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A 4-year survey is presented on the alpha-ketoglutarate (KG) values in whole blood of 200 patients with malignant neoplasms mainly of the gastroinstestinal tract and the female breast. A group of patients with benign surgical diseases served as control. KG showed an association with the extent of the primary tumor classified according to the TNM-scheme. The percentages of single values above the 2s-range of the control were as follows: T2:18%, T3: 41%, and T 4: 64%. The mean value of KG had highly significantly increased already in stage T 2 as compared to the control group. Rats bearing the Yoshida sarcoma showed a significant correlation between the tumor size and the daily excretion of KG into urine during the early stage of tumor growth. The results suggested that KG cannot be regarded as an early tumor marker in humans, but may be of some value as an aid for the differential diagnosis in advanced tumor stages. [ABSTRACT FROM AUTHOR]

The continuous administration of physiological doses of the branched-chain amino acids leucine, isoleucine, and valine (Leu-Ile-Val) to Yoshida sarcoma-bearing rats caused a significant increase in the survival time by 32% and a significant reduction of tumor size after 3 weeks of growth by 33%. The shift of the nitrogen balance to negative values during the cachectic stage was delayed but not prevented. On the average, less nitrogen (-47 mg/day) were lost by Leu-Ile-Val treated rats compared with untreated tumor-bearing animals (-91 mg N/day). It appeared that Leu-Ile-Val increased the synthesis of carcass proteins, while it left the proteolysis rate unchanged, since the excretion of urea and creatinine was unaffected by these amino acids. The daily excretion of α-ketoglutarate, which is correlated with tumor size during the early stage of growth, was decreased during the first 2 weeks by Leu-Ile-Val, but remained for a longer period on a high level than in untreated tumor bearers. The results point to an improvement of the metabolic resistance against carcass protein depletion of the tumor-bearing host by the administration of branched-chain amino acids. [ABSTRACT FROM AUTHOR]

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Referring to the increased α-ketoglutarate (I) concentration in blood of cancer-bearing humans, the concentration of (I) in blood and of albumin (II) in serum were measured in 85 tumor-bearing and control rats by fluorimetry and cellulose acetate electrophoresis, respectively. (I) was elevated on the average by a factor of 1.82 and (II) was decreased by a factor of 0.73 in rats bearing the solid Rhabdomyosarcoma BA 1112 and the ascitic or solid forms of Walker-carcinoma 256 and Yoshida-sarcoma compared with tumor-free animals. All deviations were found to be statistically significant for the different tumor types, with the exception of the increase of (I) in Walker-ascites tumor-bearing rats. For rats bearing the ascitic and solid Yoshida-sarcoma and the ascitic Walker-carcinoma 256 and for tumor-free rats a negative correlation could be observed between increase of (I) and decrease of (II). The increase of (I) in blood was paralleled by an increased excretion rate of (I) into urine. In rats bearing the solid Yoshida-sarcoma the daily excreted amount of (I) was significantly increased by a factor of 1.86 compared with tumor-free animals. The excretion of (I) was already increased in the first week after tranplantation, remained constant for two more weeks and decreased sharply below normal values prior to death. The results suggest that rats bearing the inquested tumors are suitable models for metabolic studies on causes of pathological concentrations of (I) in blood of cancer patients. [ABSTRACT FROM AUTHOR]

Sepsis has emerged as a global health burden associated with multiple organ dysfunction and 20% mortality rate in patients. Numerous clinical studies over the past two decades have correlated the disease severity and mortality in septic patients with impaired heart rate variability (HRV), as a consequence of impaired chronotropic response of sinoatrial node (SAN) pacemaker activity to vagal/parasympathetic stimulation. However, the molecular mechanism(s) downstream to parasympathetic inputs have not been investigated yet in sepsis, particularly in the SAN. Based on electrocardiography, fluorescence Ca 2+ imaging, electrophysiology, and protein assays from organ to subcellular level, we report that impaired muscarinic receptor subtype 2-G protein-activated inwardly-rectifying potassium channel (M2R-GIRK) signaling in a lipopolysaccharide-induced proxy septic mouse model plays a critical role in SAN pacemaking and HRV. The parasympathetic responses to a muscarinic agonist, namely I KACh activation in SAN cells, reduction in Ca 2+ mobilization of SAN tissues, lowering of heart rate and increase in HRV, were profoundly attenuated upon lipopolysaccharide-induced sepsis. These functional alterations manifested as a direct consequence of reduced expression of key ion-channel components (GIRK1, GIRK4, and M2R) in the mouse SAN tissues and cells, which was further evident in the human right atrial appendages of septic patients and likely not mediated by the common proinflammatory cytokines elevated in sepsis.

