The Structure-Function Relationship of Prestin: From an Evolutionary Perspective.

機譯：普雷斯汀的結構-功能關系：從進化的角度。

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Prestin is the motor protein of cochlear outer hair cells. It belongs to a distinct anion transporter family called solute carrier protein 26A, or SLC26A. Members of this family serve two fundamentally distinct functions. While most members transport different anion substrates across a variety of epithelia, prestin (SLC26A5) uniquely functions as a voltage-dependent motor protein. This voltage-dependent response of prestin is accompanied by a charge movement, which is reflected in nonlinear capacitance (NLC). Prestin is assumed to contain a tunnel-like structure which is only accessible from intracellular side, based on a hypothetical working model as a partial anion transporter proposed by Oliver et al. (2001). Intracellular anions such as Cl- act as the external voltage sensor of prestin and trigger the conformational change in the molecule, which in turn alters the surface area of plasma membrane. This study tends to gain insight on the functionally critical structures of prestin using comparative approach.;Recent evidence suggests that prestin orthologs from zebrafish and chicken retain transporter function without motile capability. Mammalian prestin does not appear to transport anions across the cell membrane, while controversial studies suggest that mammalian prestin may also be able to transport anions and this transporter function is independent with its electromotility capability. These studies suggest that prestin is evolved from an anion transporter. In this study, I first examined the motor and transport functions of prestin and its orthologs from four different vertebrate species (zebrafish, chicken, platypus and gerbil), to gain insight regarding how these two physiological functions might have distinctly evolved. Somatic motility, voltage-dependent NLC and transporter function were measured in transfected human embryonic kidney (HEK) cells using voltage-clamp and anion uptake techniques. Zebrafish and chicken prestins both exhibit weak NLC with peaks significantly shifted in the depolarization (right) direction. This is contrasted by robust NLC with leftshifted peaks for both platypus and gerbil prestins. Platypus and gerbil prestins may only retain little transporter function in comparison with robust anion transport capacities in the zebrafish and chicken orthologs. Somatic motility is only detected in the platypus and gerbil prestins. There appears to be an inverse relationship between NLC and anion transport functions, whereas the motor function appears to have only emerged in mammalian prestin. Our results suggest that motor function is an innovation of mammalian prestin and is concurrent with diminished transporter capability.;Evolutionary studies combined with comparative genomic and bioinformatic analyses have identified highly conserved sequences among mammalian prestins that show significant variability among nonmammalian vertebrate prestin orthologs and other SLC26A paralogs. Among these sequences is one segment of 11 residues. In the second part of this study, I investigated whether this region represents the minimal essential motif for the motor function. Chimeric proteins swapping corresponding residues of prestin orthologs from zebrafish and chicken with those from gerbil prestin (zebrafish prestin with gerbil sites, Zf(g), and chicken prestin with gerbil sites, Ck(g), respectively) were constructed. Motility, NLC and anion transport were examined. A gain of motor function was observed with two hallmarks (NLC and motility) in both Zf(g) and Ck(g) without loss of transport function. These results show that the substitution of only 11 amino acids is sufficient to confer motor function upon the electrogenic anion transporters of zebrafish and chicken prestins. Therefore, this segment represents the minimal essential motif for the motor.;The regions or amino acids within prestin that are essential for voltage sensing are still unclear. Charged amino acids are likely to play an important role in voltage sensing because of their potential capability to serve as anion binding sites. Previous studies focused mostly on the property of charged side chains. In the third part of this study, the roles of three positively charged amino acids in voltage sensing of prestin were examined. I hypothesize that the size or charge location of the amino acid side chain is also an important factor for prestin function. Three positive amino acids in the putative tunnel region were selected based on molecular dynamics simulation and sequence alignment of prestin paralogs and orthologs, namely R197, K227 and K449. A series of substitutions using similarly charged amino acids (R to K and K to R) are constructed, assuming that R and K substitutions only affect size and charge orientation of the side chain. Negative (R / K to E), neutral (R / K to A) substitutions and combinations of substitutions at these three sites (double and triple mutations) were also constructed assuming that there are multiple anion binding sites in the molecule. The results show that all three sites are important for voltage sensing and that the size or charge orientation of the side chain is also a critical factor. Furthermore, negative correlations between the peak voltage of NLC or the total charge movement and the slope factor are observed, suggesting that other electrical features such as dielectric properties were changed by these substitutions rather than the number of charges or their traveling distance.

