微生物学报  2022, Vol. 62 Issue (9): 3529-3541   DOI: 10.13343/j.cnki.wsxb.20220039.
http://dx.doi.org/10.13343/j.cnki.wsxb.20220039
中国科学院微生物研究所,中国微生物学会

文章信息

王晓敏, 张怡, 吕梦丹, 王志坚, 应瑾瑶, 殷乐依, 吴宇涛, 刘胜兵, 敖雷, 郑永霞, 徐营, 潘巍巍, 李月舟. 2022
WANG Xiaomin, ZHANG Yi, LV Mengdan, WANG Zhijian, YING Jinyao, YIN Leyi, WU Yutao, LIU Shengbing, AO Lei, ZHENG Yongxia, XU Ying, PAN Weiwei, LI Yuezhou.
大肠杆菌机械敏感性离子通道MscS失活特性分析
Analysis of inactivation characteristics of Escherichia coli mechanosensitive ion channel MscS
微生物学报, 62(9): 3529-3541
Acta Microbiologica Sinica, 62(9): 3529-3541

文章历史

收稿日期:2022-01-21
修回日期:2022-04-08
网络出版日期:2022-05-26
大肠杆菌机械敏感性离子通道MscS失活特性分析
王晓敏1 #, 张怡1 #, 吕梦丹1 #, 王志坚1 , 应瑾瑶1 , 殷乐依1 , 吴宇涛1 , 刘胜兵1 , 敖雷1 , 郑永霞1 , 徐营1 , 潘巍巍1 , 李月舟2     
1. 嘉兴学院医学院, 浙江 嘉兴 314000;
2. 浙江大学医学院附属儿童医院, 浙江 杭州 310000
摘要[目的] 细菌机械敏感性离子通道MscS能够在细菌周围环境渗透压急剧降低时,打开并释放胞内内容物,平衡内外渗透压差,使细菌存活。鉴于其广泛分布在各种细菌中,而在哺乳动物中未发现其同源体,MscS被认为是一种新型抗生素靶点。MscS一个独特的开放特征是具有失活特性,即在持续的机械刺激条件下,MscS从开放状态进入一种非离子通透的失活状态,从而避免因通道持续开放引起大量内容物流失导致细菌死亡。该研究的目的是鉴定影响MscS失活的关键氨基酸,为靶向MscS的药物设计提供思路。[方法] 采用分子克隆方法制备MscS Cyto-helix(P166−I170)半胱氨酸突变体,利用巯基化合物MTSET+结合半胱氨酸从而对其侧链基团进行修饰,并通过低渗刺激实验,检测表达MscS半胱氨酸突变体的大肠杆菌分别在无或有MTSET+处理下,低渗刺激诱发通道开放后的存活率筛选显著影响通道功能的突变体。利用电生理膜片钳方法检测突变体在MTSET+处理前后通道失活特性的变化,结合定点突变手段进一步探讨失活机制。[结果] MTSET+处理导致表达半胱氨酸突变体G168C-MscS的大肠杆菌在低渗刺激后存活率极大降低;G168C- MscS在结合MTSET+后失去失活特性,保持持续开放,是导致细菌胞内内容物大量流失并死亡的重要原因;酪氨酸突变G168Y-MscS、亮氨酸突变G168L-MscS和赖氨酸突变G168K-MscS的失活特性与野生型WT-MscS一致,而天冬氨酸突变G168D、缬氨酸突变G168V和异亮氨酸突变G168I的失活速率显著降低,尤其是G168I-MscS失去失活特性,表明MscS 168位点是影响通道失活的关键位点,并且通道失活特性与该位点氨基酸侧链基团的大小及电荷性质相关。[结论] G168位点甘氨酸是影响MscS通道失活的关键氨基酸。
关键词机械敏感性离子通道    MscS    大肠杆菌    低渗刺激    膜片钳    
Analysis of inactivation characteristics of Escherichia coli mechanosensitive ion channel MscS
WANG Xiaomin1 #, ZHANG Yi1 #, LV Mengdan1 #, WANG Zhijian1 , YING Jinyao1 , YIN Leyi1 , WU Yutao1 , LIU Shengbing1 , AO Lei1 , ZHENG Yongxia1 , XU Ying1 , PAN Weiwei1 , LI Yuezhou2     
1. College of Medicine, Jiaxing University, Jiaxing 314000, Zhejiang, China;
2. The Children's Hospital, Zhejiang University School of Medicine, Hangzhou 310000, Zhejiang, China
Abstract: [Objective] The mechanosensitive channel of small conductance (MscS) in bacteria releases solutes and water when a hypo-osmotic shock raises the pressure in the cells, thereby enabling the survival of bacteria. Given its wide distribution in various bacteria and no homologues found in mammals, MscS is considered a novel antibiotic target. A hallmark of MscS is that it enters a tension-insensitive inactivated state upon prolonged mechanical stimulation, thereby avoiding the loss of a large amount of cell content and preventing cell death. This study aims to identify the key residues related to the inactivation of MscS, which is expected to serve as a reference for the development of MscS-targeting drugs. [Methods] The cysteine mutants of MscS Cyto-helix (P166−I170) were prepared with molecular cloning method. The thiol compound MTSET+ binds to cysteine and thus modify cysteine's side chain group. In this study, osmotic downshock assay was used to examine the viability of Escherichia coli expressing cysteine mutants of MscS Cyto-helix (P166−I170) upon hypotonic stimulation without or with MTSET+ treatment and screened for cysteine mutant that significantly affected the channel function. The inactivation of MscS mutants before and after MTSET+ treatment was examined by electrophysiological experiments. In addition, the inactivation mechanism of MscS was further explored by eletrophysiology combined with site-directed mutagenesis. [Results] MTSET+ led to a great decrease in the survival rate of E. coli expressing G168C-MscS upon hypotonic stimulation. G168C-MscS lost its inactivation property after binding to MTSET+ and remained open, resulting in great loss of intracellular contents and bacterial death. The inactivation properties of G168Y-MscS, G168L-MscS, and G168K-MscS mutants were consistent with WT-MscS, while the inactivation rates of the three mutants G168D, G168V, and G168I were significantly reduced, especially G168I-MscS which lost the inactivation properties. Therefore, MscS G168 affected channel inactivation, and the channel inactivation characteristics were related to the size and charge of the residue side chain group at 168 site. [Conclusion] MscS G168 is a key residue that affects the inactivation of MscS.
Keywords: mechanosensitive ion channel    MscS    Escherichia coli    osmotic downshock    patch clamp    