A search in the GDC Data Portal revealed 304 documented somatic mutations of the KCNJ3 gene in primary tumors (out of 10.202 cases). Most affected tumor types were carcinomas from uterus, skin and lung, while breast cancer exerted the lowest number of somatic mutations. We focused our research on 15 missense mutations within the region between TM1 and TM2, comprising the pore helix and ion selectivity signature. Expression was measured by confocal laser scan microscopy of eGFP tagged GIRK1 subunits, expressed with and without GIRK4 in oocytes of Xenopus laevis . GIRK ion currents were activated via coexpressed m 2 Rs and measured by the Two Electrode Voltage Clamp technique. Magnitude of the total GIRK current, as well as the fraction of current inducible by the agonist, were measured. Ion selectivity was gauged by assessment of the P Na+ /P K+ ratio, calculated by the GIRK current reversal potential in extracellular media at different Na + and K + concentrations. None of the tested mutations was able to form functional GIRK1 homooligomeric ion channels. One of the mutations, G145A, which locates directly to the ion selectivity signature, exerted an increased P Na+ /P K+ ratio. Generally, the missense mutations studied can be categorized into three groups: (i) normal/reduced expression accompanied by reduced/absent function (S132Y, F136L, E139K, G145A, R149Q, R149P, G178D, S185Y, Q186R), (ii) normal/increased expression as well as increased function (E140M, A142T, M184I) and (iii) miniscule expression but increased function relative to expression levels (I151N, G158S). We conclude, that gain of function mutations, identical or similar to categories (ii) and (iii), may potentially be involved in genesis and progression of malignancies in tissues that exert a high rate of occurrence of somatic mutations of KCNJ3., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Pelzmann, Hatab, Scheruebel, Langthaler, Rienmueller, Sokolowski, Gorischek, Platzer, Zorn-Pauly, Jahn, Bauernhofer and Schreibmayer.)

Lung cancer is still a leading cause of death worldwide. In recent years, knowledge has been obtained of the mechanisms modulating ion channel kinetics and thus of cell bioelectric properties, which is promising for oncological biomarkers and targets. The complex interplay of channel expression and its consequences on malignant processes, however, is still insufficiently understood. We here introduce the first approach of an in-silico whole-cell ion current model of a cancer cell, in particular of the A549 human lung adenocarcinoma, including the main functionally expressed ion channels in the plasma membrane as so far known. This hidden Markov-based model represents the electrophysiology behind proliferation of the A549 cell, describing its rhythmic oscillation of the membrane potential able to trigger the transition between cell cycle phases, and it predicts membrane potential changes over the cell cycle provoked by targeted ion channel modulation. This first A549 in-silico cell model opens up a deeper insight and understanding of possible ion channel interactions in tumor development and progression, and is a valuable tool for simulating altered ion channel function in lung cancer electrophysiology., Competing Interests: The authors have declared that no competing interests exist.

Excessive expression of subunit 1 of GIRK1 in ER + breast tumors is associated with reduced survival times and increased lymph node metastasis in patients. To investigate possible tumor-initiating properties, benign MCF10A and malign MCF7 mammary epithelial cells were engineered to overexpress GIRK1 neoplasia associated vital parameters and resting potentials were measured and compared to controls. The presence of GIRK1 resulted in resting potentials negative to the controls. Upon GIRK1 overexpression, several cellular pathways were regulated towards pro-tumorigenic action as revealed by comparison of transcriptomes of MCF10A GIRK1 with the control (MCF10A eGFP ). According to transcriptome analysis, cellular migration was promoted while wound healing and extracellular matrix interactions were impaired. Vital parameters in MCF7 cells were affected akin the benign MCF10A lines, but to a lesser extent. Thus, GIRK1 regulated cellular pathways in mammary epithelial cells are likely to contribute to the development and progression of breast cancer.

