Photosensitive small extracellular vesicles regulate the immune microenvironment of triple negative breast cancer.

Currently, the treatment of triple-negative breast cancer (TNBC) is limited by the special pathological characteristics of this disease. In recent years, photodynamic therapy (PDT) has created new hope for the treatment of TNBC. Moreover, PDT can induce immunogenic cell death (ICD) and improve tumor immunogenicity. However, even though PDT can improve the immunogenicity of TNBC, the inhibitory immune microenvironment of TNBC still weakens the antitumor immune response. Therefore, we used the neutral sphingomyelinase inhibitor GW4869 to inhibit the secretion of small extracellular vesicles (sEVs) by TNBC cells to improve the tumor immune microenvironment and enhance antitumor immunity. In addition, bone mesenchymal stem cell (BMSC)-derived sEVs have good biological safety and a strong drug loading capacity, which can effectively improve the efficiency of drug delivery. In this study, we first obtained primary BMSCs and sEVs, and then the photosensitizers Ce6 and GW4869 were loaded into the sEVs by electroporation to produce immunomodulatory photosensitive nanovesicles (Ce6-GW4869/sEVs). When administered to TNBC cells or orthotopic TNBC models, these photosensitive sEVs could specifically target TNBC and improve the tumor immune microenvironment. Moreover, PDT combined with GW4869-based therapy showed a potent synergistic antitumor effect mediated by direct killing of TNBC and activation of antitumor immunity. Here, we designed photosensitive sEVs that could target TNBC and regulate the tumor immune microenvironment, providing a potential approach for improving the effectiveness of TNBC treatment. STATEMENT OF SIGNIFICANCE: We designed an immunomodulatory photosensitive nanovesicle (Ce6-GW4869/sEVs) with the photosensitizer Ce6 to achieve photodynamic therapy and the neutral sphingomyelinase inhibitor GW4869 to inhibit the secretion of small extracellular vesicles (sEVs) by triple-negative breast cancer (TNBC) cells to improve the tumor immune microenvironment and enhance antitumor immunity. In this study, the immunomodulatory photosensitive nanovesicle could target TNBC cells and regulate the tumor immune microenvironment, thus providing a potential approach for improving the treatment effect in TNBC. We found that the reduction in tumor sEVs secretion induced by GW4869 improved the tumor-suppressive immune microenvironment. Moreover, similar therapeutic strategies can also be applied in other kinds of tumors, especially immunosuppressive tumors, which is of great value for the clinical translation of tumor immunotherapy.

Activation function 1 of progesterone receptor is required for progesterone antagonism of oestrogen action in the uterus.

BACKGROUND: Progesterone receptor (PGR) is a master regulator of uterine function through antagonistic and synergistic interplays with oestrogen receptors. PGR action is primarily mediated by activation functions AF1 and AF2, but their physiological significance is unknown. RESULTS: We report the first study of AF1 function in mice. The AF1 mutant mice are infertile with impaired implantation and decidualization. This is associated with a delay in the cessation of epithelial proliferation and in the initiation of stromal proliferation at preimplantation. Despite tissue selective effect on PGR target genes, AF1 mutations caused global loss of the antioestrogenic activity of progesterone in both pregnant and ovariectomized models. Importantly, the study provides evidence that PGR can exert an antioestrogenic effect by genomic inhibition of Esr1 and Greb1 expression. ChIP-Seq data mining reveals intermingled PGR and ESR1 binding on Esr1 and Greb1 gene enhancers. Chromatin conformation analysis shows reduced interactions in these genes' loci in the mutant, coinciding with their upregulations. CONCLUSION: AF1 mediates genomic inhibition of ESR1 action globally whilst it also has tissue-selective effect on PGR target genes.

Immunotherapy-based novel nanoparticles in the treatment of gastrointestinal cancer: Trends and challenges.