对机体内外机械刺激的感知和应答是生命体的重要能力。从细菌分裂,植物生长,到高等动物触觉、听觉、平衡等感觉的产生,机械响应涉及众多的生理过程[1]。机械敏感性离子通道作为一类特殊的膜蛋白,通过通道分子构象的变化将机械刺激转化为化学信号或生物电信号,在机械响应中起着重要作用[2]

最早发现的是细菌中的机械敏感性离子通道,包括大电导机械敏感性离子通道(mechanosensitive channel of large conductance,MscL)和小电导机械敏感性离子通道(mechanosensitive channel of small conductance,MscS)。它们主要作为细胞的“紧急释放阀门”,能够在周围环境渗透压急剧降低导致细菌体积膨胀时,感受到膜张力的变化、打开并释放内容物,从而平衡细胞内外渗透压差,防止细菌因过度膨胀而破裂死亡[3]。MscL和MscS高度保守,广泛分布在细菌中,包括许多致病菌株,如结核分枝杆菌[4]、金黄色葡萄球菌[5]、淋病奈瑟氏球菌[6]等,在真菌、植物中也存在MscS的同源物,然而在高等哺乳动物中未发现其家族成员[7]。多项研究表明MscL和MscS可作为一类新型抗生素药物靶点[8]。近来研究人员采用计算机分析方法设计了一种靶向金黄色葡萄球菌MscL的抗菌剂,并且该抗菌剂在秀丽隐杆线虫的感染模型中表现出抗感染功效[9]。Wang等研究显示MscS对于淋病奈瑟菌在小鼠阴道感染模型中的定殖和存活很重要,指出MscS有可能成为治疗淋病的一种药物靶点[6]。因此研究MscL和MscS结构和功能的关系将有助于理解影响通道开放特性的关键结构域或氨基酸,从而为靶向药物的设计打下基础。

MscS开放时形成一个直径约为16 Å,电导约为1 nS的孔道,具有轻微的阴离子选择性(PCl-/PK+约为1.5–3)[1011]。区别于MscL,MscS一个独特的开放特征是具有失活特性,即在持续的机械刺激条件下,MscS从开放状态进入一种非离子通透的失活状态,不再响应机械刺激[12]。当在无机械刺激的“休息”后,通道能够从失活状态恢复,再次给予机械刺激时,通道再次开放。MscS的失活特性可能是维持细胞稳态的一种保护机制。细菌周围环境渗透压降低时,MscS感受到膜张力的变化开放,一方面将胞内内容物释放到胞外,平衡细胞内外渗透压;另一方面,大量内容物流出胞外,对细胞无疑是一种损耗,而且一些离子顺浓度梯度运输,比如H+,Na+从胞外流向胞内,导致细胞难以维持正常的膜电位,而MscS的失活特性避免了这些弊端,从而使细胞行驶正常的生理功能。研究表明,通过改变MscS关键区域氨基酸性质,将引起通道持续开放,内容物大量流失导致细胞死亡[1314]。这提示我们可以鉴定得到影响MscS失活的关键氨基酸,通过化合物修饰使得MscS失去失活特性引起细菌死亡。然而关于MscS失活特性的研究很少。

