TMEM16F/ANO6, a Ca²⁺-activated anion channel, is negatively regulated by the actin cytoskeleton and intracellular MgATP

Lin, Haiyue,Roh, Jaewon,Woo, Joo Han,Kim, Sung Joon,Nam, Joo Hyun Elsevier 2018 Biochemical and biophysical research communication Vol.503 No.4

<P><B>Abstract</B></P> <P>Anoctamin 6 (ANO6/TMEM16F) is a recently identified membrane protein that has both phospholipid scramblase activity and anion channel function activated by relatively high [Ca<SUP>2+</SUP>]<SUB>i</SUB>. In addition to the low sensitivity to Ca<SUP>2+</SUP>, the activation of ANO6 Cl<SUP>−</SUP> conductance is very slow (>3–5 min to reach peak level at 10 μM [Ca<SUP>2+</SUP>]<SUB>i</SUB>), with subsequent inactivation. In a whole-cell patch clamp recording of ANO6 current (I<SUB>ANO6,w-c</SUB>), disruption of the actin cytoskeleton with cytochalasin-D (cytoD) significantly accelerated the activation kinetics, while actin filament-stabilizing agents (phalloidin and jasplakinolide) commonly inhibited I<SUB>ANO6,w-c</SUB>. Inside-out patch clamp recording of ANO6 (I<SUB>ANO6,i-o</SUB>) showed immediate activation by raising [Ca<SUP>2+</SUP>]<SUB>i</SUB>. We also found that intracellular ATP (3 mM MgATP in pipette solution) decelerated the activation of I<SUB>ANO6,w-c</SUB>, and also prevented the inactivation of I<SUB>ANO6,w-c</SUB>. However, the addition of cytoD still accelerated both activation and inactivation of I<SUB>ANO6,w-c</SUB>. We conclude that the actin cytoskeleton and intracellular ATP play major roles in the Ca<SUP>2+</SUP>-dependent activation and inactivation of I<SUB>ANO6,w-c</SUB>, respectively.</P> <P><B>Highlights</B></P> <P> <UL> <LI> ANO6 current shows sluggish activation even with high [Ca<SUP>2+</SUP>]<SUB>i</SUB>, and inactivation. </LI> <LI> Actin cytoskeleton disruption accelerate both activation and inactivation of ANO6. </LI> <LI> Intracellular MgATP further delays ANO6 activation, and prevent inactivation. </LI> <LI> The effects of MgATP on ANO6 were overcome by actin inhibitor, cytochalasin-D. </LI> <LI> State actin cytoskeleton is crucial for determining ANO6 activity in intact cells. </LI> </UL> </P>

Enhancement of 5-HT2A receptor function and blockade of Kv1.5 by MK801 and ketamine: implications for PCP derivativeinduced disease models

Haiyue Lin,김재곤,박상웅,노현주,김정민,윤창용,우남식,김보경,조성일,최복희,성동준,배영민 생화학분자생물학회 2018 Experimental and molecular medicine Vol.50 No.-

MK801 and ketamine, which are phencyclidine (PCP) derivative N-methyl-d-aspartate receptor (NMDAr) blockers, reportedly enhance the function of 5-hydroxytryptamine (HT)-2A receptors (5-HT2ARs). Both are believed to directly affect the pathogenesis of schizophrenia, as well as hypertension. 5-HT2AR signaling involves the inhibition of Kv conductance. This study investigated the interaction of these drugs with Kv1.5, which plays important roles in 5-HT2AR signaling and in regulating the excitability of the cardiovascular and nervous system, and the potential role of this interaction in the enhancement of the 5-HT2AR-mediated response. Using isometric organ bath experiments with arterial rings and conventional whole-cell patch-clamp recording of Chinese hamster ovary (CHO) cells ectopically overexpressing Kv1.5, we examined the effect of ketamine and MK801 on 5-HT2AR-mediated vasocontraction and Kv1.5 channels. Both ketamine and MK801 potentiated 5-HT2AR-mediated vasocontraction. This potentiation of 5- HT2AR function occurred in a membrane potential-dependent manner, indicating the involvement of ion channel(s). Both ketamine and MK801 rapidly and directly inhibited Kv1.5 channels from the extracellular side independently of NMDArs. The potencies of MK801 in facilitating the 5-HT2AR-mediated response and blocking Kv1.5 were higher than those of ketamine. Our data demonstrated the direct inhibition of Kv1.5 channels by MK801/ketamine and indicated that this inhibition may potentiate the functions of 5-HT2ARs. We suggest that 5-HT2AR-Kv1.5 may serve as a receptoreffector module in response to 5-HT and is a promising target in the pathogenesis of MK801-/ketamine-induced disease states such as hypertension and schizophrenia.

