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Background Slick, a sodium-activated potassium channel, has been recently identified in somatosensory pathways, but its functional role is poorly understood. The authors of this study hypothesized that Slick is involved in processing sensations of pain and itch. Methods Immunostaining, in situ hybridization, Western blot, and real-time quantitative reverse transcription polymerase chain reaction were used to investigate the expression of Slick in dorsal root ganglia and the spinal cord. Mice lacking Slick globally (Slick–/–) or conditionally in neurons of the spinal dorsal horn (Lbx1-Slick–/–) were assessed in behavioral models. Results The authors found Slick to be enriched in nociceptive Aδ-fibers and in populations of interneurons in the spinal dorsal horn. Slick–/– mice, but not Lbx1-Slick–/– mice, showed enhanced responses to noxious heat in the hot plate and tail-immersion tests. Both Slick–/– and Lbx1-Slick–/– mice demonstrated prolonged paw licking after capsaicin injection (mean ± SD, 45.6 ± 30.1 s [95% CI, 19.8 to 71.4]; and 13.1 ± 16.1 s [95% CI, 1.8 to 28.0]; P = 0.006 [Slick–/– {n = 8} and wild-type {n = 7}, respectively]), which was paralleled by increased phosphorylation of the neuronal activity marker extracellular signal–regulated kinase in the spinal cord. In the spinal dorsal horn, Slick is colocalized with somatostatin receptor 2 (SSTR2), and intrathecal preadministration of the SSTR2 antagonist CYN-154806 prevented increased capsaicin-induced licking in Slick–/– and Lbx1-Slick–/– mice. Moreover, scratching after intrathecal delivery of the somatostatin analog octreotide was considerably reduced in Slick–/– and Lbx1-Slick–/– mice (Slick–/– [n = 8]: 6.1 ± 6.7 bouts [95% CI, 0.6 to 11.7]; wild-type [n =8]: 47.4 ± 51.1 bouts [95% CI, 4.8 to 90.2]; P = 0.039). Conclusions Slick expressed in a subset of sensory neurons modulates heat-induced pain, while Slick expressed in spinal cord interneurons inhibits capsaicin-induced pain but facilitates somatostatin-induced itch. Editor’s Perspective What We Already Know about This Topic What This Article Tells Us That Is New

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The transient receptor potential (TRP) ankyrin type 1 (TRPA1) channel is highly expressed in a subset of sensory neurons where it acts as an essential detector of painful stimuli. However, the mechanisms that control the activity of sensory neurons upon TRPA1 activation remain poorly understood. Here, using in situ hybridization and immunostaining, we found TRPA1 to be extensively co-localized with the potassium channel Slack (KNa1.1, Slo2.2, or Kcnt1) in sensory neurons. Mice lacking Slack globally (Slack−/−) or conditionally in sensory neurons (SNS-Slack−/−) demonstrated increased pain behavior after intraplantar injection of the TRPA1 activator allyl isothiocyanate. By contrast, pain behavior induced by the TRP vanilloid 1 (TRPV1) activator capsaicin was normal in Slack-deficient mice. Patch-clamp recordings in sensory neurons and in a HEK cell line transfected with TRPA1 and Slack revealed that Slack-dependent potassium currents (IKS) are modulated in a TRPA1-dependent manner. Taken together, our findings highlight Slack as a modulator of TRPA1-mediated, but not TRPV1-mediated, activation of sensory neurons.

Vascular smooth muscle cells (VSMCs) can switch from their contractile state to a synthetic phenotype resulting in high migratory and proliferative capacity and driving atherosclerotic lesion formation. The cysteine-rich LIM-only protein 4 (CRP4) reportedly modulates VSM-like transcriptional signatures, which are perturbed in VSMCs undergoing phenotypic switching. Thus, we hypothesized that CRP4 contributes to adverse VSMC behaviours and thereby to atherogenesis in vivo. The atherogenic properties of CRP4 were investigated in plaque-prone apolipoprotein E (ApoE) and CRP4 double-knockout (dKO) as well as ApoE-deficient CRP4 wildtype mice. dKO mice exhibited lower plaque numbers and lesion areas as well as a reduced content of α-smooth muscle actin positive cells in the lesion area, while lesion-associated cell proliferation was elevated in vessels lacking CRP4. Reduced plaque volumes in dKO correlated with significantly less intra-plaque oxidized low-density lipoprotein (oxLDL), presumably due to upregulation of the antioxidant factor peroxiredoxin-4 (PRDX4). This study identifies CRP4 as a novel pro-atherogenic factor that facilitates plaque oxLDL deposition and identifies the invasion of atherosclerotic lesions by VSMCs as important determinants of plaque vulnerability. Thus, targeting of VSMC CRP4 should be considered in plaque-stabilizing pharmacological strategies.

Background and purpose Pore-forming α subunits of the voltage- and Ca2+ -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.

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.

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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.

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.

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.

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.

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.

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.

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.

// 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.

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.

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.

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: 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.

