Determinants of ion channel versus transporter mechanism in the K(+) transporter superfamily

K( ) 转运蛋白超家族中离子通道与转运蛋白机制的决定因素

• 批准号: 266161834
• 负责人: Professorin Dr. Inga Hänelt
• 金额: --
• 依托单位: Abteilung Biophysikalische Chemie
• 依托单位国家: 德国
• 项目类别: Independent Junior Research Groups
• 财政年份: 2014
• 资助国家: 德国
• 起止时间: 2013-12-31 至 2020-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/266161834?language=en
• 关键词: Determinants; ion; channel; versus; transporter

The constant traffic of substrates across membranes is controlled by two classes of proteins: channels and transporters. Open channels selectively allow a fast flux of substrates down an electrochemical gradient, whereas transporters are able to slowly pump substrates thermodynamically uphill by consuming energy. Due to these strictly opposing functions and the divergent speeds of substrate translocation, channels and transporters have traditionally been viewed as completely different entities. However, new structural and mechanistic data on both classes challenge this clear-cut separation. This proposal aims at elucidating the differences and commonalities of channels and transporters by studying the uniquely chimeric K(+) transport systems KtrAB and KdpFABC. In both systems, the translocating subunits KtrB and KpdA, respectively, are members of the superfamily of K(+) transporters, which are suggested to perform channel-like activities by themselves. However, in the presence of additional regulatory subunits both complexes are likely converted into transporters: The KdpFABC complex functions as a P-type ATPase while KtrAB probably exhibits Na(+)/K(+) symporter activity. By use of a multidisciplinary approach including transport studies, electrophysiology, EPR spectroscopy and X-ray crystallography, we will address the structure-function relationships underlying the mechanisms of K(+) translocation in both the isolated subunits KtrB and KdpA, respectively, and the assembled complexes. In particular, we will answer the question whether KtrAB in fact functions as symporter, how substrate translocation via the Ktr system is achieved and which structural changes in presence of the regulatory subunit KtrA cause the altered function. For the KdpFABC system we will focus on elucidating the mechanisms of how ATP hydrolysis in subunit KdpB is coupled to the active transport via KdpA and thus how structurally a former channel is converted into a primary active transporter. Taken together, this work will lead to basic understanding of what is requiered to make a transporter or channel.

底物跨膜的持续运输由两类蛋白质控制：通道和转运蛋白。开放的通道选择性地允许基材沿电化学梯度快速流动，而传送器能够通过消耗能量在热力学上缓慢地泵送基材。由于这些严格相反的功能和底物转运的不同速度，通道和转运体传统上被视为完全不同的实体。然而，这两个类别的新的结构性和机械性数据都挑战了这种明确的区分。这一建议旨在通过研究独一无二的嵌合K(+)转运系统KtrAB和KdpFABC来阐明通道和转运蛋白的区别和共同点。在这两个系统中，转运亚单位KtrB和KpdA分别是K(+)转运体超家族的成员，建议它们自己执行通道样活动。然而，在存在额外的调节亚基的情况下，这两个复合体都可能被转换为转运体：KdpFABC复合体的功能是P型ATPase，而KtrAB可能具有Na(+)/K(+)转运体活性。我们将利用多学科的方法，包括运输研究、电生理学、EPR光谱和X射线结晶学，研究K(+)在分离的亚基KtrB和KdpA以及组装的复合体中转运机制的结构-功能关系。特别是，我们将回答KtrAB是否真的作为符号转运体发挥作用，底物是如何通过KTR系统实现转运的，以及在调节亚单位KtrA存在的情况下，哪些结构变化会导致功能改变。对于KdpFABC系统，我们将重点阐明KdpB亚基中的ATP水解如何通过KdpA偶联到主动转运的机制，从而从结构上将以前的通道转换为初级活性转运体。综上所述，这项工作将导致对建立传送器或渠道所需的基本理解。