Mutation of a single residue within the recently identified lipid (diacylglycerol) recognition window of TRPC3 (G652A) was found to abolish channel activation via endogenous lipid mediators while retaining sensitivity to the non-lipid activator GSK1702934A (abb. GSK). The mechanism of this change in chemical sensing by TRPC3 was analysed by whole-cell and single channel electrophysiology as well as Ca 2+ imaging. Currents initiated by GSK or the structural (benzimidazole) analog BI-2 were significantly larger in cells expressing the G652A mutant as compared to wild type (WT) channels. Whole cell patch-clamp experiments revealed that enhanced sensitivity to benzimidazoles was not due to augmented potency but reflected enhanced efficacy of benzimidazoles. Single channel analysis demonstrated that neither unitary conductance nor I-V characteristics were altered by the G652A mutation, precluding altered pore architecture as the basis of enhanced efficacy. These experiments uncovered a distinct gating pattern of BI-2-activated G652A mutant channels, featuring a unique, long-lived open state. Moreover, G652A mutant channels lacked PLC/diacylglycerol mediated cross-desensitization for GSK activation as typically observed for TRPC3. Lack of desensitization in G652A channels enabled large GSK/BI-2-induced Ca 2+ signals in conditions that fully desensitized TRPC3 WT channels. We demonstrate that the lipid-recognition window of TRPC3 determines both sensitivity to lipid mediators and chemical gating by benzimidazoles. TRPC3 mutations within this lipid interaction site are suggested as a basis for chemogenetic targeting of TRPC3-signaling., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Transient receptor potential canonical (TRPC) channels TRPC3, TRPC6 and TRPC7 are able to sense the lipid messenger diacylglycerol (DAG). The DAG-sensing and lipid-gating processes in these ion channels are still unknown. To gain insights into the lipid-sensing principle, we generated a DAG photoswitch, OptoDArG, that enabled efficient control of TRPC3 by light. A structure-guided mutagenesis screen of the TRPC3 pore domain unveiled a single glycine residue behind the selectivity filter (G652) that is exposed to lipid through a subunit-joining fenestration. Exchange of G652 with larger residues altered the ability of TRPC3 to discriminate between different DAG molecules. Light-controlled activation-deactivation cycling of TRPC3 channels by an OptoDArG-mediated optical 'lipid clamp' identified pore domain fenestrations as pivotal elements of the channel´s lipid-sensing machinery. We provide evidence for a novel concept of lipid sensing by TRPC channels based on a lateral fenestration in the pore domain that accommodates lipid mediators to control gating.

Numerous studies showed abnormal expression of ion channels in different cancer types. Amongst these, the potassium channel gene KCNJ3 (encoding for GIRK1 proteins) has been reported to be upregulated in tumors of patients with breast cancer and to correlate with positive lymph node status. We aimed to study KCNJ3 levels in different breast cancer subtypes using gene expression data from the TCGA, to validate our findings using RNA in situ hybridization in a validation cohort (GEO ID GSE17705), and to study the prognostic value of KCNJ3using survival analysis. In a total of > 1000 breast cancer patients of two independent data sets we showed a) that KCNJ3 expression is upregulated in tumor tissue compared to corresponding normal tissue (p < 0.001), b) that KCNJ3 expression is associated with estrogen receptor (ER) positive tumors (p < 0.001), but that KCNJ3 expression is variable within this group, and c) that ER positive patients with high KCNJ3 levels have worse overall (p < 0.05) and disease free survival probabilities (p < 0.01), whereby KCNJ3 is an independent prognostic factor (p <0.05). In conclusion, our data suggest that patients with ER positive breast cancer might be stratified into high risk and low risk groups based on the KCNJ3 levels in the tumor.

Increased expression levels of KCNJ3 have been correlated with lymph node metastases and poor prognosis in patients with breast cancer, suggesting a prognostic role of KCNJ3 We aimed to establish protocols for the detection of KCNJ3 in formalin-fixed, paraffin-embedded (FFPE) breast cancer tissue. Several antibodies were tested for sensitivity and specificity by western blot, followed by optimisation of the immunohistochemistry (IHC) procedure and establishment of KCNJ3 mRNA in situ hybridisation (ISH). Methods were validated by processing 15 FFPE breast cancer samples for which microarray data were available. Spearman's rank correlation analysis resulted in borderline significant correlation for IHC versus ISH (r S : 0.625; p<0.05) and IHC versus microarray (r S : 0.668; p<0.01), but in significant correlation for ISH versus microarray (r S : 0.861; p<0.001). The ISH method was superior to IHC, regarding robustness, sensitivity and specificity and will aid to further study expression levels of KCNJ3 in both malignant and physiological conditions., Competing Interests: Conflicts of Interest: None declared., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.)

Mechanical interaction between cells - specifically distortion of tensional homeostasis-emerged as an important aspect of breast cancer genesis and progression. We investigated the biophysical characteristics of mechanosensitive ion channels (MSCs) in the malignant MCF-7 breast cancer cell line. MSCs turned out to be the most abundant ion channel species and could be activated by negative pressure at the outer side of the cell membrane in a saturable manner. Assessing single channel conductance (GΛ) for different monovalent cations revealed an increase in the succession: Li(+) < Na(+) < K(+) ≈Rb(+) ≈ Cs(+). Divalent cations permeated also with the order: Ca(2+) < Ba(2+). Comparison of biophysical properties enabled us to identify MSCs in MCF-7 as ion channels formed by the Piezo1 protein. Using patch clamp technique no functional MSCs were observed in the benign MCF-10A mammary epithelial cell line. Blocking of MSCs by GsMTx-4 resulted in decreased motility of MCF-7, but not of MCF-10A cells, underscoring a possible role of Piezo1 in invasion and metastatic propagation. The role of Piezo1 in biology and progression of breast cancer is further substantiated by markedly reduced overall survival in patients with increased Piezo1 mRNA levels in the primary tumor.