Platelets have been shown to migrate and thus to invade the vascular wall. Platelet migration is stimulated by SDF-1. In other cell types, migration is dependent on Ca(2+) entry via Ca(2+) channels. Ca(2+) influx is sensitive to cell membrane potential which is maintained by K(+) channel activity and/or Cl(-) channel activity. The present study explored the role of ion channels in the regulation of SDF-1 induced migration. Platelets were isolated from human volunteers as well as from gene targeted mice lacking the Ca(2+) activated K(+) channel SK4 (sk4(-/-)) and their wild type littermates (sk4(+/+)). According to confocal microscopy human platelets expressed the Ca(2+) channel Orai1 and the Ca(2+)-activated K(+) channel K(Ca)3.1 (SK4). SDF-1 (100 ng/ml) stimulated migration in human platelets, an effect blunted by Orai1 inhibitors 2-aminoethoxydiphenyl borate 2-APB (10 μM) and SKF-96365 (10 μM), by unspecific K(+) channel inhibitor TEA (30 mM), by SK4 specific K(+) channel blocker clotrimazole (10 μM), but not by Cl(-) channel inhibitor 5-nitro-2-(3-phenylpropylamino) benzoic acid NPPB (100 μM). Significant stimulation of migration by SDF-1 was further observed in sk4(+/+) platelets but was virtually absent in sk4(-/-) platelets. In conclusion, platelet migration requires activity of the Ca(2+) channel Orai1 and of the Ca(2+) activated K(+) channel SK4, but not of NPPB-sensitive Cl(-) channels.

Evidence is accumulating that Ca2+-regulated K+ (K-Ca) channels are important for beta cell function. We used BK channel knockout (BK-KO) mice to examine the role of these K-Ca channels for glucose homeostasis, beta cell function and viability.Glucose and insulin tolerance were tested with male wild-type and BK-KO mice. BK channels were detected by single-cell RT-PCR, cytosolic Ca2+ concentration ([Ca2+](c)) by fura-2 fluorescence, and insulin secretion by radioimmunoassay. Electrophysiology was performed with the patch-clamp technique. Apoptosis was detected via caspase 3 or TUNEL assay.BK channels were expressed in murine pancreatic beta cells. BK-KO mice were normoglycaemic but displayed markedly impaired glucose tolerance. Genetic or pharmacological deletion of the BK channel reduced glucose-induced insulin secretion from isolated islets. BK-KO and BK channel inhibition (with iberiotoxin, 100 nmol/l) broadened action potentials and abolished the after-hyperpolarisation in glucose-stimulated beta cells. However, BK-KO did not affect action potential frequency, the plateau potential at which action potentials start or glucose-induced elevation of [Ca2+](c). BK-KO had no direct influence on exocytosis. Importantly, in BK-KO islet cells the fraction of apoptotic cells and the rate of cell death induced by oxidative stress (H2O2, 10-100 mu mol/l) were significantly increased compared with wild-type controls. Similar effects were obtained with iberiotoxin. Determination of H2O2-induced K+ currents revealed that BK channels contribute to the hyperpolarising K+ current activated under conditions of oxidative stress.Ablation or inhibition of BK channels impairs glucose homeostasis and insulin secretion by interfering with beta cell stimulus-secretion coupling. In addition, BK channels are part of a defence mechanism against apoptosis and oxidative stress.

S-Palmitoylation is rapidly emerging as an important post-translational mechanism to regulate ion channels. We have previously demonstrated that large conductance calcium-and voltage-activated potassium (BK) channels are palmitoylated within an alternatively spliced (STREX) insert. However, these studies also revealed that additional site(s) for palmitoylation must exist outside of the STREX insert, although the identity or the functional significance of these palmitoylated cysteine residues are unknown. Here, we demonstrate that BK channels are palmitoylated at a cluster of evolutionary conserved cysteine residues (Cys-53, Cys-54, and Cys-56) within the intracellular linker between the S0 and S1 transmembrane domains. Mutation of Cys-53, Cys-54, and Cys-56 completely abolished palmitoylation of BK channels lacking the STREX insert (ZERO variant). Palmitoylation allows the S0-S1 linker to associate with the plasma membrane but has no effect on single channel conductance or the calcium/voltage sensitivity. Rather, S0-S1 linker palmitoylation is a critical determinant of cell surface expression of BK channels, as steady state surface expression levels are reduced by similar to 55% in the C53:54:56 A mutant. STREX variant channels that could not be palmitoylated in the S0-S1 linker also displayed significantly reduced cell surface expression even though STREX insert palmitoylation was unaffected. Thus our work reveals the functional independence of two distinct palmitoylation-dependent membrane interaction domains within the same channel protein and demonstrates the critical role of S0-S1 linker palmitoylation in the control of BK channel cell surface expression.

The large-conductance voltage- and calcium-activated potassium (BK) channels are ubiquitously expressed in the brain and play an important role in the regulation of neuronal excitation. Previous work has shown that the total deletion of these channels causes an impaired motor behavior, consistent with a cerebellar dysfunction. Cellular analyses showed that a decrease in spike firing rate occurred in at least two types of cerebellar neurons, namely in Purkinje neurons (PNs) and in Golgi cells. To determine the relative role of PNs, we developed a cell-selective mouse mutant, which lacked functional BK channels exclusively in PNs. The behavioral analysis of these mice revealed clear symptoms of ataxia, indicating that the BK channels of PNs are of major importance for normal motor coordination. By using combined two-photon imaging and patch-clamp recordings in these mutant mice, we observed a unique type of synaptic dysfunction in vivo, namely a severe silencing of the climbing fiber–evoked complex spike activity. By performing targeted pharmacological manipulations combined with simultaneous patch-clamp recordings in PNs, we obtained direct evidence that this silencing of climbing fiber activity is due to a malfunction of the tripartite olivo-cerebellar feedback loop, consisting of the inhibitory synaptic connection of PNs to the deep cerebellar nuclei (DCN), followed by a projection of inhibitory DCN afferents to the inferior olive, the origin of climbing fibers. Taken together, our results establish an essential role of BK channels of PNs for both cerebellar motor coordination and feedback regulation in the olivo-cerebellar loop.