Gastrointestinal cancer (GIC) is the most common cancer with a poor prognosis. Currently, surgery is the main treatment for GIC. However, the high rate of postoperative recurrence leads to a low five-year survival rate. In recent years, immunotherapy has received much attention. As the only immunotherapy drugs approved by the Food and Drug Administration (FDA), immune checkpoint blockade (ICB) drugs have great potential in cancer therapy. Nevertheless, the efficacy of ICB treatment is greatly limited by the low immunogenicity and immunosuppressive microenvironment of GIC. Therefore, the targets of immunotherapy have expanded from ICB to increasing tumor immunogenicity, increasing the recruitment and maturation of immune cells and reducing the proportion of inhibitory immune cells, such as M2-like macrophages, regulatory T cells and myeloid-derived suppressor cells. Moreover, with the development of nanotechnology, a variety of nanoparticles have been approved by the FDA for clinical therapy, so novel nanodrug delivery systems have become a research focus for anticancer therapy. In this review, we summarize recent advances in the application of immunotherapy-based nanoparticles in GICs, such as gastric cancer, hepatocellular carcinoma, colorectal cancer and pancreatic cancer, and described the existing challenges and future trends.

The APP intracellular domain promotes LRRK2 expression to enable feed-forward neurodegenerative mechanisms in Parkinson's disease.

Gain-of-function mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are common in familial forms of Parkinson's disease (PD), which is characterized by progressive neurodegeneration that impairs motor and cognitive function. We previously demonstrated that LRRK2-mediated phosphorylation of ß-amyloid precursor protein (APP) triggers the production and nuclear translocation of the APP intracellular domain (AICD). Here, we connected LRRK2 to AICD in a feed-forward cycle that enhanced LRRK2-mediated neurotoxicity. In cooperation with the transcription factor FOXO3a, AICD promoted LRRK2 expression, thus increasing the abundance of LRRK2 that promotes AICD activation. APP deficiency in LRRK2G2019S mice suppressed LRRK2 expression, LRRK2-mediated mitochondrial dysfunction, α-synuclein accumulation, and tyrosine hydroxylase (TH) loss in the brain, phenotypes associated with toxicity and loss of dopaminergic neurons in PD. Conversely, AICD overexpression increased LRRK2 expression and LRRK2-mediated neurotoxicity in LRRK2G2019S mice. In LRRK2G2019S mice or cultured dopaminergic neurons from LRRK2G2019S patients, treatment with itanapraced reduced LRRK2 expression and was neuroprotective. Itanapraced showed similar effects in a neurotoxin-induced PD mouse model, suggesting that inhibiting the AICD may also have therapeutic benefits in idiopathic PD. Our findings reveal a therapeutically targetable, feed-forward mechanism through which AICD promotes LRRK2-mediated neurotoxicity in PD.

Loss of CaV1.3 RNA editing enhances mouse hippocampal plasticity, learning, and memory.

L-type CaV1.3 calcium channels are expressed on the dendrites and soma of neurons, and there is a paucity of information about its role in hippocampal plasticity. Here, by genetic targeting to ablate CaV1.3 RNA editing, we demonstrate that unedited CaV1.3ΔECS mice exhibited improved learning and enhanced long-term memory, supporting a functional role of RNA editing in behavior. Significantly, the editing paradox that functional recoding of CaV1.3 RNA editing sites slows Ca2+-dependent inactivation to increase Ca2+ influx but reduces channel open probability to decrease Ca2+ influx was resolved. Mechanistically, using hippocampal slice recordings, we provide evidence that unedited CaV1.3 channels permitted larger Ca2+ influx into the hippocampal pyramidal neurons to bolster neuronal excitability, synaptic transmission, late long-term potentiation, and increased dendritic arborization. Of note, RNA editing of the CaV1.3 IQ-domain was found to be evolutionarily conserved in mammals, which lends support to the importance of the functional recoding of the CaV1.3 channel in brain function.

Active p38α causes macrovesicular fatty liver in mice.

