Search

Human mutations of the Na+-activated K+ channel Slack (KCNT1) are associated with epilepsy and intellectual disability. Accordingly, Slack knockout mice (Slack−/−) exhibit cognitive flexibility deficits in distinct behavioral tasks. So far, however, the underlying causes as well as the role of Slack in hippocampus-dependent memory functions remain enigmatic. We now report that infant (P6–P14) Slack−/− lack both hippocampal LTD and LTP, likely due to impaired NMDA receptor (NMDAR) signaling. Postsynaptic GluN2B levels are reduced in infant Slack−/−, evidenced by lower amplitudes of NMDAR-meditated excitatory postsynaptic potentials. Low GluN2B affected NMDAR-mediated Ca2+-influx, rendering cultured hippocampal Slack−/−neurons highly insensitive to the GluN2B-specific inhibitor Ro 25-6981. Furthermore, dephosphorylation of the AMPA receptor (AMPAR) subunit GluA1 at S845, which is involved in AMPAR endocytosis during homeostatic and neuromodulator-regulated plasticity, is reduced after chemical LTD (cLTD) in infant Slack−/−. We additionally detect a lack of mGluR-induced LTD in infant Slack−/−, possibly caused by upregulation of the recycling endosome-associated small GTPase Rab4 which might accelerate AMPAR recycling from early endosomes. Interestingly, LTP and mGluR LTD, but not LTD and S845 dephosphorylation after cLTD are restored in adult Slack−/−. This together with normalized expression levels of GluN2B and Rab4 hints to developmental “restoration” of LTP expression despite Slack ablation, whereas in infant and adult brain, NMDAR-dependent LTD induction depends on this channel. Based on the present findings, NMDAR and vesicular transport might represent novel targets for the therapy of intellectual disability associated with Slack mutations. Consequently, careful modulation of hippocampal Slack activity should also improve learning abilities. Supplementary Information The online version contains supplementary material available at 10.1007/s00018-021-03953-0.

Additional file 3. Comparison of the justification categories between 2016 and 2018 in calves with diarrhea. Absolute numbers used to calculate proportions and 95% confidence intervals.

Background Sodium-activated potassium channels (KNa) Slack (sequence like a calcium-activated potassium channel, aka Slo 2.2, KNa1.1 or KCNT1) are widely expressed throughout neuronal tissue, whereas their presence and function in the cardiovascular system is not well understood. Due to high intracellular sodium concentrations ([Na+]i) necessary to induce half-maximal channel activation, we hypothesized that Slack function is attributed to pathophysiological conditions such as myocardial ischemia. Purpose To elucidate the putative functions of Slack in the murine heart and in cardiomyocytes (CMs) and to explore whether the ischemia and reperfusion (I/R)-induced cardiac damage is affected by endogenous Slack channel activity. Methods I/R injury was evaluated in global and CM-specific Slack knockout mice (Slack gKO, CM Slack KO) and compared to litter-matched controls (Slack gWT, CM Slack CTR) by applying an in vivo model of acute myocardial infarction (MI). Infarct size (IS) was assessed at baseline, after ischemic pre- (iPre) and postconditioning (iPost) and in response to cinaciguat (CIN), a cGMP-elevating agent. Moreover, Slack expression and function in CMs was studied by biochemical and electrophysiological means and by utilizing the newly developed FRET-based K+ probe GEPII 1.0. Results IS in Slack gKO mice was increased in comparison to gWT littermates. In addition, the cardioprotection afforded by iPost was attenuated in the absence of Slack. To test if the increased vulnerability to I/R injury of the Slack gKO mouse model was originating from Slack activity in CMs, we subjected CM-specific Slack CTR and KO mutants to an identical MI procedure. IS measurements confirmed increased cardiac damage at baseline and reduced cardioprotective effects afforded by iPre and iPost in CM Slack KO mice. Interestingly, CIN (i.p., 30 min prior to I/R) reduced IS to a similar extent in both genotypes, suggesting that Slack functions in a cGMP-independent manner. Whole-cell patch clamp experiments on CMs demonstrated a reduction of the KNa-inhibitors clofilium- and chinidine-sensitive K+ outward currents in Slack gKO CM. Extracellular potassium ([K+]ex) accumulation measured with GEPII 1.0 was lower in Slack gKO versus gWT CM pools exposed to membrane permeabilizing agent digitonin. Accordingly, [K+]ex evoked by the Slack activators niclosamide and bithionol was lower in the absence of functional Slack in CMs. Conclusion The presented findings establish an important role of Slack channels for cardioprotective signalling mechanisms during I/R in vivo and for mediating beneficial effects of mechanical conditioning on IS. Corroborating in vitro studies on adult CMs exhibit an impaired [K+]ex dynamic in response to genetic or pharmacological modulation of Slack activity. Thus, we conclude that Slack-dependent K+ signalling pathways in CMs may represent a promising drug target that renders the heart muscle less vulnerable to the I/R-induced damage. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Work in the authors' laboratories is supported by grants from the Deutsche Forschungsgemeinschaft (DFG) (to R.L.) and the DFG Research Unit 2060, “cGMP Signaling in Cell Growth and Survival” (to R.L. and P.R.).

BACKGROUND Antimicrobial stewardship programs (ASPs) are important tools to foster prudent antimicrobial use. OBJECTIVE To evaluate antimicrobial prescriptions by Swiss veterinarians before and after introduction of the online ASP AntibioticScout.ch in December 2016. ANIMALS Dogs presented to 2 university hospitals and 14 private practices in 2016 or 2018 for acute diarrhea (AD; n = 779), urinary tract infection (UTI; n = 505), respiratory tract infection (RTI; n = 580), or wound infection (WI; n = 341). METHODS Retrospective study. Prescriptions of antimicrobials in 2016 and 2018 were compared and their appropriateness assessed by a justification score. RESULTS The proportion of dogs prescribed antimicrobials decreased significantly between 2016 and 2018 (74% vs 59%; P