Large conductance voltage- and Ca 2+ -activated potassium channels (BK channels) are important feedback regulators in excitable cells and are potently regulated by protein kinases. The present study reveals a dual role of protein kinase C (PKC) on BK channel regulation. Phosphorylation of S 695 by PKC, located between the two regulators of K + conductance (RCK1/2) domains, inhibits BK channel open-state probability. This PKC-dependent inhibition depends on a preceding phosphorylation of S 1151 in the C terminus of the channel α-subunit. Phosphorylation of only one α-subunit at S 1151 and S 695 within the tetrameric pore is sufficient to inhibit BK channel activity. We further detected that protein phosphatase 1 is associated with the channel, constantly counteracting phosphorylation of S 695 . PKC phosphorylation at S 1151 also influences stimulation of BK channel activity by protein kinase G (PKG) and protein kinase A (PKA). Though the S 1151 A mutant channel is activated by PKA only, the phosphorylation of S 1151 by PKC renders the channel responsive to activation by PKG but prevents activation by PKA. Phosphorylation of S 695 by PKC or introducing a phosphomimetic aspartate at this position (S 695 D) renders BK channels insensitive to the stimulatory effect of PKG or PKA. Therefore, our findings suggest a very dynamic regulation of the channel by the local PKC activity. It is shown that this complex regulation is not only effective in recombinant channels but also in native BK channels from tracheal smooth muscle.

Calcium-activated potassium channels have been shown to be critically involved in neuronal function, but an elucidation of their detailed roles awaits identification of the microdomains where they are located. This study was undertaken to unravel the precise subcellular distribution of the large-conductance calcium-activated potassium channels (called BK, KCa1.1, or Slo1) in the somatodendritic compartment of cerebellar Purkinje cells by means of postembedding immunogold cytochemistry and SDS-digested freeze-fracture replica labeling (SDS-FRL). We found BK channels to be unevenly distributed over the Purkinje cell plasma membrane. At distal dendritic compartments, BK channels were scattered over the plasma membrane of dendritic shafts and spines but absent from postsynaptic densities. At the soma and proximal dendrites, BK channels formed two distinct pools. One pool was scattered over the plasma membrane, whereas the other pool was clustered in plasma membrane domains overlying subsurface cisterns. The labeling density ratio of clustered to scattered channels was about 60:1, established in SDS-FRL. Subsurface cisterns, also called hypolemmal cisterns, are subcompartments of the endoplasmic reticulum likely representing calciosomes that unload and refill Ca2+ independently. Purkinje cell subsurface cisterns are enriched in inositol 1,4,5-triphosphate receptors that mediate the effects of several neurotransmitters, hormones, and growth factors by releasing Ca2+ into the cytosol, generating local Ca2+ sparks. Such increases in cytosolic [Ca2+] may be sufficient for BK channel activation. Clustered BK channels in the plasma membrane may thus participate in building a functional unit (plasmerosome) with the underlying calciosome that contributes significantly to local signaling in Purkinje cells. J. Comp. Neurol. 515:215–230, 2009. © 2009 Wiley-Liss, Inc.

OBJECTIVE Ca2+-regulated K+ channels are involved in numerous Ca2+-dependent signaling pathways. In this study, we investigated whether the Ca2+-activated K+ channel of intermediate conductance SK4 (KCa3.1, IK1) plays a physiological role in pancreatic β-cell function. RESEARCH DESIGN AND METHODS Glucose tolerance and insulin sensitivity were determined in wild-type (WT) or SK4 knockout (SK4-KO) mice. Electrophysiological experiments were performed with the patch-clamp technique. The cytosolic Ca2+ concentration ([Ca2+]c) was determined by fura-2 fluorescence. Insulin release was assessed by radioimmunoassay, and SK4 protein was detected by Western blot analysis. RESULTS SK4-KO mice showed improved glucose tolerance, whereas insulin sensitivity was not altered. The animals were not hypoglycemic. Isolated SK4-KO β-cells stimulated with 15 mmol/l glucose had an increased Ca2+ action potential frequency, and single-action potentials were broadened. These alterations were coupled to increased [Ca2+]c. In addition, glucose responsiveness of membrane potential, [Ca2+]c, and insulin secretion were shifted to lower glucose concentrations. SK4 protein was expressed in WT islets. An increase in K+ currents and concomitant membrane hyperpolarization could be evoked in WT β-cells by the SK4 channel opener DCEBIO (100 μmol/l). Accordingly, the SK4 channel blocker TRAM-34 (1 μmol/l) partly inhibited KCa currents and induced electrical activity at a threshold glucose concentration. In stimulated WT β-cells, TRAM-34 further increased [Ca2+]c and broadened action potentials similar to those seen in SK4-KO β-cells. SK4 channels were found to substantially contribute to Kslow (slowly activating K+ current). CONCLUSIONS SK4 channels are involved in β-cell stimulus-secretion coupling. Deficiency of SK4 current induces elevated β-cell responsiveness and coincides with improved glucose tolerance in vivo. Therefore, pharmacologic modulation of these channels might provide an interesting approach for the development of novel insulinotropic drugs.

