在血清饥饿条件下CHP2调节NHE活性减少细胞死亡

钠氢离子交换蛋白（NHE）是维持细胞内pH值等内环境稳定的重要蛋白;钙调磷酸酶B同源蛋白（CHP）是NHE的一个活性调节亚单位。研究CHP2对NHE1的调节作用时发现,在血清饥饿的条件下,PS120细胞依赖于CHP2的表达来调节外源性NHE1的活性,使细胞维持必要的钠氢交换生理活性和较高水平的细胞内pH值（pHi 7．4）,明显减少细胞因自身的胞浆酸性化而死亡,延长细胞存活时间（70％以上的细胞存活时间超过7天）。实验结果提示,通过研究减少CHP2表达或抑制其活性,可望找到加速细胞死亡的新方法。     

钠氢交换蛋白的研究进展

钠氢交换蛋白是一类存在于细胞膜表面的离子转运泵蛋白家族.它负责将细胞内H 与胞外Na 按照1:1的比例进行交换来调控细胞内pH的动态平衡,影响细胞的容积、运动、分化、凋亡和营养吸收,从而参与许多复杂的生理和病理过程.迄今为止,钠氢交换蛋白家族已发现有9个成员,各亚型间具有结构相似性和组织分布特异性.深入研究NHE的结构、功能及基因表达调控,将为人和哺乳动物的营养生理、疾病治疗提供新的思路和方法.     

NHE1的结构研究及其在缺血再灌注中的作用

钠氢交换体1(sodium/hydrogen exchanger 1,NHE1)是已知唯一一种在心肌细胞膜上显著表达的钠氢交换体(NHE)亚型,由N端的介导离子转运的跨膜结构域和C端胞内调节结构域两部分构成,对于维持正常心肌细胞内的pH值具有重要的作用,并且在代偿心肌缺血再灌注造成的细胞内pH值的变化中发挥主要作用。本文主要论述NHE1的结构以及其在心肌缺血再灌注中的研究进展。     

丁酸钠至少部分通过NF-Y依赖的TXNIP表达诱导人肺癌细胞A549死亡北大核心CSCD

组蛋白去乙酰化酶(HDACs)抑制剂丁酸钠调节细胞分化、增殖和抑制肿瘤发生。硫氧还蛋白相互作用蛋白(thioredoxin-interacting protein,TXNIP)通过负性调控硫氧还蛋白的活性,调控细胞内的氧化还原平衡,抑制细胞生长。本研究证明,丁酸钠可通过激活依赖于转录因子NF-Y的TXNIP表达,诱导人非小细胞肺癌细胞A549死亡。MTT法显示,5 mmol/L丁酸钠处理A549细胞72 h可显著诱导其死亡;流式细胞分析发现,其中大部分细胞以凋亡形式死亡。表达芯片分析表明,在丁酸钠处理的A549细胞中,TXNIP的mRNA水平显著提高30~50倍;实时定量PCR、免疫细胞化学和蛋白质印迹结果进一步证明,丁酸钠可显著上调TXNIP表达。荧光素酶报告基因分析证明,与对照细胞比较,丁酸钠刺激的细胞内报告酶活性可提高约10倍,提示丁酸钠可激活TXNIP启动子的转录活性。TXNIP启动子删除突变分析显示,删除NF-Y结合的DNA序列显著降低丁酸钠对TXNIP启动子的激活能力,表明NF-Y转录因子参与丁酸钠介导的TXNIP基因转录激活。为分析TXNIP在A549细胞中的定位和部分功能,在A549细胞中过表达GFP-TXNIP融合蛋白及其截短突变体融合蛋白;结果显示,野生型和保留N端1-281aa的截短突变体定位在细胞核,而删除N端1-200aa时,其定位在细胞核和细胞质,提示N端1-200aa可调节该蛋白质的定位。然而,丁酸钠刺激未发现表达的GFP-TXNIP在细胞内定位改变。以上结果表明,丁酸钠可通过激活转录因子NF-YC依赖的TXNIP激活,诱导A549细胞死亡,但不能改变TXNIP蛋白在细胞内的定位。上述结果还提示,TXNIP的N端1-200aa可能在调节TXNIP的细胞定位中发挥作用。是否丁酸钠刺激TXNIP表达导致的细胞死亡系通过改变细胞氧化压力,以及TXNIP在细胞中定位的详尽调节机制尚待进一步研究证明。     