Background Antimicrobial resistance is an increasing problem in human and veterinary medicine and is closely linked to the use of antimicrobials. The objective of this study was to describe antimicrobial prescriptions for selected canine diseases in Switzerland during 2016. Methods Dogs presented to two university hospitals and 14 private practices for acute diarrhoea (AD; n=371), suspected or confirmed urinary tract infections (UTIs; n=245), respiratory tract infections (RTIs; n=274) or wound infections (WIs; n=175) were included. Clinical history, diagnostic work-up and antimicrobial prescription (class, dosage and duration) were retrospectively assessed. A justification score was applied to evaluate appropriateness of antimicrobial therapy based on available national and international consensus guidelines. Results Antimicrobials were prescribed in 65 per cent of dogs with AD, 88 per cent with UTI, 62 per cent with RTI and 90 per cent with WI. The most prescribed antimicrobial classes (monotherapy and combination therapy) were potentiated aminopenicillins (59 per cent), nitroimidazoles (22 per cent), non-potentiated aminopenicillins (16 per cent) and fluoroquinolones (13 per cent). Overall, 38 per cent (95 per cent CI 0.35 to 0.41) of the prescriptions were in accordance with consensus guidelines. In dogs with AD, antimicrobial therapy was associated with the presence of haemorrhagic diarrhoea (P

International audience; 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.

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 2+ -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.

Intermediate conductance calcium-activated potassium channels (KCa3.1) have been recently implicated in pain processing. However, the functional role and localization of KCa3.1 in the nociceptive system are largely unknown. We here characterized the behavior of mice lacking KCa3.1 (KCa3.1-/-) in various pain models and analyzed the expression pattern of KCa3.1 in dorsal root ganglia (DRG) and the spinal cord. KCa3.1-/- 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 KCa3.1 inhibitor TRAM-34. In situ hybridization experiments detected KCa3.1 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 KCa3.1 for the processing of noxious chemical stimuli.

Nitric oxide (NO) activates the NO-sensitive soluble guanylate cyclase (NO-GC, sGC) and triggers intracellular signaling pathways involving cGMP. For survival of cochlear hair cells and preservation of hearing, NO-mediated cascades have both protective and detrimental potential. Here we examine the cochlear function of mice lacking one of the two NO-sensitive guanylate cyclase isoforms [NO-GC1 knockout (KO) or NO-GC2 KO]. The deletion of NO-GC1 or NO-GC2 did not influence electromechanical outer hair cell (OHC) properties, as measured by distortion product otoacoustic emissions, neither before nor after noise exposure, nor were click- or noise-burst-evoked auditory brainstem response thresholds different from controls. Yet inner hair cell (IHC) ribbons and auditory nerve responses showed significantly less deterioration in NO-GC1 KO and NO-GC2 KO mice after noise exposure. Consistent with a selective role of NO-GC in IHCs, NO-GC β1 mRNA was found in isolated IHCs but not in OHCs. Using transgenic mice expressing the fluorescence resonance energy transfer-based cGMP biosensor cGi500, NO-induced elevation of cGMP was detected in real-time in IHCs but not in OHCs. Pharmacologic long-term treatment with a NO-GC stimulator altered auditory nerve responses but did not affect OHC function and hearing thresholds. Interestingly, NO-GC stimulation exacerbated the loss of auditory nerve response in aged animals but attenuated the loss in younger animals. We propose NO-GC2 and, to some degree, NO-GC1 as targets for early pharmacologic prevention of auditory fiber loss (synaptopathy). Both isoforms provide selective benefits for hearing function by maintaining the functional integrity of auditory nerve fibers in early life rather than at old age.

// Dominik Klumpp 1 , Stephanie C. Frank 2, 3 , Lukas Klumpp 1, 4 , 5 , Efe C. Sezgin 1 , Marita Eckert 1 , Lena Edalat 1, 6 , Martin Bastmeyer 2, 3 , Daniel Zips 1 , Peter Ruth 6 and Stephan M. Huber 1 1 Department of Radiation Oncology, University of Tubingen, Tubingen, Germany 2 Zoological Institute, Department of Cell- and Neurobiology, Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany 3 Institute of Functional Interfaces, Karlsruhe Institute of Technology (KIT), Eggenstein-Leopoldshafen, Germany 4 Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology, Stuttgart, Germany 5 University of Tubingen, Tubingen, Germany 6 Department of Pharmacology, Toxicology and Clinical Pharmacy, University of Tubingen, Tubingen, Germany Correspondence to: Stephan M. Huber, email: stephan.huber@uni-tuebingen.de Keywords: glioblastoma; radioresistance; TRPM8 channels; Ca 2+ signaling; cell migration Received: July 26, 2017 Accepted: August 25, 2017 Published: September 30, 2017 ABSTRACT TRPM8 is a Ca 2+ -permeable nonselective cation channel belonging to the melastatin sub-group of the transient receptor potential (TRP) family. TRPM8 is aberrantly overexpressed in a variety of tumor entities including glioblastoma multiforme where it reportedly contributes to tumor invasion. The present study aimed to disclose further functions of TRPM8 in glioma biology in particular upon cell injury by ionizing radiation. To this end, TCGA data base was queried to expose the TRPM8 mRNA abundance in human glioblastoma specimens and immunoblotting was performed to analyze the TRPM8 protein abundance in primary cultures of human glioblastoma. Moreover, human glioblastoma cell lines were irradiated with 6 MV photons and TRPM8 channels were targeted pharmacologically or by RNA interference. TRPM8 abundance, Ca 2+ signaling and resulting K + channel activity, chemotaxis, cell migration, clonogenic survival, DNA repair, apoptotic cell death, and cell cycle control were determined by qRT-PCR, fura-2 Ca 2+ imaging, patch-clamp recording, transfilter migration assay, wound healing assay, colony formation assay, immunohistology, flow cytometry, and immunoblotting. As a result, human glioblastoma upregulates TRPM8 channels to variable extent. TRPM8 inhibition or knockdown slowed down cell migration and chemotaxis, attenuated DNA repair and clonogenic survival, triggered apoptotic cell death, impaired cell cycle and radiosensitized glioblastoma cells. Mechanistically, ionizing radiation activated and upregulated TRPM8-mediated Ca 2+ signaling that interfered with cell cycle control probably via CaMKII, cdc25C and cdc2. Combined, our data suggest that TRPM8 channels contribute to spreading, survival and radioresistance of human glioblastoma and, therefore, might represent a promising target in future anti-glioblastoma therapy.