Homozygous deletion of the gene of the neuronal glucose transporter GLUT3 ( Slc2a3) in mice results in embryonic lethality, whereas heterozygotes ( Slc2a3+/−) are viable. Here, we describe the characterization of heterozygous mice with regard to neuronal function, glucose homeostasis, and, since GLUT3 might be a component of the neuronal glucose-sensing mechanism, food intake and energy balance. Levels of GLUT3 mRNA and protein in brain were reduced by 50% in Slc2a3+/−mice. Electrographic features examined by electroencephalographic recordings give evidence for slightly but significantly enhanced cerebrocortical activity in Slc2a3+/−mice. In addition, Slc2a3+/−mice were slightly more sensitive to an acoustic startle stimulus (elevated startle amplitude and reduced prepulse inhibition). However, systemic behavioral testing revealed no other functional abnormalities, e.g., in coordination, reflexes, motor abilities, anxiety, learning, and memory. Furthermore, no differences in body weight, blood glucose, and insulin levels were detected between wild-type and Slc2a3+/−littermates. Food intake as monitored randomly or after intracerebroventricular administration of 2-deoxyglucose or d-glucose, or food choice for carbohydrates/fat was not affected in Slc2a3+/−mice. Taken together, our data indicate that, in contrast to Slc2a1, a single allele of Slc2a3 is sufficient for maintenance of neuronal energy supply, motor abilities, learning and memory, and feeding behavior.

Mast cell stimulation by Ag is followed by the opening of Ca(2+)-activated K(+) channels, which participate in the orchestration of mast cell degranulation. The present study has been performed to explore the involvement of the Ca(2+)-activated K(+) channel K(Ca)3.1 in mast cell function. To this end mast cells have been isolated and cultured from the bone marrow (bone marrow-derived mast cells (BMMCs)) of K(Ca)3.1 knockout mice (K(Ca)3.1(-/-)) and their wild-type littermates (K(Ca)3.1(+/+)). Mast cell number as well as in vitro BMMC growth and CD117, CD34, and FcepsilonRI expression were similar in both genotypes, but regulatory cell volume decrease was impaired in K(Ca)3.1(-/-) BMMCs. Treatment of the cells with Ag, endothelin-1, or the Ca(2+) ionophore ionomycin was followed by stimulation of Ca(2+)-activated K(+) channels and cell membrane hyperpolarization in K(Ca)3.1(+/+), but not in K(Ca)3.1(-/-) BMMCs. Upon Ag stimulation, Ca(2+) entry but not Ca(2+) release from intracellular stores was markedly impaired in K(Ca)3.1(-/-) BMMCs. Similarly, Ca(2+) entry upon endothelin-1 stimulation was significantly reduced in K(Ca)3.1(-/-) cells. Ag-induced release of beta-hexosaminidase, an indicator of mast cell degranulation, was significantly smaller in K(Ca)3.1(-/-) BMMCs compared with K(Ca)3.1(+/+) BMMCs. Moreover, histamine release upon stimulation of BMMCs with endothelin-1 was reduced in K(Ca)3.1(-/-) cells. The in vivo Ag-induced decline in body temperature revealed that IgE-dependent anaphylaxis was again significantly (by approximately 50%) blunted in K(Ca)3.1(-/-) mice. In conclusion, K(Ca)3.1 is required for Ca(2+)-activated K(+) channel activity and Ca(2+)-dependent processes such as endothelin-1- or Ag-induced degranulation of mast cells, and may thus play a critical role in anaphylactic reactions.

The cGMP/cGMP-dependent protein kinase I (cGKI) signaling pathway plays an important role in spinal nociceptive processing. However, downstream targets of cGKI in this context have not been identified to date. Using a yeast two-hybrid screen, we isolated cysteine-rich protein 2 (CRP2) as a novel cGKI interactor in the spinal cord. CRP2 is expressed in laminas I and II of the mouse spinal cord and is colocalized with cGKI, calcitonin gene-related peptide, and isolectin B4. Moreover, the majority of CRP2 mRNA-positive dorsal root ganglion (DRG) neurons express cGKI and peripherin. CRP2 is phosphorylated in a cGMP-dependent manner, and its expression increases in the spinal cord and in DRGs after noxious stimulation of a hindpaw. To elucidate the functional role of CRP2 in nociception, we analyzed mice with a targeted deletion of CRP2. CRP2-deficient (CRP2−/−) mice demonstrate normal behavioral responses to acute nociception and after axonal injury of the sciatic nerve, but increased nociceptive behavior in models of inflammatory hyperalgesia compared with wild-type mice. Intrathecal administration of cGMP analogs increases the nociceptive behavior in wild-type but not in CRP2−/−mice, indicating that the presence of CRP2 is important for cGMP-mediated nociception. These data suggest that CRP2 is a new downstream effector of cGKI-mediated spinal nociceptive processing and point to an inhibitory role of CRP2 in the generation of inflammatory pain.

Aim: Colonic crypts are the site of Cl - secretion. Basolateral K + channels provide the driving force for luminal cystic fibrosis transmembrane regulator-mediated Cl - exit. Relevant colonic epithelial K + channels are the intermediate conductance Ca 2+ -activated K ca 3.1 (SK4) channel and the cAMP-activated K v 7.1 (KCNQ1) channel. In addition, big conductance Ca 2+ -activated K ca 1.1 (BK) channels may play a role in Ca 2+ -activated Cl - secretion. Here we use K ca 1.1 and K ca 3.1 knock-out mice, and the K v 7.1 channel inhibitor 293B (10 μM) to investigate the role of K ca 1.1, K ca 3.1 and K v 7.1 channels in cholinergic-stimulated Cl - secretion. Methods: A Ussing chamber was used to quantify agonist-stimulated increases in short circuit current (I sc ) in distal colon. Chloride secretion was activated by bl. forskolin (FSK, 2 μM) followed by bl. carbachol (CCH, 100 μM). Luminal Ba 2+ (5 mM) was used to inhibit K ca 1.1 channels. Results: K ca 1.1 WT and KO mice displayed identical FSK and CCH-stimulated I sc changes, indicating that K ca 1.1 channels are not involved in FSK- and cholinergic-stimulated Cl - secretion. CCH-stimulated ∇I sc was significantly reduced in K ca 3.1 KO mice, underscoring the known relevance of this channel in the activation of Cl - secretion by an intracellular Ca 2+ increasing agonist. The residual CCH effect observed in K ca 3.1 KO mice suggests that yet another K + channel is driving the CCH-stimulated Cl - secretion. In the presence of the specific K v 7.1 channel blocker 293B, the residual CCH effect was abolished. Conclusions: This demonstrates that both K ca 3.1 and K v 7.1 channels are activated by cholinergic agonists and drive Cl - secretion. In contrast, K ca 1.1 channels are not involved in stimulated electrogenic Cl - secretion.