丁酸钠至少部分通过NF-Y 依赖的TXNIP 表达诱导人肺癌细胞A549死亡

组蛋白去乙酰化酶（HDACs）抑制剂丁酸钠调节细胞分化、增殖和抑制肿瘤发生。硫氧还蛋白相互作用蛋白( thioredoxin-interacting protein,TXNIP)通过负性调控硫氧还蛋白的活性,调控细胞内的氧化还原平衡,抑制细胞生长。本研究证明,丁酸钠可通过激活依赖于转录因子NF-Y的TXNIP 表达,诱导人非小细胞肺癌细胞A549死亡。MTT法显示,5 mmol/L丁酸钠处理A549 细胞72 h可显著诱导其死亡;流式细胞分析发现,其中大部分细胞以凋亡形式死亡。表达芯片分析表明,在丁酸钠处理的A549 细胞中,TXNIP 的mRNA 水平显著提高30～50倍;实时定量PCR、免疫细胞化学和蛋白质印迹结果进一步证明,丁酸钠可显著上调TXNIP 表达。荧光素酶报告基因分析证明,与对照细胞比较,丁酸钠刺激的细胞内报告酶活性可提高约10 倍,提示丁酸钠可激活TXNIP 启动子的转录活性。TXNIP 启动子删除突变分析显示,删除NF-Y 结合的DNA 序列显著降低丁酸钠对TXNIP 启动子的激活能力, 表明NF-Y转录因子参与丁酸钠介导的TXNIP基因转录激活。为分析TXNIP 在A549 细胞中的定位和部分功能,在A549细胞 中过表达GFP TXNIP 融合蛋白及其截短突变体融合蛋白;结果显示,野生型和保留N 端1-281aa的截短突变体定位在细胞核,而删除N 端1-200aa 时,其定位在细胞核和细胞质,提示N 端1 200aa 可调节该蛋白质的定位。然而,丁酸钠刺激未发现表达的GFP TXNIP在细胞内定位改变。以上结果表明,丁酸钠可通过激活转录因子NF YC 依赖的TXNIP激活,诱导A549 细胞死亡,但不能改变TXNIP蛋白在细胞内的定位。上述结果还提示,TXNIP 的N 端1-200aa 可能在调节TXNIP 的细胞定位中发挥作用。是否丁酸钠刺激TXNIP表达导致的细胞死亡系通过改变细胞氧化压力,以及TXNIP在细胞中定位的详尽调节机制尚待进一步研究证明。     

抑制eIF3I蛋白的泛素化降解促进人宫颈癌细胞增殖

目的:研究eIF3I蛋白在细胞中的泛素化修饰,阐明其对人宫颈癌细胞系Hela增殖的影响.方法:通过点突变技术获得突变体K282R,与野生型eIF3I比较泛素化的水平,研究细胞内的泛素化修饰调控.经流式细胞仪分析细胞周期,研究野生型蛋白eIF3I和突变体K282R对Hela细胞的细胞周期影响.再从周期蛋白水平研究eIF3I对细胞增殖的调控作用.结果:突变体K282R比野生型eIF3I蛋白的外源表达量大.在Hela细胞中K282R突变体的泛素化水平低,抑制了该蛋白的泛素-蛋白酶体途径降解.过表达eIF3I能上调周期蛋白Cyclin D1的表达量,促进细胞进入由G1期进入S期.同时,泛素化程度低的突变体K282R具有较强的促进细胞增殖的作用.结论:抑制eIF3I的泛素-蛋白酶体途径降解能上调周期蛋白CyclinD1的表达,促进肿瘤细胞增殖,提示eIF3I在细胞增殖和肿瘤发生发展中发挥作用.     