Oncogenic signalling via Ca2+-activated K+ channels of intermediate conductance (SK4, also known as KCa3.1 or IK) has been implicated in different cancer entities including breast cancer. Yet, the role of endogenous SK4 channels for tumourigenesis 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 promotor MMTV. Survival parameters and tumour progression were studied in cancer-prone SK4 KO in comparison to wildtype (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 [Ca2+]i oscillations. Ablation of SK4 and TRAM-34 treatment reduced the SK4-generated current fraction, growth factor-dependent Ca2+ entry, cell cycle progression and the proliferation rate of MMTV-PyMT tumour cells. In vivo, PyMT oncogene-driven tumourigenesis 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 anti-tumour strategies in human breast cancer.

KCa3.1 K+ channels reportedly contribute to the proliferation of breast tumor cells and may serve pro-tumor functions in the microenvironment. The putative interaction of KCa3.1 with major anti-cancer treatment strategies, which are based on cytotoxic drugs or radiotherapy, remains largely unexplored. We employed KCa3.1-proficient and -deficient breast cancer cells derived from breast cancer-prone MMTV-PyMT mice, pharmacological KCa3.1 inhibition, and a syngeneic orthotopic mouse model to study the relevance of functional KCa3.1 for therapy response. The KCa3.1 status of MMTV-PyMT cells did not determine tumor cell proliferation after treatment with different concentrations of docetaxel, doxorubicin, 5-fluorouracil, or cyclophosphamide. KCa3.1 activation by ionizing radiation (IR) in breast tumor cells in vitro, however, enhanced radioresistance, probably via an involvement of the channel in IR-stimulated Ca2+ signals and DNA repair pathways. Consistently, KCa3.1 knockout increased survival time of wildtype mice upon syngeneic orthotopic transplantation of MMTV-PyMT tumors followed by fractionated radiotherapy. Combined, our results imply that KCa3.1 confers resistance to radio- but not to chemotherapy in the MMTV-PyMT breast cancer model. Since KCa3.1 is druggable, KCa3.1 targeting concomitant to radiotherapy seems to be a promising strategy to radiosensitize breast tumors.

Gain-of-function mutations in KCNT1, the gene encoding Slack (KNa1.1) 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 Kcnt1R455H, 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 Kcnt1R455H/R455H mice were embryonic lethal. Video-EEG monitoring of heterozygous Kcnt1+/R455H animals revealed persistent interictal spikes, spontaneous seizures and a substantially decreased threshold for pentylenetetrazole-induced seizures. Surprisingly, Kcnt1+/R455H 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.

Mammalian inner hair cells (IHCs) transduce sound into depolarization and transmitter release. Big conductance and voltage- and Ca

Staphylococcus aureus is a facultative intracellular pathogen that invades a wide range of professional and nonprofessional phagocytes by triggering internalisation by interaction of surface‐bound adhesins with corresponding host cell receptors. Here, we identified a new concept of host cell internalisation in animal‐pathogenic staphylococcal species. This new mechanism exemplified by Staphylococcus pseudintermedius ED99 is not based on surface‐bound adhesins but is due to excreted small neurochemical compounds, such as trace amines (TAs), dopamine (DOP), and serotonin (SER), that render host cells competent for bacterial internalisation. The neurochemicals are produced by only one enzyme, the staphylococcal aromatic amino acid decarboxylase (SadA). Here, we unravelled the mechanism of how neurochemicals trigger internalisation into the human colon cell line HT‐29. We found that TAs and DOP are agonists of the α2‐adrenergic receptor, which, when activated, induces a cascade of reactions involving a decrease in the cytoplasmic cAMP level and an increase in F‐actin formation. The signalling cascade of SER follows a different pathway. SER interacts with 5HT receptors that trigger F‐actin formation without decreasing the cytoplasmic cAMP level. The neurochemical‐induced internalisation in host cells is independent of the fibronectin‐binding protein pathway and has an additive effect. In a sadA deletion mutant, ED99ΔsadA, internalisation was decreased approximately threefold compared with that of the parent strain, and treating S. aureus USA300 with TAs increased internalisation by approximately threefold.

KCa3.1 K+ channels reportedly contribute to the proliferation of breast tumor cells and may serve pro-tumor functions in the microenvironment. The putative interaction of KCa3.1 with major anti-cancer treatment strategies, which are based on cytotoxic drugs or radiotherapy, remains largely unexplored. We employed KCa3.1-proficient and -deficient breast cancer cells derived from breast cancer-prone MMTV-PyMT mice, pharmacological KCa3.1 inhibition, and a syngeneic orthotopic mouse model to study the relevance of functional KCa3.1 for therapy response. The KCa3.1 status of MMTV-PyMT cells did not determine tumor cell proliferation after treatment with different concentrations of docetaxel, doxorubicin, 5-fluorouracil, or cyclophosphamide. KCa3.1 activation by ionizing radiation (IR) in breast tumor cells in vitro, however, enhanced radioresistance, probably via an involvement of the channel in IR-stimulated Ca2+ signals and DNA repair pathways. Consistently, KCa3.1 knockout increased survival time of wildtype mice upon syngeneic orthotopic transplantation of MMTV-PyMT tumors followed by fractionated radiotherapy. Combined, our results imply that KCa3.1 confers resistance to radio- but not to chemotherapy in the MMTV-PyMT breast cancer model. Since KCa3.1 is druggable, KCa3.1 targeting concomitant to radiotherapy seems to be a promising strategy to radiosensitize breast tumors.