機譯：Prestin是耳蝸外毛細胞的運動蛋白。它屬于一個獨特的陰離子轉運蛋白家族，稱為溶質載體蛋白26A或SLC26A。這個家庭的成員具有兩個根本不同的功能。雖然大多數成員跨各種上皮細胞運輸不同的陰離子底物，但Prestin（SLC26A5）獨特地充當電壓依賴性運動蛋白。 Prestin的這種電壓依賴性響應伴隨著電荷運動，這反映在非線性電容（NLC）中。基于一個假設的工作模型，奧利弗（Oliver）等人提出的部分陰離子轉運蛋白，假定Prestin含有只能從細胞內側進入的隧道狀結構。（2001）。細胞內陰離子（例如Cl-）充當了prestin的外部電壓傳感器，并觸發了分子中的構象變化，進而改變了質膜的表面積。這項研究傾向于使用比較方法來了解蛋白素的功能關鍵結構。最新證據表明，來自斑馬魚和雞的蛋白素直系同源物保留了轉運蛋白的功能而沒有運動能力。哺乳動物的prestin似乎不能跨細胞膜轉運陰離子，而有爭議的研究表明，哺乳動物的prestin也許也能轉運陰離子，并且這種轉運蛋白功能與其電動力能力無關。這些研究表明，prestin是從陰離子轉運蛋白進化而來的。在這項研究中，我首先檢查了四種不同脊椎動物物種（斑馬魚，雞，鴨嘴獸和沙鼠）的Prestin及其直系同源物的運動和轉運功能，以了解這兩種生理功能可能如何發生明顯的進化。使用電壓鉗和陰離子攝取技術在轉染的人胚腎（HEK）細胞中測量體細胞運動性，電壓依賴性NLC和轉運蛋白功能。斑馬魚和雞肉的prestins均顯示弱的NLC，峰在去極化（右）方向上有明顯偏移。與之相比，穩健的NLC則有鴨嘴獸和沙鼠的前庭素左移峰。與斑馬魚和雞肉直系同源物中強健的陰離子轉運能力相比，鴨嘴獸和沙鼠的Prestins只能保留很少的轉運功能。體細胞運動僅在鴨嘴獸和沙鼠前體中檢測到。在NLC和陰離子轉運功能之間似乎存在反比關系，而運動功能似乎只出現在哺乳動物的prestin中。我們的研究結果表明運動功能是哺乳動物Prestin的一項創新，并且與轉運蛋白能力下降同時進行;進化研究與比較基因組和生物信息學分析相結合，已經確定了哺乳動物Prestin中高度保守的序列，這些序列顯示出非哺乳動物脊椎動物Prestin Orthologs和其他SLC26A之間的顯著變異性。旁系同源物。在這些序列中是11個殘基的一個片段。在本研究的第二部分中，我研究了該區域是否代表了運動功能的最小基本主題。構建了將斑馬魚和雞肉中的Prestin直系同源物的相應殘基與沙鼠Prestin的相應殘基交換的嵌合蛋白（分別具有Zb（g）的沙鼠魚Prestin和具有Cb（g）的沙鼠位點）。檢查了動力，NLC和陰離子轉運。在Zf（g）和Ck（g）中均具有兩個標記（NLC和運動性）的運動功能得到了改善，而轉運功能沒有損失。這些結果表明，僅11個氨基酸的取代足以賦予斑馬魚和雞肉prestins的電動陰離子轉運蛋白以運動功能。因此，該片段代表了馬達的最小基本基序。尚不清楚電壓感測中必需的區域或氨基酸。帶電荷的氨基酸由于其潛在的充當陰離子結合位點的能力而可能在電壓感測中起重要作用。先前的研究主要集中于帶電側鏈的性質。在這項研究的第三部分中，研究了三種帶正電荷的氨基酸在普雷斯汀電壓感測中的作用。我假設氨基酸側鏈的大小或電荷位置也是影響prestin功能的重要因素。基于分子動力學模擬和Prestin直系同源物和直系同源物的序列比對，在推定的隧道區域中選擇了三個正氨基酸，即R197，K227和K449。假設R和K取代僅影響側鏈的大小和電荷取向，則使用相似電荷的氨基酸（R至K和K至R）構建一系列取代。負數（R / K到E）假設分子中有多個陰離子結合位點，則還構建了這三個位點（雙突變和三突變）的中性（R / K到A）取代和取代組合。結果表明，所有三個位點對于電壓感測都很重要，并且側鏈的大小或電荷取向也是關鍵因素。此外，觀察到NLC的峰值電壓或總電荷運動與斜率因子之間呈負相關，這表明通過這些替代方法（而不是電荷數量或其行進距離）改變了其他電學特性（例如介電特性）。

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作者
Tan, Xiaodong.;
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Creighton University.;

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授予單位 Creighton University.;
學科 Biology Neuroscience.
學位 Ph.D.
年度 2010
頁碼 108 p.
總頁數 108
原文格式 PDF
正文語種 eng
中圖分類
關鍵詞
入庫時間 2022-08-17 11:36:56

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The Structure-Function Relationship of Prestin: From an Evolutionary Perspective.

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