大肠杆菌野生型MscS X衍射晶体结构[15]显示MscS是同源七聚体,由跨膜结构域和胞质结构域两部分组成(图 1)。跨膜结构域含有3个跨膜α螺旋,TM1 (29−57)、TM2 (68−91)和TM3 (96−127)。TM3在113位甘氨酸(Gly113)处发生转折,分为TM3a (96−113)和TM3b (114−127)。TM3a形成孔道区域。TM3b位于膜脂质双分子层与胞质的交界处,与膜平面几乎平行,起着连接跨膜结构域和胞质结构域的作用。胞质区由两个结构域组成:β结构域(132−177)和C端结构域(188−280)。β结构域处存在一个小的α螺旋Cyto-helix (166−170),在空间位置上与TM3b相靠近。研究表明,Cyto-helix和TM3b在维持MscS通道功能中起着重要作用,位于MscS TM3b的G113和G121两个氨基酸影响通道的失活[12, 16]。另有研究表明将Cyto-helix与TM3b通过半胱氨酸二硫键交联引起通道进入一种非离子通透状态[17],提示Cyto-helix可能参与MscS的失活过程。然而Cyto-helix如何参与MscS失活过程并不清楚。

本研究中,为探究Cyto-helix是否影响MscS失活,我们将166−170位氨基酸逐个替换为半胱氨酸,进而利用巯基化合物MTSET+结合半胱氨酸侧链巯基基团对其带电性质和侧链结构进行进一步修饰,通过结合低渗实验和电生理实验鉴定得到了影响大肠杆菌MscS失活的关键氨基酸, 即位于Cyto-helix的第168位甘氨酸G168 (图 1)。该研究揭示了MscS胞质结构域Cyto-helix在通道失活过程中起重要作用,并为以MscS为靶点的抗生素药物的设计提供了思路。

图 1 大肠杆菌MscS晶体结构模式图[15](PDB ID: 2OAU) Figure 1 Schematic diagram of the crystal structure of Escherichia coli MscS[15] (PDB ID: 2OAU). A: side view of MscS heptamer. MscS is composed of transmembrane domain and cytoplasmic domain; B: side view of an individual MscS subunit. The transmembrane domain contains three membrane-spanning helices (TM1, TM2 and TM3). TM3 is divided into two helical segments (TM3a and TM3b) with a kink at G113. The Cyto-helix in the cytoplasmic region is colored in green. The position of G168 in Cyto-helix is colored in blue. The TM3b and Cyto-helix were enlarged in the box.

1 材料与方法 1.1 菌株,质粒,细菌培养

本研究所用菌株与质粒见表 1。大肠杆菌菌株MJF465 (Frag1, ΔmscL: : Cm, ΔmscS, ΔmscK: : Kan)用于携载质粒。DH10β感受态细胞用于克隆质粒。PB10b质粒载有大肠杆菌WT-MscS基因(PB10b-Ec-MscS)。所有菌株储存于冻存液(65% glycerol,0.1 mol/L MgSO4,0.025 mol/L Tris·Cl)中,−80 ℃冻存。细菌生长于新鲜LB培养液(5 g/L NaCl,5 g/L yeast extract,10 g/L trptone),并适当添加氨苄青霉素(100 µg/mL)。对于高渗培养基,需要在LB培养基中再添加1 mol/L NaCl。1 mmol/L异丙基硫代半乳糖苷(isopropyl β-d-thiogalactoside,IPTG)诱导通道表达。