Increased Expression of miR-146a in Children With Allergic Rhinitis After Allergen-Specific Immunotherapy

Xi Luo,Haiyu Hong,Jun Tang,Xingmei Wu,Zhibin Lin,Renqiang Ma,Yunping Fan,Geng Xu,Dabo Liu,Huabin Li 대한천식알레르기학회 2016 Allergy, Asthma & Immunology Research Vol.8 No.2

Purpose: MicroRNAs (miRs) were recently recognized to be important for immune cell differentiation and immune regulation. However, whether miRs were involved in allergen-specific immunotherapy (SIT) remains largely unknown. This study sought to examine changes in miR-146a and T regulatory cells in children with persistent allergic rhinitis (AR) after 3 months of subcutaneous immunotherapy (SCIT) and sublingual immunotherapy (SLIT). Methods: Twenty-four HDM-sensitized children with persistent AR were enrolled and treated with SCIT (n=13) or SLIT (n=11) for 3 months. Relative miR-146a and Foxp3 mRNA expression, the TRAF6 protein level, and the ratio of post-treatment to baseline IL-10+CD4+ T cells between the SCIT and SLIT groups were examined in the peripheral blood mononuclear cells (PBMCs) of AR patients using quantitative reverse transcription polymerase chain reaction (qRT-PCR), flow cytometry, and Western blot analysis, respectively. Serum levels of IL-5 and IL-10 were determined using ELISA. Results: After 3 months of SIT, both the TNSS and INSS scores were significantly decreased compared to the baseline value (P<0.01). The relative expression of miR-146a and Foxp3 mRNA was significantly increased after both SCIT and SLIT (P<0.01). The ratio of post-treatment to baseline IL-10+CD4+ T cells and the serum IL-10 level were significantly increased in both the SCIT and SLIT groups (P<0.01), whereas the TRAF6 protein level and serum IL-5 level were significantly decreased (P<0.01). No significant differences in these biomarkers were observed between the SCIT and SLIT groups. Conclusions: Our findings suggest that miR-146a and its related biomarkers may be comparably modulated after both SCIT and SLIT, highlighting miR-146a as a potential therapeutic target for the improved management of AR.

Rare KCNQ4 variants found in public databases underlie impaired channel activity that may contribute to hearing impairment

Jinsei Jung,Haiyue Lin,Young Ik Koh,Kunhi Ryu,Joon Suk Lee,John Hoon Rim,Hye Ji Choi,Hak Joon Lee,Hye-Youn Kim,Seyoung Yu,Hyunsoo Jin,Ji Hyun Lee,Min Goo Lee,Wan Namkung,Jae Young Choi,Heon Yung Gee 생화학분자생물학회 2019 Experimental and molecular medicine Vol.51 No.-

KCNQ4 is frequently mutated in autosomal dominant non-syndromic hearing loss (NSHL), a typically late-onset, initially high-frequency loss that progresses over time (DFNA2). Most KCNQ4 mutations linked to hearing loss are clustered around the pore region of the protein and lead to loss of KCNQ4-mediated potassium currents. To understand the contribution of KCNQ4 variants to NSHL, we surveyed public databases and found 17 loss-of-function and six missense KCNQ4 variants affecting amino acids around the pore region. The missense variants have not been reported as pathogenic and are present at a low frequency (minor allele frequency < 0.0005) in the population. We examined the functional impact of these variants, which, interestingly, induced a reduction in potassium channel activity without altering expression or trafficking of the channel protein, being functionally similar to DFNA2-associated KCNQ4 mutations. Therefore, these variants may be risk factors for late-onset hearing loss, and individuals harboring any one of these variants may develop hearing loss during adulthood. Reduced channel activity could be rescued by KCNQ activators, suggesting the possibility of medical intervention. These findings indicate that KCNQ4 variants may contribute more to late-onset NSHL than expected, and therefore, genetic screening for this gene is important for the prevention and treatment of NSHL.