The protective effects of 5′-AMP–activated protein kinase (AMPK) on the metabolic syndrome may include direct effects on resistance artery vasomotor function. However, the precise actions of AMPK on microvessels and their potential interaction are largely unknown. Thus, we set to determine the effects of AMPK activation on vascular smooth muscle tone and the underlying mechanisms. Resistance arteries isolated from hamster and mouse exhibited a pronounced endothelium-independent dilation on direct pharmacological AMPK activation by 2 structurally unrelated compounds (PT1 and A769662). The dilation was associated with a decrease of intracellular-free calcium [Ca 2+ ] i in vascular smooth muscle cell. AMPK stimulation induced activation of BK Ca channels as assessed by patch clamp studies in freshly isolated hamster vascular smooth muscle cell and confirmed by direct proof of membrane hyperpolarization in intact arteries. The BK Ca channel blocker iberiotoxin abolished the hyperpolarization but only partially reduced the dilation and did not affect the decrease of [Ca 2+ ] i . By contrast, the sarcoplasmic/endoplasmic Ca 2+ -ATPase (SERCA) inhibitor thapsigargin largely reduced these effects, whereas combined inhibition of SERCA and BK Ca channels virtually abolished them. AMPK stimulation significantly increased the phosphorylation of the SERCA modulator phospholamban at the regulatory T17 site. Stimulation of smooth muscle AMPK represents a new, potent vasodilator mechanism in resistance vessels. AMPK directly relaxes vascular smooth muscle cell by a decrease of [Ca 2+ ] i . This is achieved by calcium sequestration via SERCA activation, as well as activation of BK Ca channels. There is in part a mutual compensation of both calcium-lowering mechanisms. However, SERCA activation which involves an AMPK-dependent phosphorylation of phospholamban is the predominant mechanism in resistance vessels.

Phosphodiesterase-6 (PDE6) is a multisubunit enzyme that plays a key role in the visual transduction cascade in rod and cone photoreceptors. Each type of photoreceptor utilizes discrete catalytic and inhibitory PDE6 subunits to fulfill its physiological tasks, i.e. the degradation of cyclic guanosine-3',5'-monophosphate at specifically tuned rates and kinetics. Recently, the human PDE6H gene was identified as a novel locus for autosomal recessive (incomplete) color blindness. However, the three different classes of cones were not affected to the same extent. Short wave cone function was more preserved than middle and long wave cone function indicating that some basic regulation of the PDE6 multisubunit enzyme was maintained albeit by a unknown mechanism. To study normal and disease-related functions of cone Pde6h in vivo, we generated Pde6h knock-out (Pde6h(-/-)) mice. Expression of PDE6H in murine eyes was restricted to both outer segments and synaptic terminals of short and long/middle cone photoreceptors, whereas Pde6h(-/-) retinae remained PDE6H-negative. Combined in vivo assessment of retinal morphology with histomorphological analyses revealed a normal overall integrity of the retinal organization and an unaltered distribution of the different cone photoreceptor subtypes upon Pde6h ablation. In contrast to human patients, our electroretinographic examinations of Pde6h(-/-) mice suggest no defects in cone/rod-driven retinal signaling and therefore preserved visual functions. To this end, we were able to demonstrate the presence of rod PDE6G in cones indicating functional substitution of PDE6. The disparities between human and murine phenotypes caused by mutant Pde6h/PDE6H suggest species-to-species differences in the vulnerability of biochemical and neurosensory pathways of the visual signal transduction system.

Slack (Slo2.2) is a sodium-activated potassium channel that regulates neuronal firing activities and patterns. Previous studies identified Slack in sensory neurons, but its contribution to acute and chronic painin vivoremains elusive. Here we generated global and sensory neuron-specific Slack mutant mice and analyzed their behavior in various animal models of pain. Global ablation of Slack led to increased hypersensitivity in models of neuropathic pain, whereas the behavior in models of inflammatory and acute nociceptive pain was normal. Neuropathic pain behaviors were also exaggerated after ablation of Slack selectively in sensory neurons. Notably, the Slack opener loxapine ameliorated persisting neuropathic pain behaviors. In conclusion, Slack selectively controls the sensory input in neuropathic pain states, suggesting that modulating its activity might represent a novel strategy for management of neuropathic pain.

