胞外酸化经ASIC1/RIP1途径抑制TFEB介导的巨噬细胞脂噬

目的 探讨胞外酸化对巨噬细胞脂噬的影响及其作用机制。方法 采用RAW264.7巨噬细胞,以pH 6.5培养液与 25 mg/L氧化低密度脂蛋白（ox-LDL）共孵育24 h构建胞外酸化诱导的泡沫细胞模型。分别以ASIC1特异性阻断剂PcTx-1和RIP1抑制剂Nec-1干预胞外酸化诱导的RAW264.7巨噬细胞24 h,油红O染色检测细胞内脂质蓄积;蛋白质印迹（Western blot）检测总ASIC1、膜ASIC1、p-RIP1 Ser166、p-TFEB Ser142、LC3和p62蛋白的表达;激光共聚焦显微镜观察脂滴（Bodipy示踪）与自噬标志物LC3II和LAMP1共定位;透射电镜观察细胞内脂滴和脂噬泡的数量变化;胆固醇荧光试剂盒检测ABCA1介导的胆固醇流出。结果 与pH 7.4组相比较,pH 6.5胞外酸化组胞内的脂质蓄积和细胞质膜上的ASIC1蛋白表达显著增加,p-RIP1Ser166、p-TFEB Ser142水平升高,LC3II蛋白减少和p62蛋白增加,脂滴与LC3II和LAMP1的共定位都分别减少,细胞内的脂滴数量显著增加,自噬体和脂噬泡的数量则明显减少,ABCA1介导的巨噬细胞内胆固醇流出显著减少。然而,胞外酸化对RAW264.7巨噬细胞的上述效应能被ASIC1特异性阻断剂PcTx-1和RIP1抑制剂Nec-1所取消。结论 胞外酸化经激活ASIC1/RIP1途径促进TFEB磷酸化抑制巨噬细胞脂噬,ASIC1可能是防治动脉粥样硬化等脂质蓄积疾病的新靶点。     

Leu85 in the β1-β2 Linker of ASIC1 Slows Activation and Decreases the Apparent Proton Affinity by Stabilizing a Closed Conformation

Acid-sensing ion channels (ASICs) are proton-activated channels expressed in neurons of the central and peripheral nervous systems where they modulate neuronal activity in response to external increases in proton concentration. The size of ASIC1 currents evoked by a given local acidification is determined by the number of channels in the plasma membrane and by the apparent proton affinities for activation and steady-state desensitization of the channel. Thus, the magnitude of the pH drop and the value of the baseline pH both are functionally important. Recent characterization of ASIC1s from an increasing number of species has made evident that proton affinities of these channels vary across vertebrates. We found that in species with high baseline plasma pH, e.g. frog, shark, and fish, ASIC1 has high proton affinity compared with the mammalian channel. The β1-β2 linker in the extracellular domain, specifically by the substitution M85L, determines the interspecies differences in proton affinities and also the time course of ASIC1 macroscopic currents. The mechanism underlying these observations is a delay in channel opening after application of protons, most likely by stabilizing a closed conformation that decreases the apparent affinity to protons and also slows the rise and decay phases of the current. Together, the results suggest evolutionary adaptation of ASIC1 to match the value of the species-specific plasma pH. At the molecular level, adaptation is achieved by substitutions of nonionizable residues rather than by modification of the channel proton sensor.     

Proteolytic Cleavage of Human Acid-sensing Ion Channel 1 by the Serine Protease Matriptase

Acid-sensing ion channel 1 (ASIC1) is a H⁺-gated channel of the amiloride-sensitive epithelial Na⁺ channel (ENaC)/degenerin family. ASIC1 is expressed mostly in the central and peripheral nervous system neurons. ENaC and ASIC function is regulated by several serine proteases. The type II transmembrane serine protease matriptase activates the prototypical αβγENaC channel, but we found that matriptase is expressed in glioma cells and its expression is higher in glioma compared with normal astrocytes. Therefore, the goal of this study was to test the hypothesis that matriptase regulates ASIC1 function. Matriptase decreased the acid-activated ASIC1 current as measured by two-electrode voltage clamp in Xenopus oocytes and cleaved ASIC1 expressed in oocytes or CHO K1 cells. Inactive S805A matriptase had no effect on either the current or the cleavage of ASIC1. The effect of matriptase on ASIC1 was specific, because it did not affect the function of ASIC2 and no matriptase-specific ASIC2 fragments were detected in oocytes or in CHO cells. Three matriptase recognition sites were identified in ASIC1 (Arg-145, Lys-185, and Lys-384). Site-directed mutagenesis of these sites prevented matriptase cleavage of ASIC1. Our results show that matriptase is expressed in glioma cells and that matriptase specifically cleaves ASIC1 in heterologous expression systems.     

