

Handb Exp Pharmacol 2021 ;267:253-275

Department of Radiation Oncology, University of Tübingen, Tübingen, Germany.

Neoplastic transformation is reportedly associated with alterations of the potassium transport across plasma and intracellular membranes. These alterations have been identified as crucial elements of the tumourigenic reprogramming of cells. Potassium channels may contribute to cancer initiation, malignant progression and therapy resistance of tumour cells. The book chapter focusses on (oncogenic) potassium channels frequently upregulated in different tumour entities, upstream and downstream signalling of these channels, their contribution to the maintenance of cancer stemness and the formation of an immunosuppressive tumour microenvironment. In addition, their role in adaptation to tumour hypoxia, metabolic reprogramming, as well as tumour spreading and metastasis is discussed. Finally, we discuss how (oncogenic) potassium channels may confer treatment resistance of tumours against radiation and chemotherapy and thus might be harnessed for new therapy strategies, for instance, by repurposing approved drugs known to target potassium channels.
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http://dx.doi.org/10.1007/164_2021_465DOI Listing
January 2021

Slack K channels attenuate NMDA-induced excitotoxic brain damage and neuronal cell death.

FASEB J 2021 05;35(5):e21568

Department of Pharmacology, Toxicology and Clinical Pharmacy, Institute of Pharmacy, University of Tübingen, Tübingen, Germany.

The neuronal Na -activated K channel Slack (aka Slo2.2, K 1.1, or Kcnt1) has been implicated in setting and maintaining the resting membrane potential and defining excitability and firing patterns, as well as in the generation of the slow afterhyperpolarization following bursts of action potentials. Slack activity increases significantly under conditions of high intracellular Na levels, suggesting this channel may exert important pathophysiological functions. To address these putative roles, we studied whether Slack K channels contribute to pathological changes and excitotoxic cell death caused by glutamatergic overstimulation of Ca - and Na -permeable N-methyl-D-aspartic acid receptors (NMDAR). Slack-deficient (Slack KO) and wild-type (WT) mice were subjected to intrastriatal microinjections of the NMDAR agonist NMDA. NMDA-induced brain lesions were significantly increased in Slack KO vs WT mice, suggesting that the lack of Slack renders neurons particularly susceptible to excitotoxicity. Accordingly, excessive neuronal cell death was seen in Slack-deficient primary cerebellar granule cell (CGC) cultures exposed to glutamate and NMDA. Differences in neuronal survival between WT and Slack KO CGCs were largely abolished by the NMDAR antagonist MK-801, but not by NBQX, a potent and highly selective competitive antagonist of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type ionotropic glutamate receptors. Interestingly, NMDAR-evoked Ca signals did not differ with regard to Slack genotype in CGCs. However, real-time monitoring of K following NMDAR activation revealed a significant contribution of this channel to the intracellular drop in K . Finally, TrkB and TrkC neurotrophin receptor transcript levels were elevated in NMDA-exposed Slack-proficient CGCs, suggesting a mechanism by which this K channel contributes to the activation of the extracellular-signal-regulated kinase (Erk) pathway and thereby to neuroprotection. Combined, our findings suggest that Slack-dependent K signals oppose the NMDAR-mediated excitotoxic neuronal injury by promoting pro-survival signaling via the BDNF/TrkB and Erk axis.
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http://dx.doi.org/10.1096/fj.202002308RRDOI Listing
May 2021

CRIP1 expression in monocytes related to hypertension.

Clin Sci (Lond) 2021 04;135(7):911-924

Clinic of Cardiology, University Heart and Vascular Center Hamburg, Germany.

Hypertension is a complex and multifactorial disorder caused by lifestyle and environmental factors, inflammation and disease-related genetic factors and is a risk factor for stroke, ischemic heart disease and renal failure. Although circulating monocytes and tissue macrophages contribute to the pathogenesis of hypertension, the underlying mechanisms are poorly understood. Cysteine rich protein 1 (CRIP1) is highly expressed in immune cells, and CRIP1 mRNA expression in monocytes associates with blood pressure (BP) and is up-regulated by proinflammatory modulation suggesting a link between CRIP1 and BP regulation through the immune system. To address this functional link, we studied CRIP1 expression in immune cells in relation to BP using a human cohort study and hypertensive mouse models. CRIP1 expression in splenic monocytes/macrophages and in circulating monocytes was significantly affected by angiotensin II (Ang II) in a BP-elevating dose (2 mg/kg/day). In the human cohort study, monocytic CRIP1 expression levels were associated with elevated BP, whereas upon differentiation of monocytes to macrophages this association along with the CRIP1 expression level was diminished. In conclusion, CRIP1-positive circulating and splenic monocytes seem to play an important role in hypertension related inflammatory processes through endogenous hormones such as Ang II. These findings suggest that CRIP1 may affect the interaction between the immune system, in particular monocytes, and the pathogenesis of hypertension.
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http://dx.doi.org/10.1042/CS20201372DOI Listing
April 2021

Corrigendum to "Tissue- and isoform-specific protein complex analysis with natively processed bait proteins" [Journal of Proteomics, Volume 231 (2021), Article 103947].

J Proteomics 2021 Apr 1;238:104165. Epub 2021 Mar 1.

Institute for Ophthalmic Research, Centre for Ophthalmology, University of Tuebingen, Elfriede-Aulhorn-Strasse 7, D-72076 Tuebingen, Germany. Electronic address:

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http://dx.doi.org/10.1016/j.jprot.2021.104165DOI Listing
April 2021

Functional Coupling of Slack Channels and P2X3 Receptors Contributes to Neuropathic Pain Processing.