Tetrahydrobiopterin Inhibits PDGF-stimulated Migration and Proliferation in Rat Aortic Smooth Muscle Cells via the Nitric Oxide Synthase-independent Pathway

Xiaowen Jiang,김보경,Haiyue Lin,이창권,김정완,강현,이필영,원경종,정성호,이환명 대한약리학회 2010 The Korean Journal of Physiology & Pharmacology Vol.14 No.3

Tetrahydrobiopterin (BH4), an essential cofactor for nitric oxide synthase (NOS) activity, is known to play important roles in modulating both NO and superoxide production during vascular diseases such as atherosclerosis. However, the role of BH4 in functions of vascular smooth muscle cells is not fully known. In this study, we tested the effects of BH4 and dihydrobiopterin (BH2), a BH4 precursor, on migration and proliferation in response to platelet-derived growth factor-BB (PDGF-BB) in rat aortic smooth muscle cells (RASMCs). Cell migration and proliferation were measured using a Boyden chamber and a 5-bromo-2'-deoxyuridine incorporation assay, respectively, and these results were confirmed with an ex vivo aortic sprout assay. Cell viability was examined by 2,3-bis [2-methoxy- 4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide assays. BH4 and BH2 decreased PDGF-BB- induced cell migration and proliferation in a dose-dependent manner. The inhibition of cell migration and proliferation by BH4 and BH2 was not affected by pretreatment with NG-nitro-L-arginine methyl ester, a NOS inhibitor. Moreover, the sprout outgrowth formation of aortic rings induced by PDGF-BB was inhibited by BH4 and BH2. Cell viability was not inhibited by BH4 and BH2 treatment. The present results suggest that BH4 and BH2 may inhibit PDGF-stimulated RASMC migration and proliferation via the NOS-independent pathway. Therefore, BH4 and its derivative could be useful for the development of a candidate molecule with an NO-independent anti-atherosclerotic function.

HIF-1α–Mediated Upregulation of TASK-2 K⁺ Channels Augments Ca²⁺ Signaling in Mouse B Cells under Hypoxia

Shin, Dong Hoon,Lin, Haiyue,Zheng, Haifeng,Kim, Kyung Su,Kim, Jin Young,Chun, Yang Sook,Park, Jong Wan,Nam, Joo Hyun,Kim, Woo Kyung,Zhang, Yin Hua,Kim, Sung Joon American Association of Immunologists 2014 Journal of Immunology Vol. No.

<P>The general consensus is that immune cells are exposed to physiological hypoxia in vivo (PhyO(2),2-5% P-O2). However, functional studies of B cells in hypoxic conditions are sparse. Recently, we reported the expression in mouse B cells of TASK-2, a member of pH-sensitive two-pore domain K+ channels with background activity. In this study, we investigated the response of K+ channels to sustained PhyO(2) (sustained hypoxia [SH], 3% P-O2 for 24 h) in WEHI-231 mouse B cells. SH induced voltage-independent background K+ conductance (SH-K-bg) and hyperpolarized the membrane potential. The pH sensitivity and the single-channel conductance of SH-K-bg were consistent with those of TASK-2. Immunoblotting assay results showed that SH significantly increased plasma membrane expressions of TASK-2. Conversely, SH failed to induce any current following small interfering (si)TASK-2 transfection. Similar hypoxic upregulation of TASK-2 was also observed in splenic primary B cells. Mechanistically, upregulation of TASK-2 by SH was prevented by si hypoxia-inducible factor-1 alpha (HIF-1 alpha) transfection or by YC-1, a pharmacological HIF-la inhibitor. In addition, TASK-2 current was increased in WEHI-231 cells overexpressed with 02-resistant HIF-1 alpha. Importantly, [Ca2+](c) increment in response to BCR stimulation was significantly higher in SH-exposed B cells, which was abolished by high K+-induced depolarization or by siTASK-2 transfection. The data demonstrate that TASK-2 is upregulated under hypoxia via HIF-1 alpha dependent manner in B cells. This is functionally important in maintaining the negative membrane potential and providing electrical driving force to control Ca2+ influx.</P>