转铁蛋白在低钾刺激Madin Darby狗肾细胞钠-钾ATP酶中的作用

体外低钾培养肾细胞能刺激细胞膜钠-钾ATP酶。本研究利用Madin Darby狗肾细胞能在无血清培养液中健康生存48h这一特征,研究体外低钾刺激细胞膜钠-钾ATP酶所依赖的血清中的活性因子,观察了表皮生长因子(EGF)、胰岛素样生长因子(IGF1)、前列腺素1(PGE1)和转铁蛋白(tranderrin)在这一过程中的作用。结果表明,在无血清培养液中低钾并不能刺激细胞膜钠—钾ATP酶,而添加转铁蛋白可模拟血清的作用。转铁蛋白能剂量依赖性地增加ouabain结合位点,对细胞膜钠-钾ATP酶作用呈良好的时间效应关系。在低钾无血清培养液中,细胞膜钠-钾ATP酶α1亚基启动子活性增强,α1与β1亚基蛋白质表达的增加依赖于转铁蛋白的存在。进一步研究结果表明,低钾在转铁蛋白的无血清培养液环境中能增加细胞对铁的摄取(^59Fe),该作用可被铁螯合剂(deferoxamine,DFO;35 μmol/L)所阻断。DFO也可阻断转铁蛋白依赖性低钾刺激细胞膜钠-钾ATP酶数目的增多,α1亚基启动子活性增强,α1与β1亚基蛋白质表达增加。以上结果表明,低钾对细胞膜钠-钾ATP酶活性的刺激作用依赖于转铁蛋白所调节的铁的摄取。     

水稻OsRacD蛋白G15V、T20N点突变对其细胞定位的影响及其与靶蛋白的相互作用

OsRacD是水稻小GTP结合蛋白Rho家族成员,功能之一是作为“分子开关",通过控制花粉管的延伸生长,参与光敏核不育水稻光周期育性转换过程. 为研究该蛋白的作用机制,采用重叠延伸PCR方法,分别在其GTPase结构域中引入G15V、T20N点突变,模拟GTP和GDP结合形式的OsRacD. 进一步构建了受控于CaMV35S的与绿色荧光蛋白融合表达的双元植物表达载体;采用农杆菌介导法转化洋葱表皮细胞,在荧光显微镜下观察蛋白在活细胞内定位的特点. 结果显示,野生型蛋白在细胞质和细胞膜都有分布,组成型激活的蛋白主要分布在细胞膜上,而失活型蛋白则大都集中到细胞核周围. 蛋白相互作用的酵母双杂交体系分析显示,OsRacD及其2个突变体具有不同的靶蛋白结合特性. 研究证实,水稻OsRacD蛋白G15V和T20N点突变不仅影响其在活细胞内的定位,而且也影响了与靶蛋白的相互作用.说明处于不同鸟苷酸结合状态的OsRacD具有不同的胞内定位方式,可能通过结合不同的靶蛋白,引发不同的细胞应答事件.     

利用染色质结构检测技术研究乳腺癌易感蛋白BRCA1转录激活区突变

乳腺癌易感基因BRCA1突变引起的遗传性乳腺癌中40％－50％,其突变引起的遗传性乳腺癌和卵巢癌的比例至少为80％,许多乳腺癌易感突变发生在BRCA1 C末端转录激活结构域（1560－1863aa),但该区域大部分突变导致何种表型（良性多态性或乳腺癌易感突变）目前还不清楚,由于染色质结构调节是基因转录调节的早期事件,该文基于lac阻遏物识别和结合lac操纵基因的原理,利用染色质结构检测技术比较BRCAI转录激活结构域不同突变体与野生型的染色质伸展活性,将1种野生型,2种良性多态型（S1613G和M16521）和4种乳腺癌易感突变型（A1708E,M1775R,W1837R和Y1853term)转录激活区片段以正确相位融合于lac阻遏物的下游,得到野生型重组质粒pwt和pS1613G,pM1652I,pA1708,pM1775R,pW1837R及pY1853tem6种突变型重组质粒,Western blot检测表明,这些重组质粒分别转染A03－1细胞后均表达了相应的融合蛋白。对这些重组质粒的染色质伸展活性检测表明：野生型pwt和两种良性多态性突变体不具有染色质伸展活性或只有极微弱的染色质伸展活性,而其他4种乳腺癌易感突变体均具有过强的染色质伸展活性,提示利用染色质伸展技术可预测BRCA1转录激活区基因型与乳腺癌发生风险的表现型的关系。     