Summary Primary electroencephalographic (EEG) features of sleep arise in part from thalamocortical neural assemblies, and cortical potassium channels have long been thought to play a critical role. We have exploited the regionally dynamic nature of sleep EEG to develop a novel screening strategy and used it to conduct an adeno-associated virus (AAV)-mediated RNAi screen for cellular roles of 31 different voltage-gated potassium channels in modulating cortical EEG features across the circadian sleep-wake cycle. Surprisingly, a majority of channels modified only electroencephalographic frequency bands characteristic of sleep, sometimes diurnally or even in specific vigilance states. Confirming our screen for one channel, we show that depletion of the KCa1.1 (or “BK”) channel reduces EEG power in slow-wave sleep by slowing neuronal repolarization. Strikingly, this reduction completely abolishes transcriptomic changes between sleep and wake. Thus, our data establish an unexpected connection between transcription and EEG power controlled by specific potassium channels. We postulate that additive dynamic roles of individual potassium channels could integrate different influences upon sleep and wake within single neurons.

K+ channels crosstalk with biochemical signaling cascades and regulate virtually all cellular processes by adjusting the intracellular K+ concentration, generating the membrane potential, mediating cell volume changes, contributing to Ca2+ signaling, and directly interacting within molecular complexes with membrane receptors and downstream effectors. Tumor cells exhibit aberrant expression and activity patterns of K+ channels. The upregulation of highly "oncogenic" K+ channels such as the Ca2+-activated IK channel may drive the neoplastic transformation, malignant progression, metastasis, or therapy resistance of tumor cells. In particular, ionizing radiation in doses used for fractionated radiotherapy in the clinic has been shown to activate K+ channels. Radiogenic K+ channel activity, in turn, contributes to the DNA damage response and promotes survival of the irradiated tumor cells. Tumor-specific overexpression of certain K+ channel types together with the fact that pharmacological K+ channel modulators are already in clinical use or well tolerated in clinical trials suggests that K+ channel targeting alone or in combination with radiotherapy might become a promising new strategy of anti-cancer therapy. The present article aims to review our current knowledge on K+ channel signaling in irradiated tumor cells. Moreover, it provides new data on molecular mechanisms of radiogenic K+ channel activation and downstream signaling events.

// Lena Edalat 1, 2, * , Benjamin Stegen 2, * , Lukas Klumpp 2, 5 , Erik Haehl 2 , Karin Schilbach 3 , Robert Lukowski 1 , Matthias Kuhnle 4 , Gunther Bernhardt 4 , Armin Buschauer 4 , Daniel Zips 2 , Peter Ruth 1 , Stephan M. Huber 2 1 Department of Pharmacology, Toxicology and Clinical Pharmacy, University of Tubingen, Tubingen, Germany 2 Department of Radiation Oncology, University of Tubingen, Tubingen, Germany 3 Department of General Pediatrics, Oncology/Hematology, University of Tubingen, Tubingen, Germany 4 Department of Pharmaceutical/Medicinal Chemistry II, University of Regensburg, Regensburg, Germany 5 Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology, University of Tubingen, Tubingen, Germany * These authors contributed equally to this work Correspondence to: Peter Ruth, e-mail: peter.ruth@uni-tuebingen.de Stephan M. Huber, e-mail: stephan.huber@uni-tuebingen.de Keywords: glioma, radiation therapy, patch-clamp recording, fura-2 Ca2 + imaging, transfilter migration Received: October 20, 2015 Accepted: January 29, 2016 Published: February 16, 2016 ABSTRACT Infiltration of the brain by glioblastoma cells reportedly requires Ca 2+ 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 Ca 2+ 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 Ca 2+ 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.

Background: Removal of nonvital tissue is an accepted method to eradicate biofilms and to stimulate wound healing. Debridement using a monofilament polyester fiber pad has clinically been shown to be effective as well as pain and trauma free. Methods: For in vitro determination of the cleansing capacity of this product compared to gauze swabs, a wound debridement model was used with glass plates coated with a bovine serum albumin solution, stained with hematoxylin. Both products were moistened and fixed to a weight connected to a regulated motor and were then pulled over the holding device with the coated glass plate under standardized conditions (power = 0.067 N/cm2, velocity = 1.6 cm/s). Results: At a low coating concentration (0.5%) both products were equally effective, but at a high concentration (1.5%) cleansing did not occur after 5 wipes. When wiping the plates 15 times, the debridement pad cleansed significantly (p < 0.001) better than gauze. When consecutively wiping 4 coated plates with a single debridement pad or swab, the pad exhibited and maintained a significantly higher cleansing capacity while gauze quickly lost its effect. Conclusion: Our in vitro test results indicated a higher cleansing capacity of the debridement pad compared to gauze swabs.

Objective— Vascular smooth muscle cells (VSMC) proliferation is a hallmark of atherosclerosis and vascular restenosis. The intermediate conductance Ca 2+ -activated K + (SK4) channel is required for pathological VSMC proliferation. In T lymphocytes, nucleoside diphosphate kinase B (NDPKB) has been implicated in SK4 channel activation. We thus investigated the role of NDPKB in the regulation of SK4 currents (I SK4 ) in proliferating VSMC and neointima formation. Approach and Results— Function and expression of SK4 channels in VSMC from injured mouse carotid arteries were assessed by patch-clamping and real-time polymerase chain reaction. I SK4 was detectable in VSMC from injured but not from uninjured arteries correlating with the occurrence of the proliferative phenotype. Direct application of NDPKB to the membrane of inside-out patches increased I SK4 , whereas NDPKB did not alter currents in VSMC obtained from injured vessels of SK4-deficient mice. The NDPKB-induced increase in I SK4 was prevented by protein histidine phosphatase 1, but not an inactive protein histidine phosphatase 1 mutant indicating that I SK4 is regulated via histidine phosphorylation in proliferating VSMC; moreover, genetic NDPKB ablation reduced I SK4 by 50% suggesting a constitutive activation of I SK4 in proliferating VSMC. In line, neointima formation after wire injury of the carotid artery was substantially reduced in mice deficient in SK4 channels or NDPKB. Conclusions— NDPKB to SK4 signaling is required for neointima formation. Constitutive activation of SK4 by NDPKB in proliferating VSMC suggests that targeting this interaction via, for example, activation of protein histidine phosphatase 1 may provide clinically meaningful effects in vasculoproliferative diseases such as atherosclerosis and post angioplasty restenosis.