表 1. 菌株与质粒 Table 1. Bacterial strains and plasmids
Strains/plasmids Features Source
Escherichia coli
MJF465 Frag1; ΔmscL: : CmΔmscSΔmscK: : Kan Our laboratory
DH10β Cloning host Our laboratory
Plasmids
PB10b-Ec-MscS Ampr; vector PB10b expressing Ec-mscS from an IPTG-inducible promotor Our laboratory
PB10b-Ec-MscS(P166C) Ampr; vector PB10b-Ec-mscS derivative containing a Ec-mscS(P166C) mutation This study
PB10b-Ec-MscS(N167C) Ampr; vector PB10b-Ec-mscS derivative containing a Ec-mscS(N167C) mutation This study
PB10b-Ec-MscS(G168C) Ampr; vector PB10b-Ec-mscS derivative containing a Ec-mscS(G168C) mutation This study
PB10b-Ec-MscS(K169C) Ampr; vector PB10b-Ec-mscS derivative containing a Ec-mscS(K169C) mutation This study
PB10b-Ec-MscS(I170C) Ampr; vector PB10b-Ec-mscS derivative containing a Ec-mscS(I170C) mutation This study

1.2 MscS突变体构建

本部分参照定点突变试剂盒步骤(Vazyme,#C214),根据突变位点设计相应上下游引物,以PB10b-Ec-MscS质粒为模板,PCR扩增获得线性化质粒DNA。采用Dpn I酶消化,去除甲基化模板质粒。利用重组酶将线性化质粒DNA环化,将重组产物转化进入感受态细胞DH10β,涂板,挑取单克隆,测序鉴定。用于制备E. coli MscS突变体的引物见表 2

表 2. 本研究中所用引物 Table 2. Primers used in this study
Primer Sequence (5ʹ→3ʹ)
Ec-MscS(P166C)-F ATTATCGTTATTtgcAACGGTAAAATTATTGCCGG
Ec-MscS(P166C)-R CCGGCAATAATTTTACCGTTgcaAATAACGATAAT
Ec-MscS(N167C)-F ATTATCGTTATTCCGtgcGGTAAAATTATTGCCGG
Ec-MscS(N167C)-R CCGGCAATAATTTTACCgcaCGGAATAACGATAAT
Ec-MscS(G168C)-F ATCGTTATTCCGAACtgcAAAATTATTGCCGG
Ec-MscS(G168C)-R CCGGCAATAATTTTgcaGTTCGGAATAACGAT
Ec-MscS(K169C)-F ATTCCGAACGGTtgcATTATTGCCGGAAATATT
Ec-MscS(K169C)-R AATATTTCCGGCAATAATgcaACCGTTCGGAAT
Ec-MscS(I170C)-F ATTCCGAACGGTAAAtgcATTGCCGGAAATATT
Ec-MscS(I170C)-R AATATTTCCGGCAATgcaTTTACCGTTCGGAAT

1.3 低渗刺激实验

挑取表达WT-MscS或MscS突变体的MJF465菌株至新鲜LB培养基中,加入100 µg/mL氨苄青霉素,37 ℃、250 r/min振荡培养过夜。将过夜培养的菌液按照1:100比例稀释至LB培养液中振荡培养。1 h后,加入等体积的含有1 mol/L NaCl的高渗培养基,继续振荡培养。当OD600至0.2时,加入IPTG至终浓度为1 mmol/L,培养1 h。将菌液按照1:10的比例分别稀释到含0.5 mol/L NaCl的LB培养基(等渗刺激)、含有1 mmol/L MTSTE+的0.5 mol/L NaCl的LB培养基(等渗刺激+MTSET+)、蒸馏水(低渗刺激)和含有1 mmol/L MTSTE+的蒸馏水(低渗刺激+1 mmol/L MTSTE+)中处理。20 min后,按照1:10比例将菌液分别进行梯度稀释6次。取2.5 µL菌液点到LB固体培养基平板上,37 ℃培养过夜。将低渗刺激下稀释104倍的菌落个数除以等渗刺激相对应稀释倍数下菌落个数,计算细菌存活率。

1.4 原生质球制备

过夜培养的菌液按1:100的比例稀释至LB液体培基中,37 ℃振荡培养。当OD600至0.2时,将菌液以1:10的比例稀释至LB液体培养基中,加入cephalexin至终浓度为60 µg/mL,继续培养1.5–2.0 h,然后加入IPTG至终浓度为1 mmol/L,诱导目的通道蛋白表达。40 min后,2 000 r/min,4 ℃离心10 min,去上清。加入0.8 mol/L蔗糖2.5 mL,轻轻摇晃,将沉淀悬浮。然后依次加入1 mol/L Tris•Cl 125 µL;5 mg/mL lysozyme 30 µL;5 mg/mL DNase 30 µL;125 mmol/L Na·EDTA 30 µL摇匀,计时共反应5 min。5 min后,加入1 mL终止反应液(0.8 mol/L蔗糖,20 mmol/L MgCl2,10 mmol/L Tris·Cl),轻轻混匀,并立即置于冰上,终止反应。将溶液慢慢滴到预冷的10 mL稀释液(0.8 mol/L蔗糖,10 mmol/L MgCl2,10 mmol/L Tris·Cl)的上层,1 800 r/min,4 ℃离心5 min。去上清,保留大约300 µL的上清液,将底部的原生质球沉淀轻轻悬浮起来,即可用于膜片钳记录。