Int J Mol Sci 2021 Jan 2;22(1). Epub 2021 Jan 2.

Institut für Pharmakologie und Klinische Pharmazie, Goethe-Universität Frankfurt am Main, 60438 Frankfurt am Main, Hessen, Germany.

The sodium-activated potassium channel Slack (K1.1, Slo2.2, or Kcnt1) is highly expressed in populations of sensory neurons, where it mediates the sodium-activated potassium current (I) and modulates neuronal activity. Previous studies suggest that Slack is involved in the processing of neuropathic pain. However, mechanisms underlying the regulation of Slack activity in this context are poorly understood. Using whole-cell patch-clamp recordings we found that Slack-mediated I in sensory neurons of mice is reduced after peripheral nerve injury, thereby contributing to neuropathic pain hypersensitivity. Interestingly, Slack is closely associated with ATP-sensitive P2X3 receptors in a population of sensory neurons. In vitro experiments revealed that Slack-mediated I may be bidirectionally modulated in response to P2X3 activation. Moreover, mice lacking Slack show altered nocifensive responses to P2X3 stimulation. Our study identifies P2X3/Slack signaling as a mechanism contributing to hypersensitivity after peripheral nerve injury and proposes a potential novel strategy for treatment of neuropathic pain.
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http://dx.doi.org/10.3390/ijms22010405DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7795269PMC
January 2021

Tissue- and isoform-specific protein complex analysis with natively processed bait proteins.

J Proteomics 2021 01 24;231:103947. Epub 2020 Aug 24.

Institute for Ophthalmic Research, Centre for Ophthalmology, University of Tuebingen, Elfriede-Aulhorn-Strasse 7, D-72076 Tuebingen, Germany. Electronic address:

Protein-protein interaction analysis is an important tool to elucidate the function of proteins and protein complexes as well as their dynamic behavior. To date, the analysis of tissue- or even cell- or compartment-specific protein interactions is still relying on the availability of specific antibodies suited for immunoprecipitation. Here, we aimed at establishing a method that allows identification of protein interactions and complexes from intact tissues independent of specific, high affinity antibodies used for protein pull-down and isolation. Tagged bait proteins were expressed in human HEK293T cells and residual interactors removed by SDS. The resulting tag-fusion protein was then used as bait to pull proteins from tissue samples. Tissue-specific interactions were reproducibly identified from porcine retina as well as from retinal pigment epithelium using the ciliary protein lebercilin as bait. Further, murine heart-specific interactors of two gene products of the 3',5'-cyclic guanosine monophosphate (cGMP)-dependent protein kinase type 1 (cGK1) were investigated. Here, specific interactions were associated with the cGK1α and β gene products, that differ only in their unique amino-terminal region comprising about 100 aa. As such, the new protocol provides a fast and reliable method for tissue-specific protein complex analysis which is independent of the availability or suitability of antibodies for immunoprecipitation. SIGNIFICANCE: Protein-protein interaction in the functional relevant tissue is still difficult due to the dependence on specific antibodies or bait production in bacteria or insect cells. Here, the tagged protein of interest is produced in a human cell line and bound proteins are gently removed using SDS. Because applying the suitable SDS concentration is a critical step, different SDS solutions were tested to demonstrate their influence on interactions and the clean-up process. The established protocol enabled a tissue-specific analysis of the ciliary proteins lebercilin and TMEM107 using pig eyes. In addition, two gene products of the 3',5'-cyclic guanosine monophosphate (cGMP)-dependent protein kinase type 1 showed distinct protein interactions in mouse heart tissue. With the easy, fast and cheap protocol presented here, deep insights in tissue-specific and functional relevant protein complex formation is possible.
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http://dx.doi.org/10.1016/j.jprot.2020.103947DOI Listing
January 2021

Subunits of BK channels promote breast cancer development and modulate responses to endocrine treatment in preclinical models.

Br J Pharmacol 2020 May 28. Epub 2020 May 28.

Department of Pharmacology, Toxicology and Clinical Pharmacy, Institute of Pharmacy, University of Tuebingen, Tuebingen, Germany.

Background And Purpose: Pore-forming α subunits of the voltage- and Ca -activated K channel with large conductance (BKα) promote malignant phenotypes of breast tumour cells. Auxiliary subunits such as the leucine-rich repeat containing 26 (LRRC26) protein, also termed BKγ1, may be required to permit activation of BK currents at a depolarized resting membrane potential that frequently occur in non-excitable tumour cells.

Experimental Approach: Anti-tumour effects of BKα loss were investigated in breast tumour-bearing MMTV-PyMT transgenic BKα knockout (KO) mice, primary MMTV-PyMT cell cultures, and in a syngeneic transplantation model of breast cancer derived from these cells. The therapeutic relevance of BK channels in the context of endocrine treatment was assessed in human breast cancer cell lines expressing either low (MCF-7) or high (MDA-MB-453) levels of BKα and BKγ1, as well as in BKα-negative MDA-MB-157.

Key Results: BKα promoted breast cancer onset and overall survival in preclinical models. Conversely, lack of BKα and/or knockdown of BKγ1 attenuated proliferation of murine and human breast cancer cells in vitro. At low concentrations, tamoxifen and its major active metabolites stimulated proliferation of BKα/γ1-positive breast cancer cells, independent of the genomic signalling controlled by the oestrogen receptor. Finally, tamoxifen increased the relative survival time of BKα KO but not of wild-type tumour cell recipient mice.