Kcnt1 encoded sodium-activated potassium channels (Slack channels) are highly expressed throughout the brain where they modulate the firing patterns and general excitability of many types of neurons. Increasing evidence suggests that Slack channels may be important for higher brain functions such as cognition and normal intellectual development. In particular, recent findings have shown that human Slack mutations produce very severe intellectual disability and that Slack channels interact directly with the Fragile X mental retardation protein (FMRP), a protein that when missing or mutated results in Fragile X syndrome (FXS), the most common form of inherited intellectual disability and autism in humans. We have now analyzed a recently developed Kcnt1 null mouse model in several behavioral tasks to assess which aspects of memory and learning are dependent on Slack. We demonstrate that Slack deficiency results in mildly altered general locomotor activity, but normal working memory, reference memory, as well as cerebellar control of motor functions. In contrast, we find that Slack channels are required for cognitive flexibility, including reversal learning processes and the ability to adapt quickly to unfamiliar situations and environments. Our data reveal that hippocampal-dependent spatial learning capabilities require the proper function of Slack channels.

Vascular tone can be regulated via nucleotides like ATP and UTP that are derived from erythrocytes or platelets or released from the endothelium (Ralevic & Burnstock 2003). It is known that ATP is an agonist of P2Y2 receptors and UTP is an agonist of P2Y2/4 receptors (Abbracchio et al. 2006). A decrease in blood pressure most likely results from ATP inducing endothelium-dependent relaxation (von Kugelgen et al. 1987, von Kugelgen & Starke 1990, Ralevic & Burnstock 1991a, 2003). Evidence suggests that this vasodilatory response can be mediated by nitric oxide (NO; Ralevic & Burnstock 1991a), endothelial-derived hyperpolarization (EDH; Malmsjo et al. 1999, 2002, Wihlborg et al. 2003) and/or prostacyclin (Hammer et al. 2003, Wihlborg et al. 2003). P2Y2 receptors are found on the endothelium and it was proposed that their activation stimulates the synthesis and release of NO (Ralevic & Burnstock 1991b, Buvinic et al. 2002, Burnstock 2009). Moreover, aortic rings from P2Y2 receptor knockout mice (P2Y2−/−) exhibit impaired vasorelaxation in response to ATP, which suggests that NO release is subsequent to P2Y2 receptor activation (Guns et al. 2005, 2006). Of note, mechanical destruction of the endothelium abolishes the ATP induced vasodilatory effect and produces a direct vasoconstrictory response on vascular smooth muscle cells (Kennedy & Burnstock 1985, Kennedy et al. 1985, Ralevic & Burnstock 1996b). Activation of P2Y4 receptors is associated with vasoconstriction (Dietrich et al. 1996, McMillan et al. 1999, Rubino et al. 1999). In contrast to ATP causing vasodilation, UTP was found to vasoconstrict mouse aortic rings (Boarder & Hourani 1998, Kauffenstein et al. 2010) and rabbit inner ear arteries (von Kugelgen et al. 1987). However, depending on route of administration, species, localization within the vascular tree or vessel type, UTP can cause vasoconstriction, vasodilation or both (Ralevic & Burnstock 1996a,b, Janigro et al. 1997, Horiuchi et al. 2001, Guns et al. 2005, Rayment et al. 2007, Inscho 2009). In recent studies, we demonstrated that P2Y2 receptors play a physiological role in blood pressure regulation and, as a consequence, P2Y2−/− mice were found to have salt-resistant hypertension (Rieg et al. 2007a, Pochynyuk et al. 2010). Direct intra-arterial blood pressure measurements indicated that the blood pressure responses to a P2Y2/4 agonist result in vasodilation via a NO-independent mechanism that possibly involves EDH release subsequently to P2Y2 receptor activation. This was concluded because endothelial NO synthase knockout mice (eNOS−/−) showed an identical blood pressure effect in response to P2Y2/4 receptor activation compared to WT mice. In contrast to WT mice, P2Y2−/− mice responded to P2Y2/4 receptor activation with an increase in blood pressure, possibly a direct effect of P2Y4 receptor activation on vascular smooth muscle cells, which is independent of P2Y2 receptors (Rieg et al. 2011). The vasodilation mediated by EDH requires activation of calcium-activated potassium channels, including KCa2.3 (small-conductance) and KCa3.1 (intermediate-conductance), which are expressed in most endothelial cells (Kohler & Ruth 2010). In contrast, KCa1.1 (big-conductance) is expressed in vascular smooth muscle cells (Feletou 2009, Kohler & Ruth 2010). The activation of calcium-activated potassium channels is speculated to produce hyperpolarization of the endothelium, which is then transmitted (possibly via connexins, see below) to underlying vascular smooth muscle cells causing vasodilation via EDH. Functional studies employing blockers of calcium-activated potassium channels have demonstrated the role of these channels in EDH and subsequent vascular smooth muscle relaxation (Adeagbo & Triggle 1993, Holzmann et al. 1994, Waldron & Garland 1994, Zygmunt & Hogestatt 1996, Eichler et al. 2003). Vascular smooth muscle cells and endothelial cells are functionally linked, and the point of contact between the two cells, the myoendothelial gap junction (MEGJ), plays a key role in the regulation of vascular function (Figueroa & Duling 2009). Initially, it was assumed that a diffusible endothelial factor was the mechanism resulting in hyperpolarization; however, this view was later questioned by experiments demonstrating the involvement of the MEGJ (Griffith et al. 2002, Dora et al. 2003, Chaytor et al. 2005, Mather et al. 2005, Sokoya et al. 2006). It was concluded from these studies that EDH is transferred from the endothelium to the smooth muscle by direct charge transfer through the MEGJ (de Wit & Wolfle 2007, Feletou & Vanhoutte 2009, Grgic et al. 2009, Edwards et al. 2010, de Wit & Griffith 2010, Garland et al. 2011). Gap junction proteins found in the vasculature include as follows: Cx37, Cx40, Cx43 and Cx45 (Figueroa et al. 2004, Lohman et al. 2012); however, the MEGJ is specifically comprised of Cx37 and Cx40 which are speculated to conduct the EDH response as an electrical signal between endothelial and vascular smooth muscle cells (Chaytor et al. 2005, Isakson & Duling 2005, Haddock et al. 2006). To further define the underlying mechanism(s) involved in the P2Y2 receptor-initiated blood pressure responses, possibly via EDH, we studied blood pressure and heart rate in WT, Cx37, Cx40, KCa1.1 and KCa3.1 knockout (−/−) mice in response to systemic application of a P2Y2/4 receptor agonist. We report that P2Y2/4 receptor activation generates a similar biphasic blood pressure response in all mice except for KCa3.1−/− and Cx37−/− mice, which show impaired vascular reactivity. This implies that both KCa3.1 and Cx37 are required for full vascular reactivity in response to P2Y2/4 receptor activation.