Tetrahydrobiopterin Inhibits PDGF-stimulated Migration and Proliferation in Rat Aortic Smooth Muscle Cells via the Nitric Oxide Synthase-independent Pathway

Jiang, Xiaowen,Kim, Bo-Kyung,Lin, Haiyue,Lee, Chang-Kwon,Kim, Jung-Hwan,Kang, Hyun,Lee, Pil-Young,Jung, Seung-Hyo,Lee, Hwan-Myung,Won, Kyung-Jong The Korean Society of Pharmacology 2010 The Korean Journal of Physiology & Pharmacology Vol.14 No.3

Tetrahydrobiopterin (BH4), an essential cofactor for nitric oxide synthase (NOS) activity, is known to play important roles in modulating both NO and superoxide production during vascular diseases such as atherosclerosis. However, the role of BH4 in functions of vascular smooth muscle cells is not fully known. In this study, we tested the effects of BH4 and dihydrobiopterin (BH2), a BH4 precursor, on migration and proliferation in response to platelet-derived growth factor-BB (PDGF-BB) in rat aortic smooth muscle cells (RASMCs). Cell migration and proliferation were measured using a Boyden chamber and a 5-bromo-2'-deoxyuridine incorporation assay, respectively, and these results were confirmed with an ex vivo aortic sprout assay. Cell viability was examined by 2,3-bis [2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide assays. BH4 and BH2 decreased PDGF-BBinduced cell migration and proliferation in a dose-dependent manner. The inhibition of cell migration and proliferation by BH4 and BH2 was not affected by pretreatment with $N^G$-nitro-L-arginine methyl ester, a NOS inhibitor. Moreover, the sprout outgrowth formation of aortic rings induced by PDGF-BB was inhibited by BH4 and BH2. Cell viability was not inhibited by BH4 and BH2 treatment. The present results suggest that BH4 and BH2 may inhibit PDGF-stimulated RASMC migration and proliferation via the NOS-independent pathway. Therefore, BH4 and its derivative could be useful for the development of a candidate molecule with an NO-independent anti-atherosclerotic function.

China Consensus Document on Allergy Diagnostics

Chen Hao,Li Jing,Cheng Lei,Gao Zhongshan,Lin Xiaoping,Zhu Rongfei,Yang Lin,Tao Ailin,Hong Haiyu,Tang Wei,Guo Yinshi,Huang Huaiqiu,Sun Jinlyu,Lai He,Lei Cheng,Liu Guanghui,Xiang Li,Chen Zhuanggui,Ma Ha 대한천식알레르기학회 2021 Allergy, Asthma & Immunology Research Vol.13 No.2

The prevalence of allergic diseases has increased dramatically in recent years in China, affecting the quality of life in 40% of the population. The identification of allergens is the key to the diagnosis of allergic diseases. Presently, several methods of allergy diagnostics are available in China, but they have not been standardized. Additionally, cross-sensitization and co-sensitization make allergy diagnostics even more complicated. Based on 4 aspects of allergic disease (mechanism, diagnosis procedures, allergen detection in vivo and in vitro as well as the distribution map of the most important airborne allergens in China) and by referring to the consensus of the European Society of Allergy and Clinical Immunology, the World Allergy Organization, and the important literature on allergy diagnostics in China in recent years, we drafted this consensus of allergy diagnostics with Chinese characteristics. It aims to standardize the diagnostic methods of allergens and provides a reference for health care givers. The current document was prepared by a panel of experts from the main stream of professional allergy associations in China.