One third of the western population suffers from nonalcoholic fatty liver disease (NAFLD), which may ultimately develop into hepatocellular carcinoma (HCC). The molecular event(s) that triggers the disease are not clear. Current understanding, known as the multiple hits model, suggests that NAFLD is a result of diverse events at several tissues (e.g., liver, adipose tissues, and intestine) combined with changes in metabolism and microbiome. In contrast to this prevailing concept, we report that fatty liver could be triggered by a single mutated protein expressed only in the liver. We established a transgenic system that allows temporally controlled activation of the MAP kinase p38α in a tissue-specific manner by induced expression of intrinsically active p38α allele. Here we checked the effect of exclusive activation in the liver. Unexpectedly, induction of p38α alone was sufficient to cause macrovesicular fatty liver. Animals did not become overweight, showing that fatty liver can be imposed solely by a genetic modification in liver per se and can be separated from obesity. Active p38α-induced fatty liver is associated with up-regulation of MUC13, CIDEA, PPARγ, ATF3, and c-jun mRNAs, which are up-regulated in human HCC. Shutting off expression of the p38α mutant resulted in reversal of symptoms. The findings suggest that p38α plays a direct causative role in fatty liver diseases and perhaps in other chronic inflammatory diseases. As p38α activity was induced by point mutations, it could be considered a proto-inflammatory gene (proto-inflammagene).

Impaired neurogenesis in the hippocampus of an adult VPS35 mutant mouse model of Parkinson's disease through interaction with APP.

Vacuolar protein sorting protein 35 (VPS35) is a core component of the retromer complex involved in regulating protein trafficking and retrieval. Recently, a missense mutation, Asp620Asn (D620N), in VPS35 (PARK17) has been identified as a pathogenic mutation for late-onset autosomal dominant Parkinson's disease (PD). Although PD is characterized by a range of motor symptoms associated with loss of dopaminergic neurons in the substantial nigra, non-motor symptoms such as impaired hippocampal neurogenesis were observed in both PD patients and animal models of PD caused by multiple PD-linked pathogenic genes such as alpha-synuclein and leucine-rich repeat kinase 2 (LRRK2). However, the role of the VPS35 D620N mutation in adult hippocampal neurogenesis remains unknown. Here, we showed that the VPS35 D620N mutation impaired hippocampal neurogenesis in adult transgenic mice expressing the VPS35 D620N gene. Specifically, we showed a reduction in the neural stem cell pool and neural proliferation and differentiation, retarded migration, and impaired neurite outgrowth in 3-month-old VPS35 D620N mutant mice. Moreover, we found that the VPS35 D620N mutant hyperphosphorylates amyloid precursor protein (APP) at Thr668and interacts with APP. Notably, by crossing the VPS35 D620N mutant mice with APP knockout (KO) mice, we showed that loss of APP function rescues VPS35 D620N-inhibited neurogenesis, neural migration, and maturation. Our study provides important evidence that APP is involved in the VPS35 D620N mutation in regulating adult neurogenesis, which sheds light on the pathogenic mechanisms in PD.

Expression of a constitutively active p38α mutant in mice causes early death, anemia, and accumulation of immunosuppressive cells.

The MAP kinase p38α is associated with numerous processes in eukaryotes, and its elevated activity is a prominent feature of inflammatory diseases, allergies, and aging. Since p38α is a nodal component of a complex signaling network, it is difficult to reveal exactly how p38α contributes to disparate outcomes. Identification of p38α -specific effects requires activation of p38α per se in vivo. We generated a transgenic mouse model that meets this requirement by allowing inducible and reversible expression of an intrinsically active p38α molecule (p38αD176A+F327S ). p38α's activation across all murine tissues resulted in a significant loss of body weight and death of about 40% of the mice within 17 weeks of activation, although most tissues were unaffected. Flow cytometric analysis of the lungs and bronchoalveolar lavage fluid detected an accumulation of 'debris' within the airways, suggesting impaired clearance. It also revealed increased numbers of alternatively activated alveolar macrophages and myeloid-derived suppressor cells within the lung, pointing at suppression and resolution of inflammation. Blood count suggested that mice expressing p38αD176A+F327S suffer from hemolytic anemia. Flow cytometry of bone marrow revealed a reduced number of hematopoietic stem cells and abnormalities in the erythroid lineage. Unexpectedly, p38α's substrate MAPKAPK2, mitogen-activated protein kinase-activated protein kinase 2 was downregulated in mice expressing p38αD176A+F327S , suggesting that constitutive activity of p38α may impose pathological phenotypes by downregulating downstream components, perhaps via a feedback inhibition mechanism. In summary, this new mouse model shows that induced p38α activity per se is hazardous to mouse vitality and welfare, although pathological parameters are apparent only in blood count, bone marrow, and lungs.

