Single-cell RNA sequencing reveals the heterogeneity and intercellular communication of hepatic stellate cells and macrophages during liver fibrosis.

Uncontrolled and excessive progression of liver fibrosis is thought to be the prevalent pathophysiological cause of liver cirrhosis and hepatocellular cancer, and there are currently no effective antifibrotic therapeutic options available. Intercellular communication and cellular heterogeneity in the liver are involved in the progression of liver fibrosis, but the exact nature of the cellular phenotypic changes and patterns of interregulatory remain unclear. Here, we performed single-cell RNA sequencing on nonparenchymal cells (NPCs) isolated from normal and fibrotic mouse livers. We identified eight main types of cells, including endothelial cells, hepatocytes, dendritic cells, B cells, natural killer/T (NK/T) cells, hepatic stellate cells (HSCs), cholangiocytes and macrophages, and revealed that macrophages and HSCs exhibit the most variance in transcriptional profile. Further analyses of HSCs and macrophage subpopulations and ligand-receptor interaction revealed a high heterogeneity characterization and tightly interregulated network of these two groups of cells in liver fibrosis. Finally, we uncovered a profibrotic Thbs1+ macrophage subcluster, which expands in mouse and human fibrotic livers, activating HSCs via PI3K/AKT/mTOR signaling pathway. Our findings decode unanticipated insights into the heterogeneity of HSCs and macrophages and their intercellular crosstalk at a single-cell level, and may provide potential therapeutic strategies in liver fibrosis.

A New Product of Bilirubin Degradation by H2O2 and Its Formation in Activated Neutrophils and in an Inflammatory Mouse Model.

Bilirubin (BR) is a tetrapyrrolic compound stemming from heme catabolism with diverse physiological functions. It can be oxidized by H2O2 to form several degradation products, some of which have been detected in vivo and may contribute to the pathogenesis of certain diseases. However, the oxidative degradation of BR is complex and the conditions that BR degradation occurs pathophysiologically remain obscure. Neutrophils are known to generate large amounts of reactive oxygen species, including H2O2, upon activation and they are mobilized to inflammatory sites; therefore, we hypothesized that activated neutrophils could cause BR degradation, which could occur at inflammatory sites. In the present study, we investigated BR degradation by H2O2 and identified hematinic acid (BHP1) and a new product BHP2, whose structure was characterized as 2,5-diformyl-4-methyl-1H-pyrrole-3-propanoic acid. An LC-MS/MS method for the quantitation of the two compounds was then established. Using the LC-MS/MS method, we observed the concentration-dependent formation of BHP1 and BHP2 in mouse neutrophils incubated with 10 and 30 µM of BR with the yields being 16 ± 3.2 and 31 ± 5.9 pmol/106 cells for BHP1, and 25 ± 4.4 and 71 ± 26 pmol/106 cells for BHP2, respectively. After adding phorbol 12-myristate 13-acetate, a neutrophil agonist, to 30 µM of BR-treated cells, the BHP1 yield increased to 43 ± 6.6 pmol/106 cells, whereas the BHP2 one decreased to 47 ± 9.2 pmol/106 cells. The two products were also detected in hemorrhagic skins of mice with dermal inflammation and hemorrhage at levels of 4.5 ± 1.9 and 0.18 ± 0.10 nmol/g tissue, respectively, which were significantly higher than those in the non-hemorrhagic skins. BHP2 was neurotoxic starting at 0.10 µM but BHP1 was not, as assessed using Caenorhabditis elegans as the animal model. Neutrophil-mediated BR degradation may be a universally pathophysiological process in inflammation and can be particularly important under pathological conditions concerning hemorrhage.

K2P1 leak cation channels contribute to ventricular ectopic beats and sudden death under hypokalemia.