Conclusion And Implications: Breast cancer initiation, progression, and tamoxifen sensitivity depend on functional BK channels thereby providing a rationale for the future exploration of the oncogenic actions of BK channels in clinical outcomes with anti-oestrogen therapy.
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http://dx.doi.org/10.1111/bph.15147DOI Listing
May 2020

Targets of cGMP/cGKI in Cardiac Myocytes.

J Cardiovasc Pharmacol 2020 06;75(6):494-507

Department of Pharmacology, Toxicology and Clinical Pharmacy, Institute of Pharmacy, University of Tuebingen, 72076 Tuebingen, Germany.

The 3',5'-cyclic guanosine monophosphate (cGMP)-dependent protein kinase type I (cGKI aka PKGI) is a major cardiac effector acting downstream of nitric oxide (NO)-sensitive soluble guanylyl cyclase and natriuretic peptides (NPs), which signal through transmembrane guanylyl cyclases. Consistent with the wide distribution of the cGMP-generating guanylyl cyclases, cGKI, which usually elicits its cellular effects by direct phosphorylation of its targets, is present in multiple cardiac cell types including cardiomyocytes (CMs). Although numerous targets of cGMP/cGKI in heart were identified in the past, neither their exact patho-/physiological functions nor cell-type specific roles are clear. Herein, we inform about the current knowledge on the signal transduction downstream of CM cGKI. We believe that better insights into the specific actions of cGMP and cGKI in these cells will help to guide future studies in the search for predictive biomarkers for the response to pharmacological cGMP pathway modulation. In addition, targets downstream of cGMP/cGKI may be exploited for refined and optimized diagnostic and therapeutic strategies in different types of heart disease and their causes. Importantly, key functions of these proteins and particularly sites of regulatory phosphorylation by cGKI should, at least in principle, remain intact, although upstream signaling through the second messenger cGMP is impaired or dysregulated in a stressed or diseased heart state.
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http://dx.doi.org/10.1097/FJC.0000000000000817DOI Listing
June 2020

Deciphering the Dynamics and Therapeutic Potential of the Cardiac cGMP Cascade: An Update on Where We Are and What We Need to Know.

J Cardiovasc Pharmacol 2020 05;75(5):368-369

Department of Pharmacology and Toxicology, School of Medicine, University of Mississippi Medical Center, Jackson, MS.

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http://dx.doi.org/10.1097/FJC.0000000000000814DOI Listing
May 2020

Impaired motor skill learning and altered seizure susceptibility in mice with loss or gain of function of the Kcnt1 gene encoding Slack (K1.1) Na-activated K channels.

Sci Rep 2020 02 21;10(1):3213. Epub 2020 Feb 21.

Departments of Pharmacology and Cellular and Molecular Physiology, Yale University, New Haven, CT, USA.

Gain-of-function mutations in KCNT1, the gene encoding Slack (K1.1) channels, result in epilepsy of infancy with migrating focal seizures (EIMFS) and several other forms of epilepsy associated with severe intellectual disability. We have generated a mouse model of this condition by replacing the wild type gene with one encoding Kcnt1, a cytoplasmic C-terminal mutation homologous to a human R474H variant that results in EIMFS. We compared behavior patterns and seizure activity in these mice with those of wild type mice and Kcnt1 mice. Complete loss of Kcnt1 produced deficits in open field behavior and motor skill learning. Although their thresholds for electrically and chemically induced seizures were similar to those of wild type animals, Kcnt1 mice were significantly protected from death after maximum electroshock-induced seizures. In contrast, homozygous Kcnt1 mice were embryonic lethal. Video-EEG monitoring of heterozygous Kcnt1 animals revealed persistent interictal spikes, spontaneous seizures and a substantially decreased threshold for pentylenetetrazole-induced seizures. Surprisingly, Kcnt1 mice were not impaired in tasks of exploratory behavior or procedural motor learning. These findings provide an animal model for EIMFS and suggest that Slack channels are required for the development of procedural learning and of pathways that link cortical seizures to other regions required for animal survival.
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http://dx.doi.org/10.1038/s41598-020-60028-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7035262PMC
February 2020

Comprehensive lipidomics of mouse plasma using class-specific surrogate calibrants and SWATH acquisition for large-scale lipid quantification in untargeted analysis.

Anal Chim Acta 2019 Dec 17;1086:90-102. Epub 2019 Aug 17.

Institute of Pharmaceutical Sciences, Pharmaceutical (Bio-)Analysis, University of Tübingen, Tübingen, Germany. Electronic address:

Lipidomics has gained rising attention in recent years. Several strategies for lipidomic profiling have been developed, with targeted analysis of selected lipid species, typically utilized for lipid quantification by low-resolution triple quadrupole MS/MS, and untargeted analysis by high-resolution MS instruments, focusing on hypothesis generation for prognostic, diagnostic and/or disease-relevant biomarker discovery. The latter methodologies generally yield relative quantification data with limited inter-assay comparability. In this work we aimed to combine untargeted analysis and absolute quantification to enhance data quality and to obtain independent results for optimum comparability to previous studies or database entries. For the lipidomic analysis of mouse plasma, RP-UHPLC hyphenated to a high-resolution quadrupole TOF mass spectrometer in comprehensive data-independent SWATH acquisition mode was employed. This way, quantifiable data on the MS and the MS/MS level were recorded, which increases assay specificity and quantitative performance. Due to the lack of an appropriate blank matrix for untargeted lipidomics, we herein established a sophisticated strategy for lipid class-specific calibration with stable isotope labeled standards (surrogate calibrants). LLOQs were in the range between 10 and 50 ng mL for LPC, LPE, PI, PS, PG, SM, PC, PE, DAG) or 100-700 ng mL (MAG, TAG), except for cholesterol and CE (1-20 μg mL). Acceptable values for accuracy and precision well below ±15% bias were reached for the majority of surrogate calibrants. However, to achieve sufficient accuracy for target lipids, response factors to corresponding surrogate calibrants are required. An approach to estimate response factors via a standard reference material (NIST SRM 1950) was therefore conducted. Furthermore, a useful workflow for post-acquisition re-calibration, involving response factor determination and iteratively built libraries, is suggested. In comparison to single-point calibration, the presented surrogate calibrant method was shown to yield results with improved accuracy that are largely in accordance with standard addition. Quantitative results of real samples (high-fat diet vs control diet) were then compared to two previously published dietary mouse plasma studies that provided absolute lipid levels and showed similar trends.
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http://dx.doi.org/10.1016/j.aca.2019.08.030DOI Listing
December 2019