Altered striatal dopamine levels in Parkinson's disease VPS35 D620N mutant transgenic aged mice.

Vacuolar protein sorting 35 (VPS35) is a major component of the retromer complex that mediates the retrograde transport of cargo proteins from endosomes to the trans-Golgi network. Mutations such as D620N in the VPS35 gene have been identified in patients with autosomal dominant Parkinson's disease (PD). However, it remains poorly understood whether and how VPS35 deficiency or mutation contributes to PD pathogenesis; specifically, the studies that have examined VPS35 thus far have differed in results and methodologies. We generated a VPS35 D620N mouse model using a Rosa26-based transgene expression platform to allow expression in a spatial manner, so as to better address these discrepancies. Here, aged (20-months-old) mice were first subjected to behavioral tests. Subsequently, DAB staining analysis of substantia nigra (SN) dopaminergic neurons with the marker for tyrosine hydroxylase (TH) was performed. Next, HPLC was used to determine dopamine levels, along with levels of its two metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), in the striatum. Western blotting was also performed to study the levels of key proteins associated with PD. Lastly, autoradiography (ARG) evaluation of [3H]FE-PE2I binding to the striatal dopamine transporter DAT was carried out. We found that VPS35 D620N Tg mice displayed a significantly higher dopamine level than NTg counterparts. All results were then compared with that of current VPS35 studies to shed light on the disease pathogenesis. Our model allows future studies to explicitly control spatial expression of the transgene which would generate a more reliable PD phenotype.

Amyloid precursor protein intracellular domain-dependent regulation of FOXO3a inhibits adult hippocampal neurogenesis.

The amyloid precursor protein (APP) intracellular domain (AICD) is a metabolic by-product of APP produced through sequential proteolytic cleavage by α-, ß-, and γ-secretases. The interaction between AICD and Fe65 has been reported to impair adult neurogenesis in vivo. However, the exact role of AICD in mediating neural stem cell fate remains unclear. To identify the role of AICD in neuronal proliferation and differentiation, as well as to clarify the molecular mechanisms underlying the role of AICD in neurogenesis, we first generated a mouse model expressing the Rosa26-based AICD transgene. AICD overexpression did not alter the spatiotemporal expression pattern of full-length APP or accumulation of its metabolites. In addition, AICD decreased the newly generated neural progenitor cell (NPC) pool, inhibited the proliferation and differentiation efficiency of NPCs, and increased cell death both in vitro and in vivo. Given that abnormal neurogenesis is often associated with depression-like behavior in adult mice, we conducted a forced swim test and tail suspension test with AICD mice and found a depression-like behavioral phenotype in AICD transgenic mice. Moreover, AICD stimulated FOXO3a transcriptional activation, which in turn negatively regulated AICD. In addition, functional loss of FOXO3a in NPCs derived from the hippocampal dentate gyrus of adult AICD transgenic mice rescued neurogenesis defects. AICD also increased the mRNA expression of FOXO3a target genes related to neurogenesis and cell death. These results suggest that FOXO3a is the functional target of AICD in neurogenesis regulation. Our study reveals the role of AICD in mediating neural stem cell fate to maintain homeostasis during brain development via interaction with FOXO3a.

Cobalt ion interaction with TMEM16A calcium-activated chloride channel: Inhibition and potentiation.