Solution structure of APETx2, a specific peptide inhibitor of ASIC3 proton-gated channels

Acid-sensing ion channels (ASIC) are proton-gated sodium channels that have been implicated in pain transduction associated with acidosis in inflamed or ischemic tissues. APETx2, a peptide toxin effector of ASIC3, has been purified from an extract of the sea anemone Anthopleura elegantissima. APETx2 is a 42-amino-acid peptide cross-linked by three disulfide bridges. Its three-dimensional structure, as determined by conventional two-dimensional 1H-NMR, consists of a compact disulfide-bonded core composed of a four-stranded beta-sheet. It belongs to the disulfide-rich all-beta structural family encompassing peptide toxins commonly found in animal venoms. The structural characteristics of APETx2 are compared with that of PcTx1, another effector of ASIC channels but specific to the ASIC1a subtype and to APETx1, a toxin structurally related to APETx2, which targets the HERG potassium channel. Structural comparisons, coupled with the analysis of the electrostatic characteristics of these various ion channel effectors, led us to suggest a putative channel interaction surface for APETx2, encompassing its N terminus together with the type I-beta turn connecting beta-strands III and IV. This basic surface (R31 and R17) is also rich in aromatic residues (Y16, F15, Y32, and F33). An additional region made of the type II'-beta turn connecting beta-strands I and II could also play a role in the specificity observed for these different ion effectors.     

A new sea anemone peptide, APETx2, inhibits ASIC3, a major acid-sensitive channel in sensory neurons

From a systematic screening of animal venoms, we isolated a new toxin (APETx2) from the sea anemone Anthopleura elegantissima, which inhibits ASIC3 homomeric channels and ASIC3-containing heteromeric channels both in heterologous expression systems and in primary cultures of rat sensory neurons. APETx2 is a 42 amino-acid peptide crosslinked by three disulfide bridges, with a structural organization similar to that of other sea anemone toxins that inhibit voltage-sensitive Na+ and K+ channels. APETx2 reversibly inhibits rat ASIC3 (IC50=63 nM), without any effect on ASIC1a, ASIC1b, and ASIC2a. APETx2 directly inhibits the ASIC3 channel by acting at its external side, and it does not modify the channel unitary conductance. APETx2 also inhibits heteromeric ASIC2b+3 current (IC50=117 nM), while it has less affinity for ASIC1b+3 (IC50=0.9 microM), ASIC1a+3 (IC50=2 microM), and no effect on the ASIC2a+3 current. The ASIC3-like current in primary cultured sensory neurons is partly and reversibly inhibited by APETx2 with an IC50 of 216 nM, probably due to the mixed inhibitions of various co-expressed ASIC3-containing channels.     

酸感受性离子通道生物学特性及其调控

酸感受离子通道(ASICs)为H -门控的阳离子通道,是一类新的配体门控性离子通道,属于钠通道超家族的新成员.作为近来研究的热点,ASICs具有许多重要的生物学功能,并很有可能成为抗癫痫、镇痛、提高学习记忆能力和保护神经元缺血损伤作用药理学新靶点.近来,ASICs各个亚基已被克隆,它们在生物体内分布、表达、功能和相关调节因素的研究正受到广泛重视.     