K3.1 Channels Confer Radioresistance to Breast Cancer Cells.

Cancers (Basel) 2019 Sep 1;11(9). Epub 2019 Sep 1.

Department of Pharmacology, Toxicology and Clinical Pharmacy, Institute of Pharmacy, University of Tuebingen, 72076 Tuebingen, Germany.

K3.1 K channels reportedly contribute to the proliferation of breast tumor cells and may serve pro-tumor functions in the microenvironment. The putative interaction of K3.1 with major anti-cancer treatment strategies, which are based on cytotoxic drugs or radiotherapy, remains largely unexplored. We employed K3.1-proficient and -deficient breast cancer cells derived from breast cancer-prone MMTV-PyMT mice, pharmacological K3.1 inhibition, and a syngeneic orthotopic mouse model to study the relevance of functional K3.1 for therapy response. The K3.1 status of MMTV-PyMT cells did not determine tumor cell proliferation after treatment with different concentrations of docetaxel, doxorubicin, 5-fluorouracil, or cyclophosphamide. K3.1 activation by ionizing radiation (IR) in breast tumor cells in vitro, however, enhanced radioresistance, probably via an involvement of the channel in IR-stimulated Ca signals and DNA repair pathways. Consistently, K3.1 knockout increased survival time of wildtype mice upon syngeneic orthotopic transplantation of MMTV-PyMT tumors followed by fractionated radiotherapy. Combined, our results imply that K3.1 confers resistance to radio- but not to chemotherapy in the MMTV-PyMT breast cancer model. Since K3.1 is druggable, K3.1 targeting concomitant to radiotherapy seems to be a promising strategy to radiosensitize breast tumors.
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http://dx.doi.org/10.3390/cancers11091285DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6770875PMC
September 2019

From in vitro to ex vivo: subcellular localization and uptake of graphene quantum dots into solid tumors.

Nanotechnology 2019 Sep 26;30(39):395101. Epub 2019 Jun 26.

Condensed Matter Physics Laboratory, Heinrich-Heine-University, D-40204 Düsseldorf, Germany.

Among various nanoparticles tested for pharmacological applications over the recent years, graphene quantum dots (GQDs) seem to be promising candidates for the construction of drug delivery systems due to their superior biophysical and biochemical properties. The subcellular fate of incorporated nanomaterial is decisive for transporting pharmaceuticals into target cells. Therefore a detailed characterization of the uptake of GQDs into different breast cancer models was performed. The demonstrated accumulation inside the endolysosomal system might be the reason for the particles' low toxicity, but has to be overcome for cytosolic or nuclear drug delivery. Furthermore, the penetration of GQDs into precision-cut mammary tumor slices was studied. These constitute a far closer to reality model system than monoclonal cell lines. The constant uptake into the depth of the tissue slices underlines the systems' potential for drug delivery into solid tumors.
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http://dx.doi.org/10.1088/1361-6528/ab2cb4DOI Listing
September 2019

Cancer-Associated Intermediate Conductance Ca-Activated K⁺ Channel K3.1.

Cancers (Basel) 2019 Jan 17;11(1). Epub 2019 Jan 17.

Department of Pharmacology, Toxicology and Clinical Pharmacy, Institute of Pharmacy, University of Tuebingen, 72076 Tuebingen, Germany.

Several tumor entities have been reported to overexpress K3.1 potassium channels due to epigenetic, transcriptional, or post-translational modifications. By modulating membrane potential, cell volume, or Ca signaling, K3.1 has been proposed to exert pivotal oncogenic functions in tumorigenesis, malignant progression, metastasis, and therapy resistance. Moreover, K3.1 is expressed by tumor-promoting stroma cells such as fibroblasts and the tumor vasculature suggesting a role of K3.1 in the adaptation of the tumor microenvironment. Combined, this features K3.1 as a candidate target for innovative anti-cancer therapy. However, immune cells also express K3.1 thereby contributing to T cell activation. Thus, any strategy targeting K3.1 in anti-cancer therapy may also modulate anti-tumor immune activity and/or immunosuppression. The present review article highlights the potential of K3.1 as an anti-tumor target providing an overview of the current knowledge on its function in tumor pathogenesis with emphasis on vasculo- and angiogenesis as well as anti-cancer immune responses.
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http://dx.doi.org/10.3390/cancers11010109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6357066PMC
January 2019

Accessory heterozygous mutations in cone photoreceptor CNGA3 exacerbate CNG channel-associated retinopathy.

J Clin Invest 2018 12 12;128(12):5663-5675. Epub 2018 Nov 12.