TMEM16A, a Ca2+-sensitive Cl- channel, plays key roles in many physiological functions related to Cl- transport across lipid membranes. Activation of this channel is mediated via binding intracellular Ca2+ to the channel with a relatively high apparent affinity, roughly in the sub-µM to low µM concentration range. Recently available high-resolution structures of TMEM16 molecules reveal that the high-affinity Ca2+ activation sites are formed by several acidic amino acids, using their negatively charged sidechain carboxylates to coordinate the bound Ca2+. In this study, we examine the interaction of TMEM16A with a divalent cation, Co2+, which by itself cannot activate current in TMEM16A. This divalent cation, however, has two effects when applied intracellularly. It inhibits the Ca2+-induced TMEM16A current by competing with Ca2+ for the aforementioned high-affinity activation sites. In addition, Co2+ also potentiates the Ca2+-induced current with a low affinity. This potentiation effect requires high concentration (mM) of Co2+, similar to our previous findings that high concentrations (mM) of intracellular Ca2+ ([Ca2+]i) can induce more TMEM16A current after the Ca2+-activation sites are saturated by tens of µM [Ca2+]i. The degrees of potentiation by Co2+ and Ca2+ also roughly correlate with each other. Interestingly, mutating a pore residue of TMEM16A, Y589, alters the degree of potentiation in that the smaller the sidechain of the replaced residue, the larger the potentiation induced by divalent cations. We suggest that the Co2+ potentiation and the Ca2+ potentiation share a similar mechanism by increasing Cl- flux through the channel pore, perhaps due to an increase of positive pore potential after the binding of divalent cations to phospholipids in the pore. A smaller sidechain of a pore residue may allow the pore to accommodate more phospholipids, thus enhancing the current potentiation caused by high concentrations of divalent cations.

Extracellular histones stimulate collagen expression in vitro and promote liver fibrogenesis in a mouse model via the TLR4-MyD88 signaling pathway.

BACKGROUND: Liver fibrosis progressing to liver cirrhosis and hepatic carcinoma is very common and causes more than one million deaths annually. Fibrosis develops from recurrent liver injury but the molecular mechanisms are not fully understood. Recently, the TLR4-MyD88 signaling pathway has been reported to contribute to fibrosis. Extracellular histones are ligands of TLR4 but their roles in liver fibrosis have not been investigated. AIM: To investigate the roles and potential mechanisms of extracellular histones in liver fibrosis. METHODS: In vitro, LX2 human hepatic stellate cells (HSCs) were treated with histones in the presence or absence of non-anticoagulant heparin (NAHP) for neutralizing histones or TLR4-blocking antibody. The resultant cellular expression of collagen I was detected using western blotting and immunofluorescent staining. In vivo, the CCl4-induced liver fibrosis model was generated in male 6-week-old ICR mice and in TLR4 or MyD88 knockout and parental mice. Circulating histones were detected and the effect of NAHP was evaluated. RESULTS: Extracellular histones strongly stimulated LX2 cells to produce collagen I. Histone-enhanced collagen expression was significantly reduced by NAHP and TLR4-blocking antibody. In CCl4-treated wild type mice, circulating histones were dramatically increased and maintained high levels during the duration of fibrosis-induction. Injection of NAHP not only reduced alanine aminotransferase and liver injury scores, but also significantly reduced fibrogenesis. Since the TLR4-blocking antibody reduced histone-enhanced collagen I production in HSC, the CCl4 model with TLR4 and MyD88 knockout mice was used to demonstrate the roles of the TLR4-MyD88 signaling pathway in CCl4-induced liver fibrosis. The levels of liver fibrosis were indeed significantly reduced in knockout mice compared to wild type parental mice. CONCLUSION: Extracellular histones potentially enhance fibrogenesis via the TLR4-MyD88 signaling pathway and NAHP has therapeutic potential by detoxifying extracellular histones.

Author Correction: WWP2 regulates pathological cardiac fibrosis by modulating SMAD2 signaling.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

WWP2 regulates pathological cardiac fibrosis by modulating SMAD2 signaling.

