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

底物跨膜的恒定运输由两类蛋白质控制：通道和转运蛋白。开放通道选择性地允许底物沿着电化学梯度快速流动，而转运体能够通过消耗能量缓慢地将底物缓慢地泵送上山。由于这些严格相反的功能和底物转运的不同速度，通道和转运蛋白传统上被视为完全不同的实体。然而，新的结构和机制的数据，这两个类的挑战，这种明确的分离。本研究的目的是通过研究独特的嵌合K（+）转运系统KtrAB和KdpFABC来阐明通道和转运蛋白的差异和共性。在这两个系统中，转运亚基KtrB和KpdA分别是K（+）转运蛋白超家族的成员，它们被认为是独立执行通道样活动的。然而，在存在额外的调节亚基的情况下，两种复合物都可能转化为转运蛋白：KdpFABC复合物作为P型ATP酶发挥功能，而KtrAB可能表现出Na（+）/K（+）同向转运蛋白活性。通过使用多学科的方法，包括运输研究，电生理学，EPR光谱和X射线晶体学，我们将解决的结构-功能关系的K（+）易位的机制，在两个孤立的亚基KtrB和KdpA，分别组装复合物。特别是，我们将回答的问题是否KtrAB实际上作为同向转运体的功能，如何通过KTR系统实现底物易位，以及在调节亚基KtrA存在下的结构变化导致功能改变。对于KdpFABC系统，我们将重点阐明亚基KdpB中的ATP水解如何通过KdpA与主动转运偶联的机制，从而在结构上如何将前通道转化为主要的主动转运蛋白。总之，这项工作将导致基本的理解是什么要求，使运输或通道。

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.