1.5 电生理分析

本研究采用inside-out记录模式,电极内液和电极外液是同一种溶液(200 mmol/L KCl,90 mmol/L MgCl2,10 mmol/L CaCl2,5 mmol/L Hepes,pH 6.0)。取电极内液加入浴槽中,加入原生质球,轻轻混匀,置于显微镜下。选取状态好的细胞,电极靠近细胞,施加负压形成高阻封接。将电极及其钳制细胞稍微提起,轻拍防震工作台,除了电极尖端钳制的膜片外,原生质球上其他部分的膜片由于浴液的振动会从电极上脱落,形成inside-out recording模式。通过给予负压和电压,激活通道,使用AxoPatch 200B放大器和Clampex以20 kHz的采样率和5 kHz滤波器采集记录通道电流,用clampfit软件分析数据。

1.6 统计分析

所有实验均重复3次以上。使用GraphPad Prism软件(GraphPad 8.0)进行统计分析。使用Student’s two-tailed unpaired t test进行差异比较。显著性水平用星号表示,* P < 0.05,** P < 0.01和*** P < 0.001。

2 结果与分析 2.1 MTSET+导致表达G168C-MscS的细菌在低渗刺激时死亡

本研究将E. coli MscS Cyto-helix (166−170)上的氨基酸逐个突变为半胱氨酸(Cys,C),首先通过低渗刺激实验,检测这些半胱氨酸突变体是否具有正常生理功能。通过将生长于高渗培养基中的表达WT、P166C、N167C、G168C、K169C、I170C MscS的大肠杆菌分别置于高渗培养基(mock,等渗刺激)或蒸馏水(shock,低渗刺激)(图 2A),计算2种处理后细菌的存活率,结果显示表达WT、P166C、N167C、K169C、I170C MscS菌株在低渗刺激后的存活率与等渗刺激一致,而表达G168C-MscS菌株在低渗刺激后的存活率明显下降(图 2B2C),说明G168C-MscS影响了通道的开放特性,使得MscS不能行驶其正常功能。

图 2 MTSET+处理导致表达G168C-MscS的细菌在低渗刺激时死亡 Figure 2 MTSET+ treatment leads to the death of Escherichia coli strain MJF465 (ΔmscL, ΔmscS, ΔmscK) expressing G168C-MscS upon hypotonic stimulation. A: schematic diagram of osmotic downshock assay. MJF465 expressing wild-type MscS or MscS mutants was first cultured in hypertonic LB medium, and then placed into hypertonic LB (mock), hypertonic LB supplemented with 1 mmol/L MTSET+ (mock+MTSET+), H2O (shock), and H2O supplemented with 1 mmol/L MTSET+ (shock+MTSET+) respectively. Mock: isotonic treatment. Shock: hypotonic treatment. MTSET+: a sulfhydryl compound. B: growth of MJF465 strain expressing wild-type MscS or MscS mutants (P166C, N167C, K169C, I170C) upon mock, mock+MTSET+, shock, shock + MTSET+ respectively. The panels show the 2.5 µL drop-plates at each tenfold dilution (10−1 to 10−6) of cells. The growth of MJF465 expressing G168C-MscS upon shock+MTSTE+ was shown in red box. C: percentage viability of cells exposed to mock+MTSET+, shock, shock+MTSET+ relative to mock. Data are represented as mean±SEM. Significant differences were identified by Student's two-tailed unpaired t test, ***: P < 0.001.

此外利用巯基化合物MTSET+特异性结合半胱氨酸,且大肠杆菌WT-MscS本身不含半胱氨酸,只有突变位点为半胱氨酸的特性,通过检测表达WT-MscS和半胱氨酸突变体MscS大肠杆菌在MTSET+处理后低渗刺激时的存活率,反映突变位点在MTSET+修饰后通道功能的变化。当我们在等渗刺激或低渗刺激时同时用MTSET+化合物处理即mock+MTSET+,shock+MTSET+,发现表达WT、P166C、N167C、K169C、I170C MscS菌株在两种处理后的存活率与等渗刺激无显著性差异,说明MTSET+和这些位点的半胱氨酸结合后并不会引起MscS通道功能的显著变化。然而尽管表达G168C-MscS菌株在mock+MTSET+处理后的存活率与等渗刺激一致,却在shock+MTSET+处理后其存活率极低(图 2B2C),说明在通道开放过程中,MTSET+通过结合G168C改变了通道开放特性引起细菌死亡。