Background: Wound infection plays an important role in compromised wound healing. A high bioburden impairs healing and leads to formation of a chronic wound. Distinctly higher pH values were observed in chronic wounds compared to acute wounds. However, there is only limited knowledge of pH dependency on the antibacterial efficacy of common antimicrobial substances. Methods: This study investigated the pH influence on the antimicrobial efficacy of povidone (PVP)-iodine, silver nitrate, chlorhexidine, octenidine and polihexanide against Staphylococcus aureus and Pseudomonas aeruginosa using the agar diffusion test and microplate laser nephelometry. Results: The bactericidal activity of chlorhexidine and octenidine was mainly pH-independent in a pH range of 5.0-9.0. In contrast, polihexanide showed a significant efficacy increase at a higher pH. It was also found that the influence of the pH on antiseptics differs among species of bacteria. For instance, S. aureus exhibited an increasing sensitivity against silver nitrate with rising pH whereas the effect on P. aeruginosa was found to be distinctly decreased. The antimicrobial effect of PVP-iodine was strongly diminished with rising pH. Conclusions: The shift towards higher pH values in chronic wounds compared to acute wounds makes it imperative to know whether the antimicrobial efficacy of applied antimicrobial substances is altered by different pH levels. The results suggest that application of polihexanide might be advantageous for the management of wound infections, as both S. aureus and P. aeruginosa exhibited an increased susceptibility with rising pH.

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.

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.

Intermediate conductance calcium-activated potassium channels (K

Background Negative pressure wound therapy (NPWT) has advanced the management of the open abdomen and other wounds. However, using the correct wound filler is essential as the material needs to convey a heterogeneous pressure distribution and create the pressure gradients required to drain interstitial fluid and remove wound exudate. This study compared the differences in fluid distribution during NPWT using a large-pore PU-foam dressing (Suprasorb® CNP foam), drainage film (Suprasorb® CNP drainage film) and a specialized NPT dressing system (KCI ABThera™ NPT system) in vitro. Methods A tissue model was prepared from gelatine, powdered milk, and tissue marking dye. Each filler/dressing was placed on a tissue model which was connected to a fluid reservoir, linked to a TRAC™ Pad and sealed. The TRAC™ Pad was connected to a vacuum pump set to −120 mmHg for 8 h. An infra-red camera was used to record fluid transport in the dressings. Results All fillers/dressings exhibited a fast fluid distribution and transport during the first hour (gradients 0.97–1.31) and steady state was reached after approximately 160 min. The NPT dressing system demonstrated a distribution limited to 70%, while the PU-foam and the drainage film presented a uniform and complete dispersal of fluid. Conclusion The results demonstrate that using an appropriate filler/dressing can facilitate effective wound fluid transport and drainage of interstitial fluid by producing heterogeneous pressures at the wound bed.

The iberiotoxin-sensitive large conductance voltage- and Ca2+-activated potassium (BK) channels (maxi-K+-channels) hyperpolarize the cell membrane thus supporting Ca2+entry through Ca2+-release activated Ca2+channels. Janus kinase-2 (JAK2) has been identified as novel regulator of ion transport. To explore whether JAK2 participates in the regulation of BK channels, cRNA encoding Ca2+-insensitive BK channels (BKM513I+Δ899–903) was injected into Xenopus oocytes with or without cRNA encoding wild-type JAK2, gain-of-functionV617FJAK2, or inactiveK882EJAK2. K+conductance was determined by dual electrode voltage clamp and BK-channel protein abundance by confocal microscopy. In A204 alveolar rhabdomyosarcoma cells, iberiotoxin-sensitive K+current was determined utilizing whole cell patch clamp. A204 cells were further transfected with JAK2 and BK-channel transcript, and protein abundance was quantified by RT-PCR and Western blotting, respectively. As a result, the K+current in BKM513I+Δ899–903-expressing oocytes was significantly increased following coexpression of JAK2 orV617FJAK2 but notK882EJAK2. Coexpression of the BK channel withV617FJAK2 but notK882EJAK2 enhanced BK-channel protein abundance in the oocyte cell membrane. Exposure of BK-channel andV617FJAK2-expressing oocytes to the JAK2 inhibitor AG490 (40 μM) significantly decreased K+current. Inhibition of channel insertion by brefeldin A (5 μM) decreased the K+current to a similar extent in oocytes expressing the BK channel alone and in oocytes expressing the BK channel andV617FJAK2. The iberiotoxin (50 nM)-sensitive K+current in rhabdomyosarcoma cells was significantly decreased by AG490 pretreatment (40 μM, 12 h). Moreover, overexpression of JAK2 in A204 cells significantly enhanced BK channel mRNA and protein abundance. In conclusion, JAK2 upregulates BK channels by increasing channel protein abundance in the cell membrane.