The unique voltage- and Ca2+-dependent K+ (BK) channel, prominently expressed in airway smooth muscle cells, has been suggested as an important effector in controlling airway contractility. Its deletion in mice depolarized resting membrane potential of tracheal cells, suggesting an increased open-probability of voltage-gated Ca2+ channels. While carbachol concentration-dependently increased the tonic tension of wild-type (WT) trachea, mutant trachea showed a different response with rapid tension development followed by phasic contractions superimposed on a tonic component. Tonic contractions were substantially more dependent on L-type Ca2+ current in mutant than in WT trachea, even though L-type Ca2+ channels were not up-regulated. In the absence of L-type Ca2+ current, half-maximal contraction of trachea was shifted from 0.51 to 1.7 microM. In agreement, cholinergic bronchoconstriction was reduced in mutant lung slices, isolated-perfused lungs and, most impressively, in mutant mice analyzed by body plethysmography. Furthermore, isoprenaline-mediated airway relaxation was enhanced in mutants. In-depth analysis of cAMP and cGMP signaling revealed up-regulation of the cGMP pathway in mutant tracheal muscle. Inhibition of cGMP kinase reestablished normal sensitivity toward carbachol, indicating that up-regulation of cGMP signaling counterbalances for BK channel ablation, pointing to a predominant role of BK channel in regulation of airway tone.

Neurons are highly specialized cells in which the integration and processing of electrical signals critically depends on the precise localization of ion channels. For large-conductance Ca(2+)- activated K(+) (BK) channels, targeting to presynaptic membranes in hippocampal pyramidal cells was reported; however, functional evidence also suggests a somatodendritic localization. Therefore we re-examined the subcellular distribution of BK channels in mouse hippocampus using a panel of independent antibodies in a combined approach of conventional immunocytochemistry on cultured neurons, pre- and postembedding electron microscopy and immunoprecipitation. In cultured murine hippocampal neurons, the colocalization of BK channels with both pre- and postsynaptic marker proteins was observed. Electron microscopy confirmed targeting of BK channels to axonal as well as dendritic membranes of glutamatergic synapses in hippocampus. A postsynaptic localization of BK channels was also supported by the finding that the channel coimmunoprecipitated with PSD95, a protein solely expressed in the postsynaptic compartment. These results thus demonstrate that BK channels reside in both post- and presynaptic compartments of hippocampal pyramidal neurons.

K(+) secretion in the kidney and distal colon is a main determinant of K(+) homeostasis. This study investigated the identity of the relevant luminal secretory K(+) ion channel in distal colon. An Ussing chamber was used to measure ion transport in the recently generated BK channel-deficient (BK(-/-)) mice. BK(-/-) mice display a significant colonic epithelial phenotype with (1) lack of Ba(2+)-sensitive resting K(+) secretion, (2) absence of K(+) secretion stimulated by luminal P2Y(2) and P2Y(4) receptors, (3) absence of luminal Ca(2+) ionophore (A23187)-stimulated K(+) secretion, (4) reduced K(+) and increased Na(+) contents in feces, and (5) an increased colonic Na(+) absorption. In contrast, resting and uridine triphosphate (UTP)-stimulated K(+) secretion was not altered in mice that were deficient for the intermediate conductance Ca(2+)-activated K(+) channel SK4. BK channels localize to the luminal membrane of crypt, and reverse transcription-PCR results confirm the expression of the BK channel alpha-subunit in isolated distal colonic crypts. It is concluded that BK channels are the responsible K(+) channels for resting and stimulated Ca(2+)-activated K(+) secretion in mouse distal colon.

An antibody against the 442 carboxy-terminal amino acids of the BK channel alpha-subunit detects high immunoreactivity within the telencephalon in cerebral cortices, olfactory bulb, basal ganglia and hippocampus, while lower levels are found in basal forebrain regions and amygdala. Within the diencephalon, high density was found in nuclei of the ventral and dorsal thalamus and the medial habenular nucleus, and low density in the hypothalamus. The fasciculus retroflexus and its termination in the mesencephalic interpeduncular nucleus are prominently stained. Other mesencephalic expression sites are periaquaeductal gray and raphe nuclei. In the rhombencephalon, BK channels are enriched in the cerebellar cortex and in the locus coeruleus. Strong immunoreactivity is also contained in the vestibular nuclei, but not in cranial nerves and their intramedullary course of their roots. On the cellular level, BK channels show pre- and postsynaptic localizations, i.e., in somata, dendrites, axons and synaptic terminals.

The synaptic vesicle protein synapsin II plays an important role in the regulation of neurotransmitter release and synaptic plasticity. Here, we investigated its involvement in the synaptic transmission of nociceptive signals in the spinal cord and the development of pain hypersensitivity. We show that synapsin II is predominantly expressed in terminals and neuronal fibers in superficial laminae of the dorsal horn (laminae I-II). Formalin injection into a mouse hindpaw normally causes an immediate and strong release of glutamate in the dorsal horn. In synapsin II deficient mice this glutamate release is almost completely missing. This is associated with reduced nociceptive behavior in the formalin test and in the zymosan-induced paw inflammation model. In addition, the formalin evoked increase in the number of c-Fos IR neurons is significantly reduced in synapsin II knockout mice. Touch perception and motor coordination, however, are normal indicating that synapsin II deficiency does not generally disrupt sensory and/or motor functions. Antisense-mediated transient knockdown of synapsin II in the spinal cord of adult animals also reduced the nociceptive behavior. As the antisense effect is independent of a potential role of synapsin II during development we suggest that the hypoalgesia in synapsin II deficient mice does involve a direct 'pain-facilitating' effect of synapsin II and is not essentially dependent on potentially occurring developmental alterations. The distinctive role of synapsin II for pain signaling probably results from its specific localization and possibly from a specific control of glutamate release.