Intracellular ASIC1a regulates mitochondrial permeability transition-dependent neuronal death

Acid-sensing ion channel 1a (ASIC1a) is the key proton receptor in nervous systems, mediating acidosis-induced neuronal injury in many neurological disorders, such as ischemic stroke. Up to now, functional ASIC1a has been found exclusively on the plasma membrane. Here, we show that ASIC1a proteins are also present in mitochondria of mouse cortical neurons where they are physically associated with adenine nucleotide translocase. Moreover, purified mitochondria from ASIC1a^−/− mice exhibit significantly enhanced Ca²⁺ retention capacity and accelerated Ca²⁺ uptake rate. When challenged with hydrogen peroxide (H₂O₂), ASIC1a^−/− neurons are resistant to cytochrome c release and inner mitochondrial membrane depolarization, suggesting an impairment of mitochondrial permeability transition (MPT) due to ASIC1a deletion. Consistently, H₂O₂-induced neuronal death, which is MPT dependent, is reduced in ASIC1a^−/− neurons. Additionally, significant increases in mitochondrial size and oxidative stress levels are detected in ASIC1a^−/− mouse brain, which also displays marked changes (>2-fold) in the expression of mitochondrial proteins closely related to reactive oxygen species signal pathways, as revealed by two-dimensional difference gel electrophoresis followed by mass spectrometry analysis. Our data suggest that mitochondrial ASIC1a may serve as an important regulator of MPT pores, which contributes to oxidative neuronal cell death.     

The bile acid-sensitive ion channel (BASIC), the ignored cousin of ASICs and ENaC

The DEG/ENaC gene family of ion channels is characterized by a high degree of structural similarity and an equally high degree of diversity concerning the physiological function. In humans and rodents, the DEG/ENaC family comprises 2 main subgroups: the subunits of the epithelial Na⁺ channel (ENaC) and the subunits of the acid sensing ion channels (ASICs). The bile acid-sensitive channel (BASIC), previously known as BLINaC or INaC, represents a third subgroup within the DEG/ENaC family. Although BASIC was identified more than a decade ago, very little is known about its physiological function. Recent progress in the characterization of this neglected member of the DEG/ENaC family, which is summarized in this focused review, includes the discovery of surprising species differences, its pharmacological characterization, and the identification of bile acids as putative natural activators.     

Acid-induced modulation of airway basal tone and contractility: role of acid-sensing ion channels (ASICs) and TRPV1 receptor

The role of extracellular acidosis in inflammatory airway diseases is not well known. One consequence of tissue acidification is the stimulation of sensory nerves via the polymodal H(+)-gated transmembrane channels ASICs and TRPV1 receptor. The present study investigated the effect of acidosis on airway basal tone and responsiveness in the guinea pig. Acidosis (pH 6.8, 10 min, 37 degrees C) significantly decreased the basal tone of tracheal rings (p<0.01 vs. paired control). Moreover, pH fall raised the maximal contraction of tracheal rings to acetylcholine (p<0.05 vs. paired control). The pH-induced relaxation of airway basal tone was inhibited by pretreatments with ASIC1a or ASIC3/ASIC2a inhibitors (0.5 mM ibuprofen, 0.1 mM gadolinium), nitric oxide synthase inhibitor (1 mM L-NAME), and guanylate cyclase inhibitor (1 microM ODQ). In contrast, the pH-induced relaxation of airway basal tone was not modified by epithelium removal or pretreatments with a TRPV1 antagonist (1 microM capsazepine), a combination of NK(1,2,3) receptor antagonists (0.1 microM each), a blocker of voltage-sensitive Na(+) channels (1 microM tetrodotoxin), a cyclooxygenase inhibitor with no activity on ASICs (1 microM indomethacin) or ASIC3 and ASIC3/ASIC2b inhibitors (10 nM diclofenac, 1 microM aspirin). Furthermore, acid-induced hyperresponsiveness to acetylcholine was inhibited by epithelium removal, capsazepine, NK(1,2,3) receptor antagonists, tetrodotoxin, amiloride, ibuprofen and diclofenac. In summary, the initial pH-induced airway relaxation seems to be independent of sensory nerves, suggesting a regulation of airway basal tone mediated by smooth muscle ASICs. Conversely, the pH-induced hyperresponsiveness involves sensory nerves-dependent ASICs and TRPV1, and an unknown epithelial component in response to acidosis.