Hypokalemia causes ectopic heartbeats, but the mechanisms underlying such cardiac arrhythmias are not understood. In reduced serum K+ concentrations that occur under hypokalemia, K2P1 two-pore domain K+ channels change ion selectivity and switch to conduct inward leak cation currents, which cause aberrant depolarization of resting potential and induce spontaneous action potential of human cardiomyocytes. K2P1 is expressed in the human heart but not in mouse hearts. We test the hypothesis that K2P1 leak cation channels contribute to ectopic heartbeats under hypokalemia, by analysis of transgenic mice, which conditionally express induced K2P1 specifically in hearts, mimicking K2P1 channels in the human heart. Conditional expression of induced K2P1 specifically in the heart of hypokalemic mice results in multiple types of ventricular ectopic beats including single and multiple ventricular premature beats as well as ventricular tachycardia and causes sudden death. In isolated mouse hearts that express induced K2P1, sustained ventricular fibrillation occurs rapidly after perfusion with low K+ concentration solutions that mimic hypokalemic conditions. These observed phenotypes occur rarely in control mice or in the hearts that lack K2P1 expression. K2P1-expressing mouse cardiomyocytes of transgenic mice much more frequently fire abnormal single and/or rhythmic spontaneous action potential in hypokalemic conditions, compared to wild type mouse cardiomyocytes without K2P1 expression. These findings confirm that K2P1 leak cation channels induce ventricular ectopic beats and sudden death of transgenic mice with hypokalemia and imply that K2P1 leak cation channels may play a critical role in human ectopic heartbeats under hypokalemia.

Bilirubin oxidation end product B prevents CoCl2-induced primary cortical neuron apoptosis by promoting cell survival Akt/mTOR/p70S6K signaling pathway.

Bilirubin oxidation end products (BOXes) are associated with the late-developing neurological deficits after subarachnoid hemorrhage (SAH) possibly by direct constricting the cerebral arteries, but their specific impacts on neurons especially in the state of hypoxia, a prominent feature during the late stage of SAH, remain unclear. Here, we explored the effects of BOXes on the primary cortical neurons subjected to CoCl2-induced hypoxia by evaluating the morphological and apoptotic changes of neurons. The present study showed that Z-BOX B but not Z-BOX A greatly alleviated CoCl2-induced neuronal cell deterioration and apoptosis. Immunocytochemical staining assay showed Z-BOX B significantly increased neurite length, the numbers of both secondary and tertiary branches, and the protein level of Synaptophysin. Caspase 3/7 apoptosis assay and DAPI staining showed that Z-BOX B markedly reduced primary cortical neurons apoptosis. The expression of cleaved Caspase-3 was suppressed by Z-BOX B treatment, while the expression of Bcl-xL was upregulated. To further discover the mechanism of the neuroprotective effect observed in Z-BOX B, we found Z-BOX B increased the expression of p-mTOR, p-Akt, and p-p70S6K. In general, our results implicated Z-BOX B may prevent CoCl2-induced primary cortical neurons apoptosis by activating sAkt/mTOR/p70S6K signaling pathway. Hence, the present data may provide new insights into the pathophysiological mechanism of delayed neurological dysfunction after SAH and novel targets for treating SAH.

Cinobufagin Is a Selective Anti-Cancer Agent against Tumors with EGFR Amplification and PTEN Deletion.

Glioblastoma multiforme (GBM) is the most common and malignant brain tumor, and almost half of the patients carrying EGFR-driven tumor with PTEN deficiency are resistant to EGFR-targeted therapy. EGFR amplification and/or mutation is reported in various epithelial tumors. This series of studies aimed to identify a potent compound against EGFR-driven tumor. We screened a chemical library containing over 600 individual compounds purified from Traditional Chinese Medicine against GBM cells with EGFR amplification and found that cinobufagin, the major active ingredient of Chansu, inhibited the proliferation of EGFR amplified GBM cells and PTEN deficiency enhanced its anti-proliferation effects. Cinobufagin also strongly inhibited the proliferation of carcinoma cell lines with wild-type or mutant EGFR expression. In contrast, the compound only weakly inhibited the proliferation of cancer cells with low or without EGFR expression. Cinobufagin blocked EGFR phosphorylation and its downstream signaling, which additionally induced apoptosis and cytotoxicity in EGFR amplified cancer cells. In vivo, cinobufagin blocked EGFR signaling, inhibited cell proliferation, and elicited apoptosis, thereby suppressing tumor growth in both subcutaneous and intracranial U87MG-EGFR xenograft mouse models and increasing the median survival of nude mice bearing intracranial U87MG-EGFR tumors. Cinobufagin is a potential therapeutic agent for treating malignant glioma and other human cancers expressing EGFR.