Cardiac fibrosis is a final common pathology in inherited and acquired heart diseases that causes cardiac electrical and pump failure. Here, we use systems genetics to identify a pro-fibrotic gene network in the diseased heart and show that this network is regulated by the E3 ubiquitin ligase WWP2, specifically by the WWP2-N terminal isoform. Importantly, the WWP2-regulated pro-fibrotic gene network is conserved across different cardiac diseases characterized by fibrosis: human and murine dilated cardiomyopathy and repaired tetralogy of Fallot. Transgenic mice lacking the N-terminal region of the WWP2 protein show improved cardiac function and reduced myocardial fibrosis in response to pressure overload or myocardial infarction. In primary cardiac fibroblasts, WWP2 positively regulates the expression of pro-fibrotic markers and extracellular matrix genes. TGFß1 stimulation promotes nuclear translocation of the WWP2 isoforms containing the N-terminal region and their interaction with SMAD2. WWP2 mediates the TGFß1-induced nucleocytoplasmic shuttling and transcriptional activity of SMAD2.

Comparison of ion transport determinants between a TMEM16 chloride channel and phospholipid scramblase.

Two TMEM16 family members, TMEM16A and TMEM16F, have different ion transport properties. Upon activation by intracellular Ca2+, TMEM16A-a Ca2+-activated Cl- channel-is more selective for anions than cations, whereas TMEM16F-a phospholipid scramblase-appears to transport both cations and anions. Under saturating Ca2+ conditions, the current-voltage (I-V) relationships of these two proteins also differ; the I-V curve of TMEM16A is linear, while that of TMEM16F is outwardly rectifying. We previously found that mutating a positively charged lysine residue (K584) in the ion transport pathway to glutamine converted the linear I-V curve of TMEM16A to an outwardly rectifying curve. Interestingly, the corresponding residue in the outwardly rectifying TMEM16F is also a glutamine (Q559). Here, we examine the ion transport functions of TMEM16 molecules and compare the roles of K584 of TMEM16A and Q559 of TMEM16F in controlling the rectification of their respective I-V curves. We find that rectification of TMEM16A is regulated electrostatically by the side-chain charge on the residue at position 584, whereas the charge on residue 559 in TMEM16F has little effect. Unexpectedly, mutation of Q559 to aromatic amino acid residues significantly alters outward rectification in TMEM16F. These same mutants show reduced Ca2+-induced current rundown (or desensitization) compared with wild-type TMEM16F. A mutant that removes the rundown of TMEM16F could facilitate the study of ion transport mechanisms in this phospholipid scramblase in the same way that a CLC-0 mutant in which inactivation (or closure of the slow gate) is suppressed was used in our previous studies.

Publisher Correction: Utf1 contributes to intergenerational epigenetic inheritance of pluripotency.

A correction to this article has been published and is linked from the HTML version of this paper. The error has been fixed in the paper.

Utf1 contributes to intergenerational epigenetic inheritance of pluripotency.

Undifferentiated embryonic cell transcription factor 1 (Utf1) is expressed in pluripotent embryonic stem cells (ESCs) and primordial germ cells (PGCs). Utf1 expression is directly controlled by pluripotency factors Oct4 and Sox2, which form a ternary complex with the Utf1 enhancer. The Utf1 protein plays a role in chromatin organization and epigenetic control of bivalent gene expression in ESCs in vitro, where it promotes effective cell differentiation during exit from pluripotency. The function of Utf1 in PGCs in vivo, however, is not known. Here, we report that proper development of Utf1 null embryos almost entirely depends on the presence of functional Utf1 alleles in the parental germline. This indicates that Utf1's proposed epigenetic role in ESC pluripotency in vitro may be linked to intergenerational epigenetic inheritance in vivo. One component - or at least facilitator - of the relevant epigenetic mark appears to be Utf1 itself, since Utf1-driven tomato reporter and Utf1 are detected in mature germ cells. We also provide initial evidence for a reduced adult testis size in Utf1 null mice. Our findings thus point at unexpected functional links between the core ESC pluripotency factor network and epigenetic inheritance of pluripotency.

Independent activation of distinct pores in dimeric TMEM16A channels.

