Urea transporters (UT) are integral membrane proteins that facilitate transport of urea across cell membrane. UTs are highly expressed in many mammalian tissues, including kidney, liver, and brain. UT function is best understood in the kidney where UT is essential in maintaining a high urea concentration in the inner medullary region so that water is absorbed to produce concentrated urine. Mice with one of the UT genes knocked out show reduced ability to absorb water, and genetic variations of human UT are directly linked to abnormal blood pressure. These results indicate that UT plays an important role in kidney physiology and in regulating blood pressure. However, the current lack of structural information on UTs hampers our understanding of their architecture and function. Our long-term goal is therefore to obtain an atomic level mechanism for UT facilitated urea permeation and UT inhibition. We have recently crystallized a UT from the bacterium Desulfovibrio vulgaris (dvUT) that has significant sequence homology to mammalian UT, and have refined the crystals to diffract to a Bragg spacing of 2.3 ?. We have also initiated functional studies on dvUT. We have developed a scintillation proximity assay to measure, for the first time, equilibrium binding between urea and UT. We have successfully expressed dvUT in Xenopus laevis oocytes, and found that it mediates urea flux through oocyte membrane. Furthermore, phloretin, a known blocker for mammalian UTs, also inhibits urea binding and flux through dvUT. These exciting new results have led us to propose a combined structure- function study with the following four aims:
Aim 1 : To build and refine a structural model of dvUT. We will solve the phase problem by using anomalous diffraction signals from heavy atoms co-crystallized with dvUT, and we will build and refine a structural model of dvUT at 2.3 ? resolution. We will further refine crystallization conditions to improve the resolution.
Aim 2 : To investigate mechanism of urea transport through dvUT. Although dvUT has high sequence similarity to mammalian UTs, and has the highly conserved UT """"""""signature sequence"""""""", the function of dvUT has never been demonstrated. We will determine if dvUT is a functional urea transporter, and if so, we will then use X-ray crystallography to identify urea binding sites. Although many functional studies suggest that UT operates by a channel-like mechanism, the transporter mechanism has not been ruled out because in certain UTs saturation of flux rate is observed. We will address this question by analyzing the structure and by measuring urea flux at different temperatures.
Aim 3 : To investigate mechanism of dvUT blockade. We will examine if known mammalian UT blockers affect urea binding and permeation on dvUT, and if so, we will identify blocker binding sites by X-ray crystallography. We will verify the binding sites by making point mutations on dvUT, and then examine both the structure and function of mutant dvUTs.
Aim 4 : To examine if the mechanisms of urea permeation and blockade are conserved between dvUT and mammalian UT. We will examine whether coordination of urea and UT blockers observed in dvUT is achieved by homologous residues on UT-A2, a mammalian UT that is highly expressed in kidney. We will make mutations on UT-A2, and examine urea permeation and blockade by an oocyte flux assay. We will also overexpress mammalian UTs with the long term goal of obtaining a high resolution structure by X-ray crystallography. Taken together, the proposed research will substantially further our understanding of both eukaryotic and prokaryotic UT, and will place structure and function relationships of mammalian UT in an atomic-resolution, three-dimensional context which eventually we hope will lead to the development of new therapeutic reagents.
In mammals, urea transporters are expressed in a wide array of organs, such as kidney, brain, heart, liver, ear, and testis, suggesting that they play an important role in physiology. In humans, loss of urea transporter causes reduced capability to concentrate urine, and genetic variations of urea transporters have been directly linked to variations in blood pressures. Mice with urea transporters knocked out showed progressive heart block and early puberty, in addition to defects in concentrating urine. Therefore, urea transporter is a potential drug target for treating a variety of conditions ranging from hypertension, congestive heart failure, to syndrome of inappropriate secretion of antidiuretic hormone compounds (SIADH). Compounds that selectively block urea transporter will have diuretic effect but will not interfere with the salt balance.
|Lee, Jumin; Ren, Zhenning; Zhou, Ming et al. (2017) Molecular Simulation and Biochemical Studies Support an Elevator-type Transport Mechanism in EIIC. Biophys J 112:2249-2252|
|McCoy, Jason G; Ren, Zhenning; Stanevich, Vitali et al. (2016) The Structure of a Sugar Transporter of the Glucose EIIC Superfamily Provides Insight into the Elevator Mechanism of Membrane Transport. Structure 24:956-64|
|Bai, Yonghong; McCoy, Jason G; Levin, Elena J et al. (2015) X-ray structure of a mammalian stearoyl-CoA desaturase. Nature 524:252-6|
|McCoy, Jason G; Levin, Elena J; Zhou, Ming (2015) Structural insight into the PTS sugar transporter EIIC. Biochim Biophys Acta 1850:577-85|
|Levin, Elena J; Zhou, Ming (2014) Recent progress on the structure and function of the TrkH/KtrB ion channel. Curr Opin Struct Biol 27:95-101|
|Zhou, Xiaoming; Levin, Elena J; Pan, Yaping et al. (2014) Structural basis of the alternating-access mechanism in a bile acid transporter. Nature 505:569-73|
|Levin, Elena J; Zhou, Ming (2014) Structure of urea transporters. Subcell Biochem 73:65-78|
|Huang, Hua; Levin, Elena J; Liu, Shian et al. (2014) Structure of a membrane-embedded prenyltransferase homologous to UBIAD1. PLoS Biol 12:e1001911|
|Coudray, Nicolas; Valvo, Salvatore; Hu, Minghui et al. (2013) Inward-facing conformation of the zinc transporter YiiP revealed by cryoelectron microscopy. Proc Natl Acad Sci U S A 110:2140-5|
|Pan, Yaping; Levin, Elena J; Quick, Matthias et al. (2013) Correction to Potentiation of the Kv1 family K(+) Channel by Cortisone Analogues. ACS Chem Biol 8:268|
Showing the most recent 10 out of 17 publications