Bilirubin Oxidation End Products (BOXes) Induce Neuronal Oxidative Stress Involving the Nrf2 Pathway.

Delayed ischemic neurological deficit (DIND) is a severe complication after subarachnoid hemorrhage (SAH). Previous studies have suggested that bilirubin oxidation end products (BOXes) are probably associated with the DIND after SAH, but there is a lack of direct evidence yet even on cellular levels. In the present study, we aim to explore the potential role of BOXes and the involved mechanisms in neuronal function. We synthesized high-purity (>97%) BOX A and BOX B isomers. The pharmacokinetics showed they are permeable to the blood-brain barrier. Exposure of a moderate concentration (10 or 30 µM) of BOX A or BOX B to isolated primary cortical neurons increased the production of reactive oxygen species. In the human neuroblastoma SH-SY5Y cells, BOX A and BOX B decreased the mitochondrial membrane potential and enhanced nuclear accumulation of the protein Nrf2 implicated in oxidative injury repair. In addition, both chemicals increased the mRNA and protein expression levels of multiple antioxidant response genes including Hmox1, Gsta3, Blvrb, Gclm, and Srxn1, indicating that the antioxidant response element (ARE) transcriptional cascade driven by Nrf2 is activated. In conclusion, we demonstrated that primary cortical neurons and neuroblastoma cells undergo an adaptive response against BOX A- and BOX B-mediated oxidative stress by activation of multiple antioxidant responses, in part through the Nrf2 pathway, which provides in-depth insights into the pathophysiological mechanism of DIND after SAH or other neurological dysfunctions related to cerebral hemorrhage.

Clonal evolution in liver cancer at single-cell and single-variant resolution.

Genetic heterogeneity of tumor is closely related to its clonal evolution, phenotypic diversity and treatment resistance, and such heterogeneity has only been characterized at single-cell sub-chromosomal scale in liver cancer. Here we reconstructed the single-variant resolution clonal evolution in human liver cancer based on single-cell mutational profiles. The results indicated that key genetic events occurred early during tumorigenesis, and an early metastasis followed by independent evolution was observed in primary liver tumor and intrahepatic metastatic portal vein tumor thrombus. By parallel single-cell RNA-Seq, the transcriptomic phenotype of HCC was found to be related with genetic heterogeneity. For the first time we reconstructed the single-cell and single-variant clonal evolution in human liver cancer, and dissection of both genetic and phenotypic heterogeneity will facilitate better understanding of their relationship.

Protein Kinase C δ (PKCδ) Attenuates Bleomycin Induced Pulmonary Fibrosis via Inhibiting NF-κB Signaling Pathway.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive and lethal interstitial lung disease characterized by consistent pulmonary inflammation. Although protein kinase C delta (PKCδ) is involved in broad scope cellular response, the role of PKCδ in IPF is complicated and has not been fully defined yet. Here, we reported that PKCδ deficiency (PKCδ-/-) aggravated bleomycin (BLM)-induced pulmonary fibrosis and inflammation. Upon challenge with BLM, the pulmonary capillary permeability, immune cell infiltration, inflammatory cytokine production, and collagen deposition were enhanced in PKCδ-/- mice compared to that in PKCδ+/+ mice. In response to poly(I:C) stimulation, PKCδ deficient macrophages displayed an increased production of IL-1ß, IL-6, TNF-α, and IL-33, which were associated with an enhanced NF-κB activation. Furthermore, we found that PKCδ could directly bind to and phosphorylate A20, an inhibitory protein of NF-κB signal. These results suggested that PKCδ may inhibit the NF-κB signaling pathway via enhancing the stability and activity of A20, which in turn attenuates pulmonary fibrosis, suggesting that PKCδ is a promising target for treating pulmonary fibrosis.