2.2 MTSET+导致G168C-MscS失去失活特性

低渗刺激实验表明MTSET+处理导致表达G168C-MscS的细菌在低渗刺激时几乎全部死亡。导致这一现象的原因很可能是MscS第168位点半胱氨酸在结合MTSET+后其侧链结构和带电性质发生了变化,以致于MscS通道开放异常(图 3A)。本研究进一步利用电生理膜片钳技术采用inside-out记录模式检测了WT-MscS和G168C-MscS在MTSET+处理前后的开放特征的变化(图 3B)。结果显示当负压达到一定阈值时,WT-MscS瞬间开放,电流达到最大值Imax,然后迅速失活,电流降低回到基线;MTSET+处理后,WT-MscS的开放特性与处理前保持一致(图 3C)。我们将从Imax下降到50% Imax所需的时间t1/2衡量通道的失活速率。MTSET+处理前后,WT-MscS t1/2无显著性差异(MTSET+处理前后WT t1/2=1.5±0.21s,1.8±0.85s,n=4)(图 3D)。G168C- MscS在负压增大到一定值时,瞬间开放,然后缓慢进入失活状态,其t1/2约34 s (33.89±7.77,n=5),显著高于WT-MscS;并且在MTSET+处理后,G168C-MscS基本不失活,保持持续开放,我们将从Imax到最终释放负压电流回到基线的记录时间算为其t1/2,约为190 s (190.06±9.14, n=3) (图 3C3D)。图 3E失活曲线同样表明G168C-MscS在MTSET+处理后失去失活特性。该部分结果表明,G168C-MscS能够失活,但其失活速率显著低于WT-MscS。而G168C-MscS半胱氨酸在结合MTSET+后,通道持续开放,失去失活特性,以致于胞内内容物持续外流,导致几乎所有细菌死亡,与低渗刺激实验结果一致(图 3CD图 2B)。

图 3 MTSET+导致G168C-MscS失去失活特性 Figure 3 G168C-MscS loses its inactivation property upon MTSET+ treatment. A: the side chain group of the 168th residue of MscS when it is glycine, cysteine, and cysteine combined with MTSET+. B: in the inside-out recording mode, a part of the membrane of the spheroplast were sealed by electrode. When MscS (the black triangle) on the membrane is opened, ions pass through to generate current. MscS opening changes were recorded by adding MTSET+ to the bath. C: representative patch clamp recordings from the same patch before and after addition of MTSET+ to the bath at 20 mV membrane potential. Lower traces: applied negative pressure. Upper traces: current curve. D: changes in t1/2 of WT-MscS or G168C-MscS before and after MTSET+ treatment. t1/2 represents the time it takes for the current to drop from the maximum Imax to 1/2 Imax and reflects inactivation rate of the channel. Data are shown as mean ± SEM. Significant differences were identified by Student's two-tailed unpaired t test, *: P < 0.05, ***: P < 0.001. E: ΔTime/Timetotal and the normalized value I/Imax were plotted with Boltzman formula to reflect inactivation curve. The total time required for channel inactivation (Timetotal) was refered as the time at which current reaches maximum value (Timemax) minus the time at which current has just dropped to the baseline (Timebaseline). ΔTime represents the time at which the current drops to a certain value minus the time at which the current reaches maxium.

2.3 MscS第168位氨基酸侧链基团带有正电荷不影响其失活特性

电生理结果表明MTSET+导致G168C-MscS保持持续开放,失去失活特性。原因是带有正电荷的巯基化合物MTSET+能够结合G168C半胱氨酸的侧链基团,改变了其结构和带电性质。为进一步探究是否是由于侧链基团带有正电荷的性质影响了通道开放特性,我们将G168位点甘氨酸突变为另一侧链基团带有正电荷的氨基酸赖氨酸(Lys,K)。电生理结果表明,G168K-MscS开放特性与WT-MscS基本一致,在负压达到一定阈值时,通道开放,然后失活(图 4A),其t1/2约为2 s (2.02±0.12, n=3),与WT-MscS无显著性差异(图 4B),说明168位氨基酸侧链基团带有正电荷不是通道失去失活特性的原因。我们又将G168位点甘氨酸突变为侧链基团较大的亮氨酸(Leu,L)和酪氨酸(Tyr,Y),发现G168L和G168Y两个MscS突变体的开放特征与WT-MscS一致,其t1/2分别为1.9 s (1.92±0.22,n=3),1.5 s (1.52±0.14,n=3),与WT-MscS无显著性差异(图 4A4B)。图 4C失活曲线表明G168L,G168Y和G168K均能够失活。