Department of Pharmacology, Toxicology and Clinical Pharmacy, Institute of Pharmacy.

Mutations in CNGA3 and CNGB3, the genes encoding the subunits of the tetrameric cone photoreceptor cyclic nucleotide-gated ion channel, cause achromatopsia, a congenital retinal disorder characterized by loss of cone function. However, a small number of patients carrying the CNGB3/c.1208G>A;p.R403Q mutation present with a variable retinal phenotype ranging from complete and incomplete achromatopsia to moderate cone dysfunction or progressive cone dystrophy. By exploring a large patient cohort and published cases, we identified 16 unrelated individuals who were homozygous or (compound-)heterozygous for the CNGB3/c.1208G>A;p.R403Q mutation. In-depth genetic and clinical analysis revealed a co-occurrence of a mutant CNGA3 allele in a high proportion of these patients (10 of 16), likely contributing to the disease phenotype. To verify these findings, we generated a Cngb3R403Q/R403Q mouse model, which was crossbred with Cnga3-deficient (Cnga3-/-) mice to obtain triallelic Cnga3+/- Cngb3R403Q/R403Q mutants. As in human subjects, there was a striking genotype-phenotype correlation, since the presence of 1 Cnga3-null allele exacerbated the cone dystrophy phenotype in Cngb3R403Q/R403Q mice. These findings strongly suggest a digenic and triallelic inheritance pattern in a subset of patients with achromatopsia/severe cone dystrophy linked to the CNGB3/p.R403Q mutation, with important implications for diagnosis, prognosis, and genetic counseling.
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http://dx.doi.org/10.1172/JCI96098DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6264655PMC
December 2018

Loss of Sodium-Activated Potassium Channel Slack and FMRP Differentially Affect Social Behavior in Mice.

Neuroscience 2018 08 31;384:361-374. Epub 2018 May 31.

Department of Pharmacology, Toxicology and Clinical Pharmacy, Institute of Pharmacy, 72076 Tübingen, Germany. Electronic address:

The sodium-activated potassium channel Slack (Slo2.2) is widely expressed in central and peripheral neurons where it is supposed to shape firing properties important for neuronal excitability. Slack activity is enhanced by interaction with the Fragile-X-Mental-Retardation-Protein (FMRP) and loss of FMRP leads to decreased sodium-activated potassium currents in medial nucleus of the trapezoid body neurons of the Fmr1-knockout (KO) mouse representing a mouse model of the human Fragile-X-Syndrome (FXS) and autism. Autism is a frequent comorbidity of FXS, but it is unclear whether Slack is involved in autistic or related conditions of FXS in vivo. By applying a wide range of behavioral tests, we compared social and autism-related behaviors in Slack- and FMRP-deficient mice. In our hands, as expected, FMRP-deficiency causes autism-related behavioral changes in nesting and in a marble-burying test. In contrast, Slack-deficient males exhibited specific abnormalities in sociability in direct and indirect social interaction tests. Hence, we show for the first time that a proper Slack channel function is mandatory for normal social behavior in mice. Nevertheless, as deficits in social behaviors seem to occur independently from each other in FMRP and Slack null mutants, we conclude that Slack is not involved in the autistic phenotype of FMRP KO mice.
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http://dx.doi.org/10.1016/j.neuroscience.2018.05.040DOI Listing
August 2018

Purkinje cell BKchannel ablation induces abnormal rhythm in deep cerebellar nuclei and prevents LTD.

Sci Rep 2018 03 9;8(1):4220. Epub 2018 Mar 9.

Laboratory of Neurophysiology and Movement Biomechanics, ULB Neuroscience Institute, Université Libre de Bruxelles, Brussels, Belgium.

Purkinje cells (PC) control deep cerebellar nuclei (DCN), which in turn inhibit inferior olive nucleus, closing a positive feedback loop via climbing fibers. PC highly express potassium BK channels but their contribution to the olivo-cerebellar loop is not clear. Using multiple-unit recordings in alert mice we found in that selective deletion of BK channels in PC induces a decrease in their simple spike firing with a beta-range bursting pattern and fast intraburst frequency (~200 Hz). To determine the impact of this abnormal rhythm on the olivo-cerebellar loop we analyzed simultaneous rhythmicity in different cerebellar structures. We found that this abnormal PC rhythmicity is transmitted to DCN neurons with no effect on their mean firing frequency. Long term depression at the parallel-PC synapses was altered and the intra-burst complex spike spikelets frequency was increased without modification of the mean complex spike frequency in BK-PC mice. We argue that the ataxia present in these conditional knockout mice could be explained by rhythmic disruptions transmitted from mutant PC to DCN but not by rate code modification only. This suggests a neuronal mechanism for ataxia with possible implications for human disease.
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http://dx.doi.org/10.1038/s41598-018-22654-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5845018PMC
March 2018

Cardioprotection by ischemic postconditioning and cyclic guanosine monophosphate-elevating agents involves cardiomyocyte nitric oxide-sensitive guanylyl cyclase.

Cardiovasc Res 2018 05;114(6):822-829

Department of Pharmacology, Toxicology and Clinical Pharmacy, Institute of Pharmacy, University of Tübingen, 72076 Tübingen, Germany.

Aims: It has been suggested that the nitric oxide-sensitive guanylyl cyclase (NO-GC)/cyclic guanosine monophosphate (cGMP)-dependent signalling pathway affords protection against cardiac damage during acute myocardial infarction (AMI). It is, however, not clear whether the NO-GC/cGMP system confers its favourable effects through a mechanism located in cardiomyocytes (CMs). The aim of this study was to evaluate the infarct-limiting effects of the endogenous NO-GC in CMs in vivo.