Activation of BK Channels Prevents Hepatic Stellate Cell Activation and Liver Fibrosis Through the Suppression of TGFß1/SMAD3 and JAK/STAT3 Profibrotic Signaling Pathways.

Large-conductance and Ca2+-activated K+ (BK) channels are expressed in human hepatic stellate cells (HSCs), where they have roles in normal hepatic microcirculation, as well as in portal hypertension in liver cirrhosis through the regulation of contractility in activated HSCs. Nevertheless, whether BK channel activity exerts protective effects against aberrant HSC activation and hepatic fibrosis is unknown. Here, we report that BK channels are expressed in activated primary rat HSCs as well as in a human HSC line. Moreover, whole-cell K+ currents recorded from activated HSCs were markedly increased by exposure to rottlerin, a BK channel-specific activator, but were inhibited by treatment with the BK channel-specific inhibitor, paxilline, suggesting that BK channels are functional in activated HSCs. Overexpression but not downregulation of the BK channel pore-forming alpha subunit, KCNMA1, led to reduced migration and collagen expression in activated HSCs. Consistently, rottlerin treatment suppressed the fibrogenic cell function both in vitro and in CCl4-induced liver fibrosis in vivo. Microarray and pathway analysis, combined with a luciferase reporter assay and western blotting, further showed that rottlerin treatment led to a significant downregulation of the profibrotic TGFß1/SMAD3 and JAK/STAT3 signaling pathways, both in vitro and in vivo. Our findings not only link BK channel function to profibrotic signaling pathways, but also provide evidence that BK channel activation represents a promising therapeutic strategy for the treatment of liver fibrosis.

GABRP regulates chemokine signalling, macrophage recruitment and tumour progression in pancreatic cancer through tuning KCNN4-mediated Ca2+ signalling in a GABA-independent manner.

BACKGROUND AND AIMS: Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer-related death worldwide. Neurotransmitter-initiated signalling pathway is profoundly implicated in tumour initiation and progression. Here, we investigated whether dysregulated neurotransmitter receptors play a role during pancreatic tumourigenesis. METHODS: The Cancer Genome Atlas and Gene Expression Omnibus datasets were used to identify differentially expressed neurotransmitter receptors. The expression pattern of gamma-aminobutyric acid type A receptor pi subunit (GABRP) in human and mouse PDAC tissues and cells was studied by immunohistochemistry and western blot analysis. The in vivo implications of GABRP in PDAC were tested by subcutaneous xenograft model and lung metastasis model. Bioinformatics analysis, transwell experiment and orthotopic xenograft model were used to identify the in vitro and in vivo effects of GABRP on macrophages in PDAC. ELISA, co-immunoprecipitation, proximity ligation assay, electrophysiology, promoter luciferase activity and quantitative real-time PCR analyses were used to identify molecular mechanism. RESULTS: GABRP expression was remarkably increased in PDAC tissues and associated with poor prognosis, contributed to tumour growth and metastasis. GABRP was correlated with macrophage infiltration in PDAC and pharmacological deletion of macrophages largely abrogated the oncogenic functions of GABRP in PDAC. Mechanistically, GABRP interacted with KCNN4 to induce Ca2+ entry, which leads to activation of nuclear factor κB signalling and ultimately facilitates macrophage infiltration by inducing CXCL5 and CCL20 expression. CONCLUSIONS: Overexpressed GABRP exhibits an immunomodulatory role in PDAC in a neurotransmitter-independent manner. Targeting GABRP or its interaction partner KCNN4 may be an effective therapeutic strategy for PDAC.

Capturing the Interaction Kinetics of an Ion Channel Protein with Small Molecules by the Bio-layer Interferometry Assay.