Negative-pressure wound therapy (NPWT) is an advantageous treatment option in wound management to promote healing and reduce the risk of complications. NPWT is mainly carried out using open-cell polyurethane (PU) foams that stimulate granulation tissue formation. However, growth of wound bed tissue into foam material, leading to disruption of newly formed tissue upon dressing removal, has been observed. Consequently, it would be of clinical interest to preserve the positive effects of open-cell PU foams while avoiding cellular ingrowth. The study presented analyzed effects of NPWT using large-pored PU foam, fine-pored PU foam, and the combination of large-pored foam with drainage film on human dermal fibroblasts grown in a collagen matrix. The results showed no difference between the dressings in stimulating cellular migration during NPWT. However, when NPWT was applied using a large-pored PU foam, the fibroblasts continued to migrate into the dressing. This led to significant breaches in the cell layers upon removal of the samples after vacuum treatment. In contrast, cell migration stopped at the collagen matrix edge when fine-pored PU foam was used, as well as with the combination of PU foam and drainage film. In conclusion, placing a drainage film between collagen matrix and the large-pored PU foam dressing reduced the ingrowth of cells into the foam significantly. Moreover, positive effects on cellular migration were not affected, and the effect of the foam on tissue surface roughness in vitro was also reduced.

Background: The role of post-translational modification of regulatory β-subunits in the control of large conductance potassium (BK) channels is largely unknown. Results: β4-subunit palmitoylation controls surface trafficking of BK channel α-subunit splice variants. Conclusion: Palmitoylation of β4 masks an α-subunit trafficking motif to control surface delivery. Significance: Palmitoylation of regulatory subunits provides a dynamic mechanism to control surface trafficking of specific BK channel variants., Regulatory β-subunits of large conductance calcium- and voltage-activated potassium (BK) channels play an important role in generating functional diversity and control of cell surface expression of the pore forming α-subunits. However, in contrast to α-subunits, the role of reversible post-translational modification of intracellular residues on β-subunit function is largely unknown. Here we demonstrate that the human β4-subunit is S-acylated (palmitoylated) on a juxtamembrane cysteine residue (Cys-193) in the intracellular C terminus of the regulatory β-subunit. β4-Subunit palmitoylation is important for cell surface expression and endoplasmic reticulum (ER) exit of the β4-subunit alone. Importantly, palmitoylated β4-subunits promote the ER exit and surface expression of the pore-forming α-subunit, whereas β4-subunits that cannot be palmitoylated do not increase ER exit or surface expression of α-subunits. Strikingly, however, this palmitoylation- and β4-dependent enhancement of α-subunit surface expression was only observed in α-subunits that contain a putative trafficking motif (… REVEDEC) at the very C terminus of the α-subunit. Engineering this trafficking motif to other C-terminal α-subunit splice variants results in α-subunits with reduced surface expression that can be rescued by palmitoylated, but not depalmitoylated, β4-subunits. Our data reveal a novel mechanism by which palmitoylated β4-subunit controls surface expression of BK channels through masking of a trafficking motif in the C terminus of the α-subunit. As palmitoylation is dynamic, this mechanism would allow precise control of specific splice variants to the cell surface. Our data provide new insights into how complex interplay between the repertoire of post-transcriptional and post-translational mechanisms controls cell surface expression of BK channels.

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. Significant 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.

K

The energy-sensing AMP-activated serine/threonine protein kinase (AMPK) confers cell survival in part by stimulation of cellular energy production and limitation of cellular energy utilization. AMPK-sensitive functions further include activities of epithelial Na+ channel ENaC and voltage-gated K+ channel KCNE1/KCNQ1. AMPK is activated by an increased cytosolic Ca2+ concentration. The present study explored whether AMPK regulates the Ca2+-sensitive large conductance and voltage-gated potassium (BK) channel. cRNA encoding BK channel was injected into Xenopus oocytes with and without additional injection of wild-type AMPK (AMPKα1+AMPKβ1+AMPKγ1), constitutively active AMPKγR70Q, or inactive AMPKαK45R. BK-channel activity was determined utilizing the 2-electrode voltage-clamp. Moreover, BK-channel protein abundance in the cell membrane was determined by confocal immunomicroscopy. As BK channels are expressed in outer hair cells (OHC) of the inner ear and lack of BK channels increases noise vulnerability, OHC BK-channel expression was examined by immunohistochemistry and hearing function analyzed by auditory brain stem response measurements in AMPKα1-deficient mice (ampk-/-) and in wild-type mice (ampk+/+). As a result, coexpression of AMPK or AMPKγR70Q but not of AMPKαK45R significantly enhanced BK-channel-mediated currents and BK-channel protein abundance in the oocyte cell membrane. BK-channel expression in the inner ear was lower in ampk-/- mice than in ampk+/+ mice. The hearing thresholds prior to and immediately after an acoustic overexposure were similar in ampk-/- and ampk+/+ mice. However, the recovery from the acoustic trauma was significantly impaired in ampk-/- mice compared to ampk+/+ mice. In summary, AMPK is a potent regulator of BK channels. It may thus participate in the signaling cascades that protect the inner ear from damage following acoustic overstimulation.

Large conductance, voltage- and Ca2+-activated K+ (BK) channels in inner hair cells (IHCs) of the cochlea are essential for hearing. However, germline deletion of BKα, the pore-forming subunit KCNMA1 of the BK channel, surprisingly did not affect hearing thresholds in the first postnatal weeks, even though altered IHC membrane time constants, decreased IHC receptor potential alternating current/direct current ratio, and impaired spike timing of auditory fibers were reported in these mice. To investigate the role of IHC BK channels for central auditory processing, we generated a conditional mouse model with hair cell-specific deletion of BKα from postnatal day 10 onward. This had an unexpected effect on temporal coding in the central auditory system: neuronal single and multiunit responses in the inferior colliculus showed higher excitability and greater precision of temporal coding that may be linked to the improved discrimination of temporally modulated sounds observed in behavioral training. The higher precision of temporal coding, however, was restricted to slower modulations of sound and reduced stimulus-driven activity. This suggests a diminished dynamic range of stimulus coding that is expected to impair signal detection in noise. Thus, BK channels in IHCs are crucial for central coding of the temporal fine structure of sound and for detection of signals in a noisy environment.—Kurt, S., Sausbier, M., Rüttiger, L., Brandt, N., Moeller, C. K., Kindler, J., Sausbier, U., Zimmermann, U., van Straaten, H., Neuhuber, W., Engel, J., Knipper, M., Ruth, P., Schulze, H. Critical role for cochlear hair cell BK channels for coding the temporal structure and dynamic range of auditory information for central auditory processing.