图 4 G168L-MscS,G168Y-MscS和G168K-MscS失活特性与WT-MscS一致 Figure 4 The inactivation properties of G168K-MscS were similar to that of WT-MscS. A: representative patch clamp recordings of WT-MscS and MscS mutants (G168L, G168Y and G168K) at 20 mV membrane potential. Lower traces: applied negative pressure. Upper traces: current curve. B: changes in t1/2 of WT-MscS or MscS mutants (G168L, G168Y and G168K). t1/2 represents the time it takes for the current to drop from the maximum Imax to 1/2 Imax. Data are shown as mean±SEM. C: ΔTime /Timetotal and the normalized value I/Imax were plotted with Boltzman formula. ΔTime represents the time at which the current drops to a certain value minus the time at which the current reaches maxium. The total time required for channel inactivation (Timetotal) was referred as the time at which current reaches maximum value (Timemax) minus the time at which current has just dropped to the baseline (Timebaseline).

2.4 G168I-MscS失去失活特性

为进一步探究G168氨基酸性质如何影响了通道的失活特性,我们将G168甘氨酸又突变为侧链带有负电荷的天冬氨酸(Asp,D),以及侧链较大的缬氨酸(Val,V)和异亮氨酸(Ile,I)。电生理结果表明G168D和G168V在负压增大到一定值时开放,然后缓慢进入失活状态,其t1/2分别为69.6 s (69.60±20.22,n=3)和222.2 s (222.02±40.62,n=3),显著高于WT-MscS,说明其失活速率降低。有趣的是,G168I-MscS保持持续开放,失去失活特性,说明G168位点氨基酸侧链电荷性质和体积大小影响通道失活(图 5A5C)。

图 5 G168D-MscS、G168V-MscS、G168I-MscS失活速率显著变慢 Figure 5 The inactivation rate of G168D-MscS, G168V-MscS and G168I-MscS was significantly slower than that of WT-MscS. A: representative patch clamp recordings of WT-MscS and MscS mutants (G168D, G168V and G168I) at 20 mV membrane potential. Lower traces: applied negative pressure. Upper traces: current curve. B: changes in t1/2 of WT-MscS or MscS mutants (G168D, G168V and G168I). t1/2 represents the time it takes for the current to drop from the maximum Imax to 1/2 Imax. Data are shown as mean±SEM. Significant differences were identified by Student's two-tailed unpaired t test, *: P < 0.05; **: P < 0.01; ***: P < 0.001. C: ΔTime /Timetotal and the normalized value I/Imax were plotted with Boltzman formula. ΔTime represents the time at which the current drops to a certain value minus the time at which the current reaches maxium. The total time required for channel inactivation (Timetotal) was referred as the time at which current reaches maximum value (Timemax) minus the time at which current has just dropped to the baseline (Timebaseline).

3 讨论

最新一项研究通过冷冻电镜解析了E. coli MscS处于失活状态时的结构,发现MscS处于开放状态时,TM1-TM2螺旋对与同一亚基的TM3b相互作用;而MscS处于失活或关闭状态时,TM1-TM2螺旋对与相邻亚基的TM3b相互作用[18]。作者认为MscS处于失活状态与TM1-TM2螺旋对是否与相邻亚基的TM3b发生相互作用紧密相关。此外,Sukharev团队通过结合分子动力学模拟和膜片钳实验结果得出MscS TM3b的G113和G121两个氨基酸影响通道失活[12]。这些说明TM3b参与通道失活过程。我们之前的研究表明MscS开放过程中,TM3b与胞质结构域Cyto-helix相互作用[17],通过半胱氨酸二硫键交联的方法将TM3b与Cyto-helix交联,则通道进入一种非离子通透状态,提示Cyto-helix可能通过TM3b参与MscS失活进程。而且Grajkowski等[19]发现在一些多聚体存在的情况下,比如将聚乙二醇、右旋糖苷、聚蔗糖等加入到MscS的胞质部分, MscS的失活速率加速,说明MscS的胞质结构域参与调节通道失活,然而MscS胞质结构域Cyto-helix是否影响通道失活并不清楚。本研究制备MscS Cyto-helix (P166−I170)半胱氨酸突变体,利用巯基化合物MTSET+能够结合半胱氨酸从而对其侧链基团修饰特性,通过低渗刺激实验检测表达MscS半胱氨酸突变体的大肠杆菌分别在无或有MTSET+处理后细菌低渗刺激后存活率筛选得到Cyto-helix G168显著影响通道功能。进一步研究发现巯基化合物MTSET+结合G168C-MscS半胱氨酸后,G168C-MscS失去失活特性,从而解释了MTSET+处理导致表达G168C-MscS大肠杆菌在低渗刺激时几乎全部死亡这一现象。早前Booth IR和Li Y团队通过将MscS孔道区域关键氨基酸亮氨酸L109突变为亲水性氨基酸丝氨酸L109S,L109S-MscS成为功能获得性突变体,通道具有自发开放特性,诱导表达L109S-MscS使内容物大量流失导致细胞死亡[1314, 17]。这些均说明MscS通道的开放异常会导致细胞死亡。