The bio-layer interferometry (BLI) assay is a valuable tool for measuring protein-protein and protein-small molecule interactions. Here, we first describe the application of this novel label-free technique to study the interaction of human EAG1 (hEAG1) channel proteins with the small molecule PIP2. hEAG1 channel has been recognized as potential therapeutic target because of its aberrant overexpression in cancers and a few gain-of-function mutations involved in some types of neurological diseases. We purified hEAG1 channel proteins from a mammalian stable expression system and measured the interaction with PIP2 by BLI. The successful measurement of the kinetics of binding between hEAG1 protein and PIP2 demonstrates that the BLI assay is a potential high-throughput approach used for novel small-molecule ligand screening in ion channel pharmacology.

CSmiRTar: Condition-Specific microRNA targets database.

MicroRNAs (miRNAs) are functional RNA molecules which play important roles in the post-transcriptional regulation. miRNAs regulate their target genes by repressing translation or inducing degradation of the target genes' mRNAs. Many databases have been constructed to provide computationally predicted miRNA targets. However, they cannot provide the miRNA targets expressed in a specific tissue and related to a specific disease at the same time. Moreover, they cannot provide the common targets of multiple miRNAs and the common miRNAs of multiple genes at the same time. To solve these two problems, we construct a database called CSmiRTar (Condition-Specific miRNA Targets). CSmiRTar collects computationally predicted targets of 2588 human miRNAs and 1945 mouse miRNAs from four most widely used miRNA target prediction databases (miRDB, TargetScan, microRNA.org and DIANA-microT) and implements functional filters which allows users to search (i) a miRNA's targets expressed in a specific tissue or/and related to a specific disease, (ii) multiple miRNAs' common targets expressed in a specific tissue or/and related to a specific disease, (iii) a gene's miRNAs related to a specific disease, and (iv) multiple genes' common miRNAs related to a specific disease. We believe that CSmiRTar will be a useful database for biologists to study the molecular mechanisms of post-transcriptional regulation in human or mouse. CSmiRTar is available at http://cosbi.ee.ncku.edu.tw/CSmiRTar/ or http://cosbi4.ee.ncku.edu.tw/CSmiRTar/.

Eag1 K+ Channel: Endogenous Regulation and Functions in Nervous System.

Ether-à-go-go1 (Eag1, Kv10.1, KCNH1) K+ channel is a member of the voltage-gated K+ channel family mainly distributed in the central nervous system and cancer cells. Like other types of voltage-gated K+ channels, the EAG1 channels are regulated by a variety of endogenous signals including reactive oxygen species, rendering the EAG1 to be in the redox-regulated ion channel family. The role of EAG1 channels in tumor development and its therapeutic significance have been well established. Meanwhile, the importance of hEAG1 channels in the nervous system is now increasingly appreciated. The present review will focus on the recent progress on the channel regulation by endogenous signals and the potential functions of EAG1 channels in normal neuronal signaling as well as neurological diseases.

Nicotine facilitates VSMC dysfunction through a miR-200b/RhoGDIA/cytoskeleton module.

Nicotine can induce the abnormal migration and proliferation of vascular smooth muscle cells (VSMCs). We have previously shown that cytoskeletal proteins and RhoGDIA, a negative regulator of the Rho GTPase pathway, are involved in the nicotine-induced dysfunction of VSMCs. Here, we found that nicotine can activate the Rho GTPase pathway and induce the synthesis of the cytoskeletal proteins in VSMCs through the activation of intracellular downstream signaling pathways, including targets such as MYPT1, PAK1 and PI3K/AKT. Upon nicotine treatment, the mRNA level of RhoGDIA is increased but protein level is decreased both in vitro and in vivo, which suggested a mechanism of post-translational regulation. By the dual luciferase reporter assay, we identified the microRNA-200b (miR-200b) as a modulator of the behavioural changes of VSMCs in response to nicotine through targeting RhoGDIA directly. Introducing miR-200b inhibitors into cultured VSMCs significantly attenuated cell proliferation and migration. Additionally, we found that hypomethylation in the CpG island shore region of miR-200b was responsible for the nicotine-induced miR-200b up-regulation in VSMCs. The study demonstrates that nicotine facilitates VSMC dysfunction through a miR-200b/RhoGDIA/cytoskeleton module through the hypomethylation of miR-200b promoter and suggests that epigenetic modifications may play an important role in the pathological progression.