Methods And Results: Ischemia/reperfusion (I/R) injury was evaluated in mice with a CM-specific deletion of NO-GC (CM NO-GC KO) and in control siblings (CM NO-GC CTR) subjected to an in vivo model of AMI. Lack of CM NO-GC resulted in a mild increase in blood pressure but did not affect basal infarct sizes after I/R. Ischemic postconditioning (iPost), administration of the phosphodiesterase-5 inhibitors sildenafil and tadalafil as well as the NO-GC activator cinaciguat significantly reduced the amount of infarction in control mice but not in CM NO-GC KO littermates. Interestingly, NS11021, an opener of the large-conductance and Ca2+-activated potassium channel (BK), an important downstream effector of cGMP/cGKI in the cardiovascular system, protects I/R-exposed hearts of CM NO-GC proficient and deficient mice.

Conclusions: These findings demonstrate an important role of CM NO-GC for the cardioprotective signalling following AMI in vivo. CM NO-GC function is essential for the beneficial effects on infarct size elicited by iPost and pharmacological elevation of cGMP; however, lack of CM NO-GC does not seem to disrupt the cardioprotection mediated by the BK opener NS11021.
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http://dx.doi.org/10.1093/cvr/cvy039DOI Listing
May 2018

cGMP-Elevating Compounds and Ischemic Conditioning Provide Cardioprotection Against Ischemia and Reperfusion Injury via Cardiomyocyte-Specific BK Channels.

Circulation 2017 Dec 19;136(24):2337-2355. Epub 2017 Oct 19.

Department of Pharmacology, Toxicology and Clinical Pharmacy, Institute of Pharmacy, University of Tuebingen, Germany (S.F., A.K., J.S., E.M., P.R., R.L.)

Background: The nitric oxide-sensitive guanylyl cyclase/cGMP-dependent protein kinase type I signaling pathway can afford protection against the ischemia/reperfusion injury that occurs during myocardial infarction. Reportedly, voltage and Ca-activated K channels of the BK type are stimulated by cGMP/cGMP-dependent protein kinase type I, and recent ex vivo studies implicated that increased BK activity favors the survival of the myocardium at ischemia/reperfusion. It remains unclear, however, whether the molecular events downstream of cGMP involve BK channels present in cardiomyocytes or in other cardiac cell types.

Methods: Gene-targeted mice with a cardiomyocyte- or smooth muscle cell-specific deletion of the BK (CMBK or SMBK knockouts) were subjected to the open-chest model of myocardial infarction. Infarct sizes of the conditional mutants were compared with litter-matched controls, global BK knockout, and wild-type mice. Cardiac damage was assessed after mechanical conditioning or pharmacological stimulation of the cGMP pathway and by using direct modulators of BK. Long-term outcome was studied with respect to heart functions and cardiac fibrosis in a chronic myocardial infarction model.

Results: Global BK knockouts and CMBK knockouts, in contrast with SMBK knockouts, exhibited significantly larger infarct sizes compared with their respective controls. Ablation of CMBK resulted in higher serum levels of cardiac troponin I and elevated amounts of reactive oxygen species, lower phosphorylated extracellular receptor kinase and phosphorylated AKT levels and an increase in myocardial apoptosis. Moreover, CMBK was required to allow beneficial effects of both nitric oxide-sensitive guanylyl cyclase activation and inhibition of the cGMP-degrading phosphodiesterase-5, ischemic preconditioning, and postconditioning regimens. To this end, after 4 weeks of reperfusion, fibrotic tissue increased and myocardial strain echocardiography was significantly compromised in CMBK-deficient mice.

Conclusions: Lack of CMBK channels renders the heart more susceptible to ischemia/reperfusion injury, whereas the pathological events elicited by ischemia/reperfusion do not involve BK in vascular smooth muscle cells. BK seems to permit the protective effects triggered by cinaciguat, riociguat, and different phosphodiesterase-5 inhibitors and beneficial actions of ischemic preconditioning and ischemic postconditioning by a mechanism stemming primarily from cardiomyocytes. This study establishes mitochondrial CMBK channels as a promising target for limiting acute cardiac damage and adverse long-term events that occur after myocardial infarction.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.117.028723DOI Listing
December 2017

K3.1 channels modulate the processing of noxious chemical stimuli in mice.

Neuropharmacology 2017 Oct 18;125:386-395. Epub 2017 Aug 18.

Pharmakologisches Institut für Naturwissenschaftler, Goethe-Universität, Fachbereich Biochemie, Chemie und Pharmazie, 60438 Frankfurt am Main, Germany; Institut für Pharmakologie und Toxikologie, Universität Witten/Herdecke, ZBAF, 58453 Witten, Germany.

Intermediate conductance calcium-activated potassium channels (K3.1) have been recently implicated in pain processing. However, the functional role and localization of K3.1 in the nociceptive system are largely unknown. We here characterized the behavior of mice lacking K3.1 (K3.1) in various pain models and analyzed the expression pattern of K3.1 in dorsal root ganglia (DRG) and the spinal cord. K3.1 mice demonstrated normal behavioral responses in models of acute nociceptive, persistent inflammatory, and persistent neuropathic pain. However, their behavioral responses to noxious chemical stimuli such as formalin and capsaicin were increased. Accordingly, formalin-induced nociceptive behavior was increased in wild-type mice after administration of the K3.1 inhibitor TRAM-34. In situ hybridization experiments detected K3.1 in most DRG satellite glial cells, in a minority of DRG neurons, and in ependymal cells lining the central canal of the spinal cord. Together, our data point to a specific inhibitory role of K3.1 for the processing of noxious chemical stimuli.
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http://dx.doi.org/10.1016/j.neuropharm.2017.08.021DOI Listing
October 2017

Slack K Channels Influence Dorsal Horn Synapses and Nociceptive Behavior.