Large conductance voltage- and calcium-activated potassium (BK Ca ) channels are important signaling molecules that are regulated by multiple protein kinases and protein phosphatases at multiple sites. The pore-forming α-subunits, derived from a single gene that undergoes extensive alternative pre-mRNA splicing, assemble as tetramers. Although consensus phosphorylation sites have been identified within the C-terminal domain of α-subunits, it is not known whether phosphorylation of all or single α-subunits within the tetramer is required for functional regulation of the channel. Here, we have exploited a strategy to study single-ion channels in which both the α-subunit splice-variant composition is defined and the number of consensus phosphorylation sites available within each tetramer is known. We have used this approach to demonstrate that cAMP-dependent protein kinase (PKA) phosphorylation of the conserved C-terminal PKA consensus site (S899) in all four α-subunits is required for channel activation. In contrast, inhibition of BK Ca channel activity requires phosphorylation of only a single α-subunit at a splice insert (STREX)-specific PKA consensus site (S4 STREX ). Thus, distinct modes of BK Ca channel regulation by PKA phosphorylation exist: an “all-or-nothing” rule for activation and a “single-subunit” rule for inhibition. This essentially digital regulation has important implications for the combinatorial and conditional regulation of BK Ca channels by reversible protein phosphorylation.

Purpose: In addition to mutated BCR-ABL1 kinase, the organic cation transporter 1 (OCT1, encoded by SLC22A1) has been considered to contribute to imatinib resistance in patients with chronic myeloid leukemia (CML). As data are conflicting as to whether OCT1 transports imatinib and may serve as a clinical biomarker, we used a combination of different approaches including animal experiments to elucidate comprehensively the impact of OCT1 on cellular imatinib uptake. Experimental Design: Transport of imatinib was studied using OCT1-expressing Xenopus oocytes, mammalian cell lines (HEK293, MDCK, V79) stably expressing OCT1, human leukemic cells, and Oct1-knockout mice. OCT1 mRNA and protein expression were analyzed in leukemic cells from patients with imatinib-naïve CML as well as in cell lines. Results: Transport and inhibition studies showed that overexpression of functional OCT1 protein in Xenopus oocytes or mammalian cell lines did not lead to an increased cellular accumulation of imatinib. The CML cell lines (K562, Meg-01, LAMA84) and leukemic cells from patients expressed neither OCT1 mRNA nor protein as demonstrated by immunoblotting and immunofluorescence microscopy, yet they showed a considerable imatinib uptake. Oct1 deficiency in mice had no influence on plasma and hepatic imatinib concentrations. Conclusions: These data clearly demonstrate that cellular uptake of imatinib is independent of OCT1, and therefore OCT1 is apparently not a valid biomarker for imatinib resistance. Clin Cancer Res; 20(4); 985–94. ©2013 AACR.

Phagocytosis of shed photoreceptor outer segments by the retinal pigment epithelium (RPE) is critical for maintenance of visual function. Because changes in intracellular Ca(2+) regulate phagocytosis, we studied in vitro the impact of different ion channels in addition to mice deficient for Cav1.3 L-type Ca(2+) channels (Ca1.3(-/-)) and maxiK Ca(2+)-dependent K(+) channels (BK(-/-)). The knockdown of Bestrophin-1 protein, a regulator of intracellular Ca(2+) homeostasis, affected phagocytosis in porcine RPE cultures. Blockage of voltage-gated L-type channels by (+)BayK8644 inhibitor reduced phagocytosis in vitro, in contrast L-type activation by (-)BayK8644 had no impact. The expression rate of Cav1.3, the predominant L-type Ca(2+) channel in RPE cells, varied at different times of day. CaV1.3(-/-) RPE lacked peak phagocytic activity following morning photoreceptor shedding in wild-type RPE and retained a higher number of phagosomes at a later time of day. The BK-channel blocker paxilline lowered phagocytosis in RPE cultures in a concentration-dependent manner. BK(-/-) RPE in vivo retained phagocytic capability but this activity, which is normally well synchronized with circadian photoreceptor shedding, shifted out of phase. Retinae of older BK(-/-) mice showed shortened photoreceptor outer segments and diminished rhodopsin content. Store-operated Ca(2+) channels Orai-1 did not affect phagocytosis in cultured RPE. TRPV channel inhibition by ruthenium-red reduced phagocytosis, whereas activation at high concentrations of 2-APB increased phagocytosis. Our data demonstrate essential roles for bestrophin-1, BK, TRPV and L-type channels in regulating retinal phagocytosis. These data indicate further the importance of BK and CaV1.3 for rhythmic phagocytic activity synchronized with photoreceptor shedding.

Genetic variations in the large-conductance, voltage- and calcium activated potassium channels (BK channels) have been recently implicated in mental retardation, autism and schizophrenia which all come along with severe cognitive impairments. In the present study we investigate the effects of functional BK channel deletion on cognition using a genetic mouse model with a knock-out of the gene for the pore forming α-subunit of the channel. We tested the F1 generation of a hybrid SV129/C57BL6 mouse line in which the slo1 gene was deleted in both parent strains. We first evaluated hearing and motor function to establish the suitability of this model for cognitive testing. Auditory brain stem responses to click stimuli showed no threshold differences between knockout mice and their wild-type littermates. Despite of muscular tremor, reduced grip force, and impaired gait, knockout mice exhibited normal locomotion. These findings allowed for testing of sensorimotor gating using the acoustic startle reflex, as well as of working memory, spatial learning and memory in the Y-maze and the Morris water maze, respectively. Prepulse inhibition on the first day of testing was normal, but the knockout mice did not improve over the days of testing as their wild-type littermates did. Spontaneous alternation in the y-maze was normal as well, suggesting that the BK channel knock-out does not impair working memory. In the Morris water maze knock-out mice showed significantly slower acquisition of the task, but normal memory once the task was learned. Thus, we propose a crucial role of the BK channels in learning, but not in memory storage or recollection.