Human EAG channels are directly modulated by PIP2 as revealed by electrophysiological and optical interference investigations.

Voltage-gated ether à go-go (EAG) K(+) channels are expressed in various types of cancer cells and also in the central nervous system. Aberrant overactivation of human EAG1 (hEAG1) channels is associated with cancer and neuronal disorders such as Zimmermann-Laband and Temple-Baraitser syndromes. Although hEAG1 channels are recognized as potential therapeutic targets, regulation of their functional properties is only poorly understood. Here, we show that the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) is a potent inhibitory gating modifier of hEAG1 channels. PIP2 inhibits the channel activity by directly binding to a short N-terminal segment of the channel important for Ca(2+)/calmodulin (CaM) binding as evidenced by bio-layer interferometry measurements. Conversely, depletion of endogenous PIP2 either by serotonin-induced phospholipase C (PLC) activation or by a rapamycin-induced translocation system enhances the channel activity at physiological membrane potentials, suggesting that PIP2 exerts a tonic inhibitory influence. Our study, combining electrophysiological and direct binding assays, demonstrates that hEAG1 channels are subject to potent inhibitory modulation by multiple phospholipids and suggests that manipulations of the PIP2 signaling pathway may represent a strategy to treat hEAG1 channel-associated diseases.

Two distinct effects of PIP2 underlie auxiliary subunit-dependent modulation of Slo1 BK channels.

Phosphatidylinositol 4,5-bisphosphate (PIP2) plays a critical role in modulating the function of numerous ion channels, including large-conductance Ca(2+)- and voltage-dependent K(+) (BK, Slo1) channels. Slo1 BK channel complexes include four pore-forming Slo1 (α) subunits as well as various regulatory auxiliary subunits (ß and γ) that are expressed in different tissues. We examined the molecular and biophysical mechanisms underlying the effects of brain-derived PIP2 on human Slo1 BK channel complexes with different subunit compositions that were heterologously expressed in human embryonic kidney cells. PIP2 inhibited macroscopic currents through Slo1 channels without auxiliary subunits and through Slo1 + γ1 complexes. In contrast, PIP2 markedly increased macroscopic currents through Slo1 + ß1 and Slo1 + ß4 channel complexes and failed to alter macroscopic currents through Slo1 + ß2 and Slo1 + ß2 Δ2-19 channel complexes. Results obtained at various membrane potentials and divalent cation concentrations suggest that PIP2 promotes opening of the ion conduction gate in all channel types, regardless of the specific subunit composition. However, in the absence of ß subunits positioned near the voltage-sensor domains (VSDs), as in Slo1 and probably Slo1 + γ1, PIP2 augments the negative surface charge on the cytoplasmic side of the membrane, thereby shifting the voltage dependence of VSD-mediated activation in the positive direction. When ß1 or ß4 subunits occupy the space surrounding the VSDs, only the stimulatory effect of PIP2 is evident. The subunit compositions of native Slo1 BK channels differ in various cell types; thus, PIP2 may exert distinct tissue- and divalent cation-dependent modulatory influences.

A point mutation in the human Slo1 channel that impairs its sensitivity to omega-3 docosahexaenoic acid.