The TMEM16 family encompasses Ca2+-activated Cl- channels (CaCCs) and lipid scramblases. These proteins are formed by two identical subunits, as confirmed by the recently solved crystal structure of a TMEM16 lipid scramblase. However, the high-resolution structure did not provide definitive information regarding the pore architecture of the TMEM16 channels. In this study, we express TMEM16A channels constituting two covalently linked subunits with different Ca2+ affinities. The dose-response curve of the heterodimer appears to be a weighted sum of two dose-response curves-one corresponding to the high-affinity subunit and the other to the low-affinity subunit. However, fluorescence resonance energy transfer experiments suggest that the covalently linked heterodimeric proteins fold and assemble as one molecule. Together these results suggest that activation of the two TMEM16A subunits likely activate independently of each other. The Ca2+ activation curve for the heterodimer at a low Ca2+ concentration range ([Ca2+] < 5 µM) is similar to that of the wild-type channel-the Hill coefficients in both cases are significantly greater than one. This suggests that Ca2+ binding to one subunit of TMEM16A is sufficient to activate the channel and that each subunit contains more than one Ca2+-binding site. We also take advantage of the I-V curve rectification that results from mutation of a pore residue to address the pore architecture of the channel. By introducing the pore mutation and the mutation that alters Ca2+ affinity in the same or different subunits, we demonstrate that activation of different subunits appears to be associated with the opening of different pores. These results suggest that the TMEM16A CaCC may also adopt a "double-barrel" pore architecture, similar to that found in CLC channels and transporters.

Protein kinase C-dependent regulation of ClC-1 channels in active human muscle and its effect on fast and slow gating.

KEY POINTS: Regulation of ion channel function during repeated firing of action potentials is commonly observed in excitable cells. Recently it was shown that muscle activity is associated with rapid, protein kinase C (PKC)-dependent ClC-1 Cl(-) channel inhibition in rodent muscle. While this PKC-dependent ClC-1 inhibition during muscle activity was shown to be important for the maintenance of contractile endurance in rat muscle it is unknown whether a similar regulation exists in human muscle. Also, the molecular mechanisms underlying the observed PKC-dependent ClC-1 inhibition are unclear. Here we present the first demonstration of ClC-1 inhibition in active human muscle fibres, and we determine the changes in ClC-1 gating that underlie the PKC-dependent ClC-1 inhibition in active muscle using human ClC-1 expressed in Xenopus oocytes. This activity-induced ClC-1 inhibition is suggested to represent a mechanism by which human muscle fibres maintain their excitability during sustained activity. ABSTRACT: Repeated firing of action potentials (APs) is known to trigger rapid, protein kinase C (PKC)-dependent inhibition of ClC-1 Cl(-) ion channels in rodent muscle and this inhibition is important for contractile endurance. It is currently unknown whether similar regulation exists in human muscle, and the molecular mechanisms underlying PKC-dependent ClC-1 inhibition are unclear. This study first determined whether PKC-dependent ClC-1 inhibition exists in active human muscle, and second, it clarified how PKC alters the gating of human ClC-1 expressed in Xenopus oocytes. In human abdominal and intercostal muscles, repeated AP firing was associated with 30-60% reduction of ClC-1 function, which could be completely prevented by PKC inhibition (1 µm GF109203X). The role of the PKC-dependent ClC-1 inhibition was evaluated from rheobase currents before and after firing 1000 APs: while rheobase current was well maintained after activity under control conditions it rose dramatically if PKC-dependent ClC-1 inhibition had been prevented with the inhibitor. This demonstrates that the ClC-1 inhibition is important for maintenance of excitability in active human muscle fibres. Oocyte experiments showed that PKC activation lowered the overall open probability of ClC-1 in the voltage range relevant for AP initiation in muscle fibres. More detailed analysis of this reduction showed that PKC mostly affected the slow gate of ClC-1. Indeed, there was no effect of PKC activation in C277S mutated ClC-1 in which the slow gate is effectively locked open. It is concluded that regulation of excitability of active human muscle fibres relies on PKC-dependent ClC-1 inhibition via a gating mechanism.