Mol Pain 2017 Jan-Dec;13:1744806917714342

Program in Neuroscience, University at Buffalo, The State University of New York, NY, USA.

Abstract: The sodium-activated potassium channel Slack (Kcnt1, Slo2.2) is highly expressed in dorsal root ganglion neurons where it regulates neuronal firing. Several studies have implicated the Slack channel in pain processing, but the precise mechanism or the levels within the sensory pathway where channels are involved remain unclear. Here, we furthered the behavioral characterization of Slack channel knockout mice and for the first time examined the role of Slack channels in the superficial, pain-processing lamina of the dorsal horn. We performed whole-cell recordings from spinal cord slices to examine the intrinsic and synaptic properties of putative inhibitory and excitatory lamina II interneurons. Slack channel deletion altered intrinsic properties and synaptic drive to favor an overall enhanced excitatory tone. We measured the amplitudes and paired pulse ratio of paired excitatory post-synaptic currents at primary afferent synapses evoked by electrical stimulation of the dorsal root entry zone. We found a substantial decrease in the paired pulse ratio at synapses in Slack deleted neurons compared to wildtype, indicating increased presynaptic release from primary afferents. Corroborating these data, plantar test showed Slack knockout mice have an enhanced nociceptive responsiveness to localized thermal stimuli compared to wildtype mice. Our findings suggest that Slack channels regulate synaptic transmission within the spinal cord dorsal horn and by doing so establishes the threshold for thermal nociception.
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http://dx.doi.org/10.1177/1744806917714342DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5486487PMC
September 2018

SK4 channels modulate Ca signalling and cell cycle progression in murine breast cancer.

Mol Oncol 2017 09 26;11(9):1172-1188. Epub 2017 Jun 26.

Department of Pharmacology, Toxicology and Clinical Pharmacy, Institute of Pharmacy, University of Tuebingen, Germany.

Oncogenic signalling via Ca -activated K channels of intermediate conductance (SK4, also known as K 3.1 or IK) has been implicated in different cancer entities including breast cancer. Yet, the role of endogenous SK4 channels for tumorigenesis is unclear. Herein, we generated SK4-negative tumours by crossing SK4-deficient (SK4 KO) mice to the polyoma middle T-antigen (PyMT) and epidermal growth factor receptor 2 (cNeu) breast cancer models in which oncogene expression is driven by the retroviral promoter MMTV. Survival parameters and tumour progression were studied in cancer-prone SK4 KO in comparison with wild-type (WT) mice and in a syngeneic orthotopic mouse model following transplantation of SK4-negative or WT tumour cells. SK4 activity was modulated by genetic or pharmacological means using the SK4 inhibitor TRAM-34 in order to establish the role of breast tumour SK4 for cell growth, electrophysiological signalling, and [Ca ] oscillations. Ablation of SK4 and TRAM-34 treatment reduced the SK4-generated current fraction, growth factor-dependent Ca entry, cell cycle progression and the proliferation rate of MMTV-PyMT tumour cells. In vivo, PyMT oncogene-driven tumorigenesis was only marginally affected by the global lack of SK4, whereas tumour progression was significantly delayed after orthotopic implantation of MMTV-PyMT SK4 KO breast tumour cells. However, overall survival and progression-free survival time in the MMTV-cNeu mouse model were significantly extended in the absence of SK4. Collectively, our data from murine breast cancer models indicate that SK4 activity is crucial for cell cycle control. Thus, the modulation of this channel should be further investigated towards a potential improvement of existing antitumour strategies in human breast cancer.
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http://dx.doi.org/10.1002/1878-0261.12087DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5579333PMC
September 2017

Amplified pathogenic actions of angiotensin II in cysteine-rich LIM-only protein 4-negative mouse hearts.

FASEB J 2017 04 30;31(4):1620-1638. Epub 2017 Jan 30.

Department of Pharmacology, Toxicology, and Clinical Pharmacy, Institute of Pharmacy, University of Tübingen, Tübingen, Germany;

LIM domain proteins have been identified as essential modulators of cardiac biology and pathology; however, it is unclear which role the cysteine-rich LIM-only protein (CRP)4 plays in these processes. In studying CRP4 mutant mice, we found that their hearts developed normally, but lack of CRP4 exaggerated multiple parameters of the cardiac stress response to the neurohormone angiotensin II (Ang II). Aiming to dissect the molecular details, we found a link between CRP4 and the cardioprotective cGMP pathway, as well as a multiprotein complex comprising well-known hypertrophy-associated factors. ignificant enrichment of the cysteine-rich intestinal protein (CRIP)1 in murine hearts lacking CRP4, as well as severe cardiac defects and premature death of CRIP1 and CRP4 morphant zebrafish embryos, further support the notion that depleting CRP4 is incompatible with a proper cardiac development and function. Together, amplified Ang II signaling identified CRP4 as a novel antiremodeling factor regulated, at least to some extent, by cardiac cGMP.-Straubinger, J., Boldt, K., Kuret, A., Deng, L., Krattenmacher, D., Bork, N., Desch, M., Feil, R., Feil, S., Nemer, M., Ueffing, M., Ruth, P., Just, S., Lukowski, R. Amplified pathogenic actions of angiotensin II in cysteine-rich LIM-only protein 4 negative mouse hearts.
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http://dx.doi.org/10.1096/fj.201601186DOI Listing
April 2017

Obesogenic and Diabetogenic Effects of High-Calorie Nutrition Require Adipocyte BK Channels.