将G168突变为侧链基团体积较大,不带电荷的半胱氨酸C,G168C-MscS具有失活特性,但其失活速率显著变慢,说明氨基酸侧链体积大小影响通道失活。利用MTSET+结合半胱氨酸特性对G168C侧链基团进行修饰使其侧链基团体积更大,并且带有正电荷。结果显示G168C-MscS在MTSET+修饰后,通道却失去失活特性,这种特性有可能是168位点侧链体积更大引起的,也有可能是带有正电荷引起的。而将G168突变为带有正电荷的赖氨酸K,G168K-MscS呈现与WT-MscS相类似的失活特征,说明该位点正电荷性质不是通道失活异常的主要机制。相反负电荷的天冬氨酸突变G168D导致失活速率降低,提示该位点负电荷性质影响通道失活。另一方面,进一步验证G168位点氨基酸侧链体积大小是影响通道失活重要因素的是G168I和G168V。I和V侧链基团体积均较大,不带电荷,并且I的侧链体积比V大。G168I失去失活特性,保持持续开放;G168V失活速率显著变慢,提示该位点氨基酸侧链体积越大,通道失活速率越低。然而同样体积较大的氨基酸G168L和G168I其侧链基团大小和疏水性相近,但只有一个甲基基团的位置不同,G168L-MscS失活特征与WT-MscS相近,而G168I-MscS却失去失活特性,这些说明即使168位点氨基酸侧链体积相近,但是其基团空间位置不同也会影响通道失活。此外,突变体G168L、G168Y及G168K的失活特性与WT相近,说明并不是168位点氨基酸被突变或者被修饰,MscS通道失活特性就会改变。因此G168位点是影响通道失活的关键位点,但其具体机制复杂,还有待研究。研究表明构成通道氨基酸并不是孤立的,在通道开放过程中需要与通道其他区域氨基酸发生相互作用,比如改变MscS跨膜区域天冬氨酸Asp-62与胞质结构域精氨酸Arg-128或Arg-131的相互作用会影响通道失活[2021],改变MscS TM3b N117与Cyto-helix N167相互作用会影响通道开放[17]等,因此G168位点影响通道失活很可能通过与其他位点发生相互作用而进行,而G168位点的氨基酸侧链基团的大小和电荷性质影响这种相互作用。总之,本研究鉴定得到胞质结构域Cyto-helix 168位点氨基酸显著影响通道失活,为理解MscS失活特性机制提供了新的视角。

鉴于多项研究指出,细菌机械敏感性离子通道可以作为一类新型抗生素靶点,例如我们之前的一项研究表明导致淋病的淋病奈瑟球菌MscS不仅保护淋球菌在低渗刺激时免遭裂解死亡,同时它对于淋病奈瑟球菌在感染阴道过程中有重要作用。本研究的结果给出一个很重要的提示,可以设计靶向淋球菌MscS的药物使通道失去失活特性,导致细菌流失大量内容物死亡,失去感染能力。而本研究发现的MscS G168位点无疑为药物的设计提供思路。某些细菌的MscS可能具有另外的作用,例如谷氨酸棒状杆菌在工业上常被用于谷氨酸生产,即味精的生产。研究表明,谷氨酸棒状杆菌C. glutamicum ATCC 13869 MscS是谷氨酸分泌到胞外的通道[22],由于该菌株中的MscS发生突变导致通道不断开放,导致在没有诱导条件下该菌株持续向胞外分泌谷氨酸,用于工业生产。因此,研究细菌机械敏感性离子通道MscS的结构和开放特性有助于其在医学和工业领域的应用。

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大肠杆菌机械敏感性离子通道MscS失活特性分析
王晓敏 , 张怡 , 吕梦丹 , 王志坚 , 应瑾瑶 , 殷乐依 , 吴宇涛 , 刘胜兵 , 敖雷 , 郑永霞 , 徐营 , 潘巍巍 , 李月舟