具有八肽重复区突变的朊蛋白其抗氧化性降低

抗氧化性被认为是细胞朊蛋白的主要生理功能之一,研究显示它的抗氧化性主要与朊蛋白序列中的八肽重复区有关．但是迄今为止它的抗氧化机制仍旧不清楚．我们构建表达了野生型朊蛋白（PrP-PG5）和它的不同八肽重复区突变体0（PrP-PG0）,9（PrP-PG9）和12（PrP-PGl2）．各种原核表达突变体蛋白在H202氧化后出现分子量的增加,并可导致羰基产生．MTT和细胞计数实验显示表达各种突变体的细胞存活率明显低于表达野生型朊蛋白（PrP—PG5）的细胞．细胞内ROS检测发现表达各种突变体的细胞内ROS水平明显高于表达野生型朊蛋白（PrP-PG5）的细胞．此外,谷胱甘肽过氧化物酶检测显示表达野生型朊蛋白（PrP-PG5）的细胞内谷胱甘肽过氧化物酶水平明显高于表达各种突变体的细胞．H2O2处理细胞后,转染突变体的细胞总的羰基产物数量明显高于转染野生型朊蛋白（PrP-PG5）的细胞,而表达突变体细胞及转染空载体的细胞较表达野生型朊蛋白（PrP-PG5）的细胞对氧化物质的抵抗性明显减弱．这些结果提示,具有正确八肽重复区数目对于朊蛋白（PrP）的抗氧化作用起关键作用,PrP的抗氧功能的丢失可能参与家族性朊病毒病的病理过程．     

Calcineurin homologous protein as an essential cofactor for Na+/H+ exchangers

The Na+/H+ exchangers (NHEs) comprise a family of transporters that catalyze cell functions such as regulation of the pH and volume of a cell and epithelial absorption of Na+ and bicarbonate. Ubiquitous calcineurin B homologous protein (CHP or p22) is co-localized and co-immunoprecipitated with expressed NHE1, NHE2, or NHE3 independently of its myristoylation and Ca2+ binding, and its binding site was identified as the juxtamembrane region within the carboxyl-terminal cytoplasmic domain of exchangers. CHP binding-defective mutations of NHE1-3 or CHP depletion by injection of the competitive CHP-binding region of NHE1 into Xenopus oocytes resulted in a dramatic reduction (>90%) in the Na+/H+ exchange activity. The data suggest that CHP serves as an essential cofactor, which supports the physiological activity of NHE family members.     

Solution structure of the cytoplasmic region of Na+/H+ exchanger 1 complexed with essential cofactor calcineurin B homologous protein 1

Na+/H+ exchanger 1 (NHE1) regulates intracellular pH, Na+ content, and cell volume. Calcineurin B homologous protein 1 (CHP1) serves as an essential cofactor that facilitates NHE1 exchange activity under physiological conditions by direct binding to the cytoplasmic juxtamembrane region of NHE1. Here we describe the solution structure of the cytoplasmic juxtamembrane region of NHE1 complexed with CHP1. The region of NHE1 forms an amphipathic helix, which is induced by CHP1 binding, and CHP1 possesses a large hydrophobic cleft formed by EF-hand helices. The apolar side of the NHE1 helix participates in extensive hydrophobic interactions with the cleft of CHP1. We suggest that helix formation of the cytoplasmic region of NHE1 by CHP1 is a prerequisite for generating the active form of NHE1. The molecular recognition detailed in this study also provides novel insight into the target binding mechanism of EF-hand proteins.     