Diabetes 2016 Dec 7;65(12):3621-3635. Epub 2016 Sep 7.

Pharmakologie, Toxikologie und Klinische Pharmazie, Institut für Pharmazie, Tübingen, Germany

Elevated adipose tissue expression of the Ca- and voltage-activated K (BK) channel was identified in morbidly obese men carrying a BK gene variant, supporting the hypothesis that K channels affect the metabolic responses of fat cells to nutrients. To establish the role of endogenous BKs in fat cell maturation, storage of excess dietary fat, and body weight (BW) gain, we studied a gene-targeted mouse model with global ablation of the BK channel (BK) and adipocyte-specific BK-deficient (adipoqBK) mice. Global BK deficiency afforded protection from BW gain and excessive fat accumulation induced by a high-fat diet (HFD). Expansion of white adipose tissue-derived epididymal BK preadipocytes and their differentiation to lipid-filled mature adipocytes in vitro, however, were improved. Moreover, BW gain and total fat masses of usually superobese ob/ob mice were significantly attenuated in the absence of BK, together supporting a central or peripheral role for BKs in the regulatory system that controls adipose tissue and weight. Accordingly, HFD-fed adipoqBK mutant mice presented with a reduced total BW and overall body fat mass, smaller adipocytes, and reduced leptin levels. Protection from pathological weight gain in the absence of adipocyte BKs was beneficial for glucose handling and related to an increase in body core temperature as a result of higher levels of uncoupling protein 1 and a low abundance of the proinflammatory interleukin-6, a common risk factor for diabetes and metabolic abnormalities. This suggests that adipocyte BK activity is at least partially responsible for excessive BW gain under high-calorie conditions, suggesting that BK channels are promising drug targets for pharmacotherapy of metabolic disorders and obesity.
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http://dx.doi.org/10.2337/db16-0245DOI Listing
December 2016

Stimulation of Slack K(+) Channels Alters Mass at the Plasma Membrane by Triggering Dissociation of a Phosphatase-Regulatory Complex.

Cell Rep 2016 08 18;16(9):2281-8. Epub 2016 Aug 18.

Department of Pharmacology, Yale School of Medicine, New Haven, CT 06520, USA; Department of Cellular and Molecular Physiology, Yale School of Medicine, New Haven, CT 06520, USA. Electronic address:

Human mutations in the cytoplasmic C-terminal domain of Slack sodium-activated potassium (KNa) channels result in childhood epilepsy with severe intellectual disability. Slack currents can be increased by pharmacological activators or by phosphorylation of a Slack C-terminal residue by protein kinase C. Using an optical biosensor assay, we find that Slack channel stimulation in neurons or transfected cells produces loss of mass near the plasma membrane. Slack mutants associated with intellectual disability fail to trigger any change in mass. The loss of mass results from the dissociation of the protein phosphatase 1 (PP1) targeting protein, Phactr-1, from the channel. Phactr1 dissociation is specific to wild-type Slack channels and is not observed when related potassium channels are stimulated. Our findings suggest that Slack channels are coupled to cytoplasmic signaling pathways and that dysregulation of this coupling may trigger the aberrant intellectual development associated with specific childhood epilepsies.
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http://dx.doi.org/10.1016/j.celrep.2016.07.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5123741PMC
August 2016

BK K+ channel blockade inhibits radiation-induced migration/brain infiltration of glioblastoma cells.

Oncotarget 2016 Mar;7(12):14259-78

Department of Radiation Oncology, University of Tübingen, Tübingen, Germany.

Infiltration of the brain by glioblastoma cells reportedly requires Ca2+ signals and BK K+ channels that program and drive glioblastoma cell migration, respectively. Ionizing radiation (IR) has been shown to induce expression of the chemokine SDF-1, to alter the Ca2+ signaling, and to stimulate cell migration of glioblastoma cells. Here, we quantified fractionated IR-induced migration/brain infiltration of human glioblastoma cells in vitro and in an orthotopic mouse model and analyzed the role of SDF-1/CXCR4 signaling and BK channels. To this end, the radiation-induced migratory phenotypes of human T98G and far-red fluorescent U-87MG-Katushka glioblastoma cells were characterized by mRNA and protein expression, fura-2 Ca2+ imaging, BK patch-clamp recording and transfilter migration assay. In addition, U-87MG-Katushka cells were grown to solid glioblastomas in the right hemispheres of immunocompromised mice, fractionated irradiated (6 MV photons) with 5 × 0 or 5 × 2 Gy, and SDF-1, CXCR4, and BK protein expression by the tumor as well as glioblastoma brain infiltration was analyzed in dependence on BK channel targeting by systemic paxilline application concomitant to IR. As a result, IR stimulated SDF-1 signaling and induced migration of glioblastoma cells in vitro and in vivo. Importantly, paxilline blocked IR-induced migration in vivo. Collectively, our data demonstrate that fractionated IR of glioblastoma stimulates and BK K+ channel targeting mitigates migration and brain infiltration of glioblastoma cells in vivo. This suggests that BK channel targeting might represent a novel approach to overcome radiation-induced spreading of malignant brain tumors during radiotherapy.
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http://dx.doi.org/10.18632/oncotarget.7423DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4924713PMC
March 2016
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