Background: β-Synuclein (β-Syn) is a member of the highly homologous synuclein protein family. The most prominent family member, α-synuclein (α-Syn), abnormally accumulates in so-called Lewy bodies, one of the major pathological hallmarks of α-synucleinopathies. Notably, parts of the peptide backbone, called the nonamyloid component, are also found in amyloid plaques. However, β-Syn seems to have beneficial effects by reducing α-Syn aggregation, and amyloid antiaggregatory activity has been described., Objective: The aim of the study was to analyze if wild-type β-Syn can counteract functional and pathological changes in a murine Alzheimer model over different time periods., Methods: At the onset of pathology, lentiviral particles expressing human β-Syn were injected into the hippocampus of transgenic mice overexpressing human amyloid precursor protein with Swedish and London mutations (APPSL). An empty vector served as the control. Behavioral analyses were performed 1, 3 and 6 months after injection followed by biochemical and histological examinations of brain samples., Results: β-Syn expression was locally concentrated and rather modest, but nevertheless changed its effect on APP expression and plaque load in a time- and concentration-dependent manner. Interestingly, the phosphorylation of glycogen synthase kinase 3 beta was enhanced in APPSL mice expressing human β-Syn, but an inverse trend was observed in wild-type animals., Conclusion: The initially reported beneficial effects of β-Syn could be partially reproduced, but locally elevated levels of β-Syn might also cause neurodegeneration. To enlighten the controversial pathological mechanism of β-Syn, further examinations considering the relationship between concentration and exposure time of β-Syn are needed., (© 2015 S. Karger AG, Basel.)

Previous attempts to measure the functional properties of recombinant nicotinic acetylcholine receptors (nAChRs) composed of known receptor subunits have yielded conflicting results. The use of knockout mice that lack α5, β2, α5β2 or α5β2α7 nAChR subunits enabled us to measure the single-channel properties of distinct α3β4, α3β4α5 and α3β4β2 receptors in superior cervical ganglion (SCG) neurons. Using this approach, we found that α3β4 receptors had a principal conductance level of 32.6 ± 0.8 pS (mean ± SEM) and both higher and lower secondary conductance levels. α3β4α5 receptors had the same conductance as α3β4 receptors, but differed from α3β4 receptors by having an increased channel open time and increased burst duration. By contrast, α3β4β2 receptors differed from α3β4 and α3β4α5 receptors by having a significantly smaller conductance level (13.6 ± 0.5 pS). After dissecting the single-channel properties of these receptors using our knockout models, we then identified these properties - and hence the receptors themselves - in wild-type SCG neurons. This study is the first to identify the single-channel properties of distinct neuronal nicotinic receptors in their native environment.

G-protein activated inwardly rectifying K(+) channels (GIRKs) of the heterotetrameric GIRK1/GIRK4 composition mediate I(K+ACh) in atrium and are regulated by cAMP dependent protein kinase (PKA). Phosphorylation of GIRK1/GIRK4 complexes promotes the activation of the channel by the G-protein Gβγ-dimer ("heterologous facilitation"). Previously we reported that 3 serines/threonines (S/Ts) within the GIRK1 subunit are phosphorylated by the catalytic subunit of PKA (PKA-cs) in-vitro and are responsible for the acute functional effects exerted by PKA on the homooligomeric GIRK1(F137S) (GIRK1(⁎)) channel. Here we report that homooligomeric GIRK4(WT) and GIRK4(S143T) (GIRK4(⁎)) channels are clearly regulated by PKA phosphorylation. Heterooligomeric channels of the GIRK1(S385CS401CT407C)/GIRK4(WT) composition, where the GIRK1 subunit is devoid of PKA mediated phosphorylation, exhibited reduced but still significant acute effects (reduction during agonist application was ≈49% compared to GIRK1(WT)/GIRK4(WT)). Site directed mutagenesis of truncated cytosolic regions of GIRK4 revealed four serines/threonines (S/Ts) that were heavily phosphorylated by PKA-cs in vitro. Two of them were found to be responsible for the acute effects exerted by PKA in vivo, since the effect of cAMP injection was reduced by ≈99% in homooligomeric GIRK4(⁎T199CS412C) channels. Coexpression of GIRK1(WT)/GIRK4(T199CS412C) reduced the acute effect by ≈65%. Only channels of the GIRK1(S385CS401CT407C)/GIRK4(T199CS412C) composition were practically devoid of PKA mediated effects (reduction by ≈97%), indicating that both subunits contribute to the heterologous facilitation of I(K+ACh)., (Copyright © 2012 Elsevier B.V. All rights reserved.)