Acute regulation of Na/H exchanger NHE3 by adenosine A(1) receptors is mediated by calcineurin homologous protein

Adenosine is an autacoid that regulates renal Na(+) transport. Activation of adenosine A(1) receptor (A(1)R) by N(6)-cyclopentidyladenosine (CPA) inhibits the Na(+)/H(+) exchanger 3 (NHE3) via phospholipase C/Ca(2+)/protein kinase C (PKC) signaling pathway. Mutation of PKC phosphorylation sites on NHE3 does not affected regulation of NHE3 by CPA, but amino acid residues 462 and 552 are essential for A(1)R-dependent control of NHE3 activity. One binding partner of the NHE family is calcineurin homologous protein (CHP). We tested the role of NHE3-CHP interaction in mediating CPA-induced inhibition of NHE3 in opossum kidney (OK) and Xenopus laevis uroepithelial (A6) cells. Both native and transfected NHE3 and CHP are present in the same immuno-complex by co-immunoprecipitation. CPA (10(-6) M) increases CHP-NHE3 interaction by 30 - 60% (native and transfected proteins). Direct CHP-NHE3 interaction is evident by yeast two-hybrid assay (bait, NHE3(C terminus); prey, CHP); the minimal interacting region is localized to the juxtamembrane region of NHE3(C terminus) (amino acids 462-552 of opossum NHE3). The yeast data were confirmed in OK cells where truncated NHE3 (NHE3(delta552)) still shows CPA-stimulated CHP interaction. Overexpression of the polypeptide from the CHP binding region (NHE3(462-552)) interferes with the ability of CPA to inhibit NHE3 activity and to increase CHPNHE3(Full-length) interaction. Reduction of native CHP expression by small interference RNA abolishes the ability of CPA to inhibit NHE3 activity. We conclude that CHPNHE3 interaction is regulated by A(1)R activation and this interaction is a necessary and integral part of the signaling pathway between adenosine and NHE3.     

Expression of calcineurin B homologous protein 2 protects serum deprivation-induced cell death by serum-independent activation of Na+/H+ exchanger

The calcineurin B homologous protein (designated CHP1) has been shown to be a common essential cofactor for the plasma membrane Na(+)/H(+) exchangers (NHEs) (Pang, T., Su, X., Wakabayashi, S., and Shigekawa, M. (2001) J. Biol. Chem. 276, 17367-17372). In this study, we characterized the function of another isoform of CHP (designated CHP2) that has a 61% amino acid identity with CHP1. CHP2, like CHP1, conferred the ability to NHEs 1-3 to express a high exchange activity by binding to the juxtamembrane region of the cytoplasmic domain of the exchanger, but it interacts more strongly (approximately 5-fold) with NHE1 than does CHP1. Although CHP1 is expressed ubiquitously at relatively high levels, CHP2 expression was extremely low in most human tissues but was higher in tumor cells. We produced stable cell clones overexpressing either CHP1 or CHP2 in which one of them is predominantly bound to NHE1. Serum (10%) induced a significant cytoplasmic alkalinization (0.1-0.2 pH unit) in cells co-expressing CHP1 and NHE1 but not in cells co-expressing CHP2 and NHE1. In the latter, pH(i) was high (7.4-7.5) even in the absence of serum, suggesting that NHE1 was already activated. Surprisingly, most (>80%) of CHP2/NHE1 cells unlike CHP1/NHE1 cells were viable even after long serum starvation (>7 days). Thus, the expression of CHP2 appears to protect cells from serum deprivation-induced death by increasing pH(i). These properties of CHP2/NHE1 cells are similar to those of malignantly transformed cells. We propose that serum-independent activation of NHE1 by bound CHP2 is one of the key mechanisms for the maintenance of high pH(i) and the resistance to serum deprivation-induced cell death in malignantly transformed cells.     