The modulatory role of large-conductance Ca2+-activated K+(BK) channels in the nervous system has been extensively studied. In the retina, it has been shown that BK channels play a pivotal role in modulating feedback from A17 amacrine cells to rod bipolar cells (RBCs). Here, we used electroretinography to examine the functional role of BK channels for rod and cone vision in the retinain vivousing a genetically engineered mouse lacking functional BK channels (Bk−/−). Under dark-adapted and light-adapted conditions, the lack of BK channels had no effect on photoreceptor activity, suggesting that these ion channels do not modulate photoreceptor responses. At the bipolar cell level, the ERG signals attributed to RBCs inBk−/−mice were not different from those in wild-type mice at low scotopic stimulus intensities. However, at high scotopic and low mesopic stimulus intensities, close to RBC saturation, a significant reduction of ERG signals reflecting RBC activity was present in theBk−/−retina. At higher mesopic stimulus intensities activating both RBCs and cone bipolar cells (CBCs), no difference in ERG signals betweenBk−/−and wild-type mice was found. In photopic stimulus paradigms, activity of ON- and OFF-CBCs inBk−/−and wild-type retinae was indistinguishable. These findings demonstrate that BK channels modulate visual responsesin vivoat the bipolar cell level at intermediate stimulus conditions.

Background: Enzymes controlling (de)palmitoylation of ion channels are poorly defined. Results: Palmitoylation of BK channels by zDHHC22 and zDHHC23 and depalmitoylation by LYPLA1 and LYPLAL1 controls BK channel cell surface expression. Conclusion: Acyl protein transferases and thioesterases display substrate specificity and control BK channel surface expression. Significance: Understanding how channels are (de)palmitoylated is essential for defining the role of palmitoylation in ion channel physiology., Protein palmitoylation is rapidly emerging as an important determinant in the regulation of ion channels, including large conductance calcium-activated potassium (BK) channels. However, the enzymes that control channel palmitoylation are largely unknown. Indeed, although palmitoylation is the only reversible lipid modification of proteins, acyl thioesterases that control ion channel depalmitoylation have not been identified. Here, we demonstrate that palmitoylation of the intracellular S0–S1 loop of BK channels is controlled by two of the 23 mammalian palmitoyl-transferases, zDHHC22 and zDHHC23. Palmitoylation by these acyl transferases is essential for efficient cell surface expression of BK channels. In contrast, depalmitoylation is controlled by the cytosolic thioesterase APT1 (LYPLA1), but not APT2 (LYPLA2). In addition, we identify a splice variant of LYPLAL1, a homolog with ∼30% identity to APT1, that also controls BK channel depalmitoylation. Thus, both palmitoyl acyltransferases and acyl thioesterases display discrete substrate specificity for BK channels. Because depalmitoylated BK channels are retarded in the trans-Golgi network, reversible protein palmitoylation provides a critical checkpoint to regulate exit from the trans-Golgi network and thus control BK channel cell surface expression.

Background: Otoferlin, a postulated calcium sensor of 230 kDa, was proposed to trigger calcium dependent fusion of vesicles with plasma membrane in the ribbon synapses of cochlear IHCs. Otoferlin’s interaction with Rab8b and Myo6, proteins involved in the intracellular membrane trafficking, extended the previous hypothesis assigning Otoferlin an additional role in trans-Golgi trafficking. Here, we present another Otoferlin binding partner, Ergic2, a protein with a still unknown function but presenting sequence homology to other proteins involved in ER/Golgi vesicle trafficking. Methods: Novel binding partners of Otoferlin were searched by yeast two-hybrid screening in a rodent cochlear cDNA library (P3-P15). RT-PCR, western blot, immunohistochemistry staining and co-immunoprecipitation were applied to analyze and confirm an interaction between Ergic2 and Otoferlin. Results: The Y-2-H screening, using baits covering parts of Otoferlin’s C2D domain, identified Ergic2 as an interacting protein for Otoferlin. Both are co-expressed (mRNA and protein level) in rodent cochlea and brain before- and after-onset of hearing. By RT-PCR Ergic2 was detected in cochlear IHCs and OHCs and in brain regions where Otoferlin is known to be present. Co-localization studies revealed an overlap of Ergic2 and Otoferlin signals in IHCs and neurons of cerebral cortical layer I making Ergic2 the promising binding candidate. However, while Ergic2 was co-precipitated by an anti-Otoferlin antibody in protein lysates from murine brain, this specific protein interaction was not detected in cochlea. Conclusion: Our new data on Otoferlin’s interactome suggest that Otoferlin can form different, tissue-specific protein complexes.

Background: Bacterial colonization and infection are important factors in compromised wound healing. Antiseptics have become an alternative for antimicrobial applications as antibiotic resistance is increasing; they have multiple targets with a broad spectrum of activity. Hence, the risk for developing resistance should be low. However, concerns have been raised that their growing use may result in bacteria that are less susceptible. Methods: The capacity of common antiseptics such as silver nitrate, polihexanide, octenidine, chlorhexidine and polyvinylpyrrolidone (PVP)-iodine to induce adaptation in a Staphylococcus aureus strain was analyzed in vitro using microplate laser nephelometry. S. aureus was repeatedly incubated with the respective half maximal inhibitory concentration (IC50) over a time period of 100 days. The influence of the continued treatment was determined by in situ monitoring of changes in the dose-response curves and calculation of the current IC50 values for the substances tested. Results: During the experiment, S. aureus quickly adapted to high concentrations of the antibiotic mupirocin during repeated treatment. Moreover, a significant increase of the IC50 for silver nitrate was observed over time. On the other hand, no significant difference was observed for polihexanide or chlorhexidine. While the IC50 for octenidine was also found to increase significantly, although the change was only marginal, reiterated incubation with PVP-iodine led to a decrease in the IC50. Conclusion: Repeated treatment of S. aureus with polihexanide, chlorhexidine, octenidine and PVP-iodine did not trigger bacterial adaptation to these substances.