Identifying the specific functions that are mediated by cyclic GMP (cGMP)-dependent protein kinases is key to our understanding of the biological role of the nitric oxide/cGMP signaling cascade. Over the last two decades, there have been numerous reports on the functional roles of cGMP kinases. However, most studies have been performed with isolated cells and organs by using specific activators and inhibitors of cGMP kinases. Under such experimental conditions, a clear dissection between the cGMP kinase pathway and other pathways, for example, that of cyclic AMP kinase, has been difficult to achieve. In order to identify biological processes in which cGMP kinases are unambiguously involved, the genes of cGMP kinase I and cGMP kinase II have been deleted in mice. This review focuses on the physiological functions that are regulated by cGMP kinases as revealed by cGMP kinase-deficient animals. For cGMP kinase I, these functions include the contractility of vascular and gastrointestinal smooth muscle and the homeostasis of platelet activity. According to its expression pattern, the Type II cGMP kinase plays a definite biological part in transepithelial Cl − and Na + transport in the intestine, longitudinal growth of long bones, and the regulation of the kidney renin-angiotensin-aldosterone system. Further, cGMP kinase I and II mutants reveal new aspects for the role of nitric oxide/cGMP in the induction of neuronal plasticity, such as hippocampal long-term potentiation and cerebellar long-term depression. In conclusion, inactivation of the cGMP kinase genes shows that cGMP kinases regulate very specifically distinct cellular functions by pathways that are separate from those used by cyclic AMP kinases. Mice deficient in cGMP kinases exhibit severe defects and, therefore, may serve as animal models for several human diseases, including hypertension, thrombosis, gastrointestinal dysmotility, and dwarfism.

Long-term potentiation (LTP) is a potential cellular mechanism for learning and memory. The retrograde messenger nitric oxide (NO) is thought to induce LTP in the CA1 region of the hippocampus via activation of soluble guanylyl cyclase (sGC) and, ultimately, cGMP-dependent protein kinase (cGK). Two genes code for the isozymes cGKI and cGKII in vertebrates. The functional role of cGKs in LTP was analyzed using mice lacking the gene(s) for cGKI, cGKII, or both. LTP was not altered in the mutant mice lineages. However, LTP was reduced by inhibition of NO synthase and NMDA receptor antagonists, respectively. The reduced LTP was not recovered by the cGK-activator 8-(4 chlorophenylthio)-cGMP. Moreover, LTP was not affected by the sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]-quiloxalin-1-one. In contrast, it was effectively suppressed by nicotinamide, a blocker of the ADP-ribosyltransferase. These results show that cGKs are not involved in LTP in mice and that NO induces LTP through an alternative cGMP-independent pathway, possibly ADP-ribosylation.

Migration of dendritic cells (DCs) plays an important role in T-cell-mediated adaptive immune responses. Lipopolysaccharide (LPS) sensed by Toll-like receptor 4 (TLR4) serves as a signal for DC migration. We analyzed LPS-induced DC volume changes preceding the directed movement towards chemoattractants. Treatment with LPS resulted in rapid, prolonged cell swelling in wild-type (WT), but not in TLR4(-/-) bone marrow-derived (BM) DCs indicating that TLR4 signaling is essential for LPS-induced swelling. As a consequence, LPS-treatment enhanced the migratory activity along a chemokine (CCL21)-gradient in WT, but not in TLR4-deficient BMDCs suggesting that the LPS/TLR4-induced swelling response facilitates DC migration. Moreover, the role of calcium-activated potassium channels (K(Ca) 3.1) as putative regulators of immune cell volume regulation and migration was analyzed in LPS-challenged BMDCs. We found that the LPS-induced swelling of K(Ca) 3.1-deficient DCs was impaired when compared to WT DCs. Accordingly, the LPS-induced increase in [Ca(2+)](i) detected in WT DCs was reduced in K(Ca) 3.1-deficient DCs. Finally, directed migration of LPS-challenged K(Ca) 3.1-deficient DCs was low compared to WT DCs indicating that activation of K(Ca) 3.1 is involved in LPS-induced DC migration. These findings suggest that both TLR4 and K(Ca) 3.1 contribute to the migration of LPS-activated DCs as an important feature of the adaptive immune response.

Summary: The in vivo function of large conductance calcium‐activated potassium channels in sensory neurons includes control of inflammatory pain, but not of acute nociceptive or nerve injury‐induced neuropathic pain. ABSTRACT: Large conductance calcium‐activated potassium (BKCa) channels are important regulators of neuronal excitability. Although there is electrophysiological evidence for BKCa channel expression in sensory neurons, their in vivo functions in pain processing have not been fully defined. Using a specific antibody, we demonstrate here that BKCa channels are expressed in subpopulations of peptidergic and nonpeptidergic nociceptors. To test a functional association of BKCa channel activity in sensory neurons with particular pain modalities, we generated mice in which BKCa channels are ablated specifically from sensory neurons and analyzed their behavior in various models of pain. Mutant mice showed increased nociceptive behavior in models of persistent inflammatory pain. However, their behavior in models of neuropathic or acute nociceptive pain was normal. Moreover, systemic administration of the BKCa channel opener, NS1619, inhibited persistent inflammatory pain. Our investigations provide in vivo evidence that BKCa channels expressed in sensory neurons exert inhibitory control on sensory input in inflammatory pain states.