Role of calcineurin B homologous protein in pH regulation by the Na+/H+ exchanger 1: tightly bound Ca2+ ions as important structural elements

We studied the role of the interaction of calcineurin homologous protein 1 (CHP1) with the Na(+)/H(+) exchanger 1 (NHE1), particularly its EF-hand Ca(2+) binding motifs, in the intracellular pH (pH(i))-dependent regulation of NHE1. We found that (45)Ca(2+) binds to two EF-hand motifs (EF3 and 4) of the recombinant CHP1 proteins with high affinity (apparent K(d) = approximately 90 nM). Complex formation between CHP1 and the CHP1 binding domain of NHE1 resulted in a marked increase in the Ca(2+) binding affinity (K(d) = approximately 2 nM) by promoting a conformational change of the EF-hands toward the tightly Ca(2+)-bound form. This suggests that CHP1 always contains two Ca(2+) ions when associated with NHE1 in cells. Interestingly, overexpression of GFP-tagged CHP1 with mutations in EF3 or EF4 significantly reduced the exchange activity in the neutral pH(i) range and partly impaired the activation of NHE1 in response to various stimuli, such as growth factors and osmotic stress. Furthermore, we found that, in addition to reducing the activity (V(max)), a CHP1 binding-defective NHE1 mutant had a marked reduction in pH(i) sensitivity ( approximately 0.7 pH unit acidic shift), which consequently abolished various regulatory responses of NHE1. These observations suggest that the association of NHE1 with CHP1 is crucial for maintenance of the pH(i) sensitivity of NHE1 and that tightly bound Ca(2+) ions may serve as important structural elements in the "pH(i) sensor" of NHE1.     

Dual functional significance of calcineurin homologous protein 1 binding to Na(+)/H(+) exchanger isoform 1

Calcineurin homologous protein 1 (CHP1) binds to the hydrophilic tail of the Na(+)/H(+) exchanger isoform 1 (NHE1). Previous gene knockout of CHP1 revealed that the loss of CHP1 caused a decrease in the total amount of NHE1, suggesting the destabilization of NHE1 molecules without CHP1 (Matsushita et al., Am J Physiol Cell Physiol 293: C246-C254, 2007). However, Pang et al. (J Biol Chem 276: 17367-17372, 2001) reported that NHE1 without a CHP1 binding site was found in the plasma membrane, suggesting no requirement of CHP1 binding for plasma membrane localization of NHE1. Here, the functional significance of CHP1 binding to NHE1 was examined to resolve these contradictory results. In CV1 cells, which overexpressed wild-type NHE1, overexpression of CHP1 caused an increase in both the total amount of NHE1 and the colocalization of NHE1 and CHP1 at the plasma membrane. This provided new visual evidence of the localization of NHE1 from endoplasmic reticulum to the plasma membrane upon CHP1 binding. An immunoprecipitation assay showed that the expression of CHP1 reduced the ubiquitination of NHE1 and/or its associated proteins. Mutant NHE1s without CHP1 binding site exhibited a modest localization to the plasma membrane. After reaching the plasma membrane, these mutant NHE1s exhibited shorter half-lives than the wild-type NHE1 with CHP1. The results suggest a dual functional significance of CHP1 and its binding region: 1) binding of CHP1 stabilizes NHE1 and increases its plasma membrane localization by masking a NHE1 disposal signal, and 2) CHP1 binding is required for the antiporter activity.     