Long-chain polyunsaturated omega-3 fatty acids such as docosahexaenoic acid (DHA) at nanomolar concentrations reversibly activate human large-conductance Ca(2+)- and voltage-gated K(+) (Slo1 BK) channels containing auxiliary ß1 or ß4 subunits in cell-free patches. Here we examined the action of DHA on the Slo1 channel without any auxiliary subunit and sought to elucidate the biophysical mechanism and the molecular determinants of the DHA sensitivity. Measurements of ionic currents through human Slo1 (hSlo1) channels reveal that the stimulatory effect of DHA does not require activation of the voltage or Ca(2+) sensors. Unlike gating of the hSlo1 channel, that of the Drosophila melanogaster Slo1 (dSlo1) channel is unaltered by DHA. Our mutagenesis study based on the differential responses of human and dSlo1 channels to DHA pinpoints that Y318 near the cytoplasmic end of S6 in the hSlo1 channel is a critical determinant of the stimulatory action of DHA. The mutation Y318S in hSlo1, which replaces Y with S as found in dSlo1, greatly diminishes the channel's response to DHA with a 22-carbon chain whether ß1 or ß4 is absent or present. However, the responses to α-linolenic acid, an omegea-3 fatty acid with an 18-carbon chain, and to arachidonic acid, an omega-6 fatty acid with a 20-carbon chain, remain unaffected by the mutation. Y318 in the S6 segment of hSlo1 is thus an important determinant of the electrophysiological response of the channel to DHA. Furthermore, the mutation Y318S may prove to be useful in dissecting out the complex lipid-mediated modulation of Slo1 BK channels.

Omega-3 fatty acids lower blood pressure by directly activating large-conductance Ca²âº-dependent K⁺ channels.

Long-chain polyunsaturated omega-3 fatty acids such as docosahexaenoic acid (DHA), found abundantly in oily fish, may have diverse health-promoting effects, potentially protecting the immune, nervous, and cardiovascular systems. However, the mechanisms underlying the purported health-promoting effects of DHA remain largely unclear, in part because molecular signaling pathways and effectors of DHA are only beginning to be revealed. In vascular smooth muscle cells, large-conductance Ca(2+)- and voltage-activated K(+) (BK) channels provide a critical vasodilatory influence. We report here that DHA with an EC50 of ∼500 nM rapidly and reversibly activates BK channels composed of the pore-forming Slo1 subunit and the auxiliary subunit ß1, increasing currents by up to ∼20-fold. The DHA action is observed in cell-free patches and does not require voltage-sensor activation or Ca(2+) binding but involves destabilization of the closed conformation of the ion conduction gate. DHA lowers blood pressure in anesthetized wild-type but not in Slo1 knockout mice. DHA ethyl ester, contained in dietary supplements, fails to activate BK channels and antagonizes the stimulatory effect of DHA. Slo1 BK channels are thus receptors for long-chain omega-3 fatty acids, and these fatty acids--unlike their ethyl ester derivatives--activate the channels and lower blood pressure. This finding has practical implications for the use of omega-3 fatty acids as nutraceuticals for the general public and also for the critically ill receiving omega-3-enriched formulas.

Mechanism of the modulation of BK potassium channel complexes with different auxiliary subunit compositions by the omega-3 fatty acid DHA.

Large-conductance Ca(2+)- and voltage-activated K(+) (BK) channels are well known for their functional versatility, which is bestowed in part by their rich modulatory repertoire. We recently showed that long-chain omega-3 polyunsaturated fatty acids such as docosahexaenoic acid (DHA) found in oily fish lower blood pressure by activating vascular BK channels made of Slo1+ß1 subunits. Here we examined the action of DHA on BK channels with different auxiliary subunit compositions. Neuronal Slo1+ß4 channels were just as well activated by DHA as vascular Slo1+ß1 channels. In contrast, the stimulatory effect of DHA was much smaller in Slo1+ß2, Slo1+LRRC26 (γ1), and Slo1 channels without auxiliary subunits. Mutagenesis of ß1, ß2, and ß4 showed that the large effect of DHA in Slo1+ß1 and Slo1+ß4 is conferred by the presence of two residues, one in the N terminus and the other in the first transmembrane segment of the ß1 and ß4 subunits. Transfer of this amino acid pair from ß1 or ß4 to ß2 introduces a large response to DHA in Slo1+ß2. The presence of a pair of oppositely charged residues at the aforementioned positions in ß subunits is associated with a large response to DHA. The Slo1 auxiliary subunits are expressed in a highly tissue-dependent fashion. Thus, the subunit composition-dependent stimulation by DHA demonstrates that BK channels are effectors of omega-3 fatty acids with marked tissue specificity.