Protein phosphatase regulation of Na+/H+ exchanger isoform I

We investigated regulation of Na(+)/H(+) exchanger isoform 1 (NHE1) by dephosphorylation. Treatment of primary cultures of cardiomyocytes with the phosphatase inhibitor okadaic acid increased the rate of recovery from an acid load, suggesting that the okadaic acid sensitive PP1 may be involved in NHE1 regulation in vivo. We examined the ability of purified protein phosphatases PP1, PP2A, and PP2B to dephosphorylate the regulatory cytoplasmic tail. NHE1 was completely dephosphorylated by PP1, poorly dephosphorylated by PP2A, and not dephosphorylated by PP2B. Examination of NHE1 binding to PP1 or PP2B revealed that an association occurs between NHE1 and PP1 both in vitro and in vivo, but NHE1 did not associate with full-length PP2B. We expressed PP1 or inhibitor 2, a specific PP1 inhibitor, in cell lines to examine the effect of PP1 on NHE1 activity in vivo. Overexpression of PP1 causes a decrease in NHE1 activity but does not affect stimulation by thrombin. Cell lines expressing the specific PP1 inhibitor, inhibitor 2, had elevated proton efflux rates and could not be further stimulated by the Na(+)/H(+) exchanger agonist thrombin. The results suggest that PP1 is an important regulatory phosphatase of NHE1, that it can bind to and dephosphorylate the protein, and that it regulates NHE1 activity in vivo.     

Evidence for involvement of the putative first extracellular loop in differential volume sensitivity of the Na+/H+ exchangers NHE1 and NHE2

We studied hyperosmolarity-induced changes in cell volume and cytoplasmic pH in PS120 cells expressing Na(+)/H(+) exchanger (NHE) isoforms and their mutants. Change in cell volume was estimated by measuring change in cell height by means of confocal microscopy. Regulatory volume increase (RVI) and cytoplasmic alkalinization were observed in cells expressing NHE1 but not in cells expressing NHE2 or NHE3. Studies using chimeric exchangers revealed that the membrane domain of the exchanger is responsible for the difference in volume sensitivity between NHE1 and NHE2. Although deletion or point mutation within the first extracellular loop of NHE1 did not affect RVI and alkalinization, point mutations within the corresponding region of NHE2, particularly a region containing aa 41-53, as well as replacement of the N-terminus of NHE2 with the corresponding region of NHE1, rendered NHE2 responsive to the activating effect of cell shrinkage. Thus, the membrane domain plays an important role in the response of the exchanger to cell shrinkage. The data suggest that the putative first extracellular loop of NHE2, but not that of NHE1, may exert an inhibitory influence on hyperosmolarity-induced activation of the exchanger and thereby block RVI.     

Calcineurin B homologous protein 3 promotes the biosynthetic maturation, cell surface stability, and optimal transport of the Na+/H+ exchanger NHE1 isoform

Calcineurin B homologous protein (CHP) 1 and 2 are Ca(2+)-binding proteins that modulate several cellular processes, including cytoplasmic pH by positively regulating plasma membrane-type Na(+)/H(+) exchangers (NHEs). Recently another CHP-related protein, termed tescalcin or CHP3, was also shown to interact with the ubiquitous NHE1 isoform, but seemingly suppressed its activity. However, the precise physical and functional nature of this association was not examined in detail. In this study, biochemical and cellular studies were undertaken to further delineate this relationship. Glutathione S-transferase-NHE1 fusion protein pulldown assays revealed that full-length CHP3 binds directly to the proximal juxtamembrane C-terminal region (amino acids 505-571) of rat NHE1 in the same region that binds CHP1 and CHP2. The interaction was further validated by coimmunoprecipitation and coimmunolocalization experiments using full-length CHP3 and wild-type NHE1 in transfected Chinese hamster ovary AP-1 cells. Simultaneous mutation of four hydrophobic residues within this region ((530)FLDHLL(535)) to either Ala, Gln, or Arg (FL-A, FL-Q, or FL-R) abrogated this interaction both in vitro and in intact cells. The NHE1 mutants were sorted properly to the cell surface but showed markedly reduced (FL-A) or minimal (FL-R and FL-Q) activity. Interestingly, and contrary to an earlier finding, ectopic coexpression of CHP3 up-regulated the cell surface activity of wild-type NHE1. This stimulation was not observed with the CHP3 binding-defective mutants. Mechanistically, overexpression of CHP3 did not alter the H(+) sensitivity of wild-type NHE1 but rather promoted its biosynthetic maturation and half-life at the cell surface, thereby increasing the steady-state abundance of functional NHE1 protein.