Mechanism of action of the ammonia gas channel AmtB in E. coli

描述(由申请人提供)：I.项目摘要：氨运输(AMT)蛋白和恒河猴(Rh)蛋白是生物体中仅有的两种已识别的生物气体通道。众所周知，RH蛋白是人类红细胞上的抗原，是二氧化碳的气体通道。它们是绿藻衣藻在高二氧化碳条件下最佳生长所必需的。AMT蛋白是NH3的气体通道，被称为Rh蛋白的祖先同系物。它们是肠道细菌大肠杆菌和鼠伤寒沙门氏菌在低NH3条件下最佳生长所必需的。由于细胞膜的磷脂双层对气体物种具有通透性，因此细胞需要气体通道的原因尚不清楚。在大肠杆菌和鼠伤寒沙门氏菌中的生理研究表明，在低NH3浓度下，AmtB转运NH3似乎与谷氨酰胺合成酶偶联，后者将NH3同化为谷氨酰胺。因此，在这个方案中，我们试图了解大肠杆菌中的AmtB是如何与谷氨酰胺合成酶功能偶联的，并假设AmtB和谷氨酰胺合成酶在物理上是关联的。AmtB和谷氨酰胺合成酶之间的物理接触可能允许NH3从AmtB的孔道直接输送到谷氨酰胺合成酶的活性部位，从而提高NH3的同化速率。这项建议的两个具体目标之一是确定AmtB的氨基酸突变，这些突变会在低NH3条件下损害生长和氨吸收活性。这些amtB突变不会导致AmtB的整体蛋白质错误折叠或AmtB孔功能的丧失，而是局部影响蛋白质的细胞质区域，这些区域是其与谷氨酰胺合成酶结合的部位。第二个具体目标是利用分子生物学和遗传学方法测试AmtB和GS之间的联系。 相关性：这项拟议的对E.ColiAmtB的研究将使我们能够更好地了解气体通道如何工作，以帮助维持健康的细胞生理。了解气体通道的功能将有助于我们识别人类疾病的功能障碍的基础，如Rh零病。

DESCRIPTION (provided by applicant): I. Project Summary: Ammonia transport (Amt) proteins and Rhesus (Rh) proteins are the only two identified biological gas channels among organisms. Rh proteins, which are best known as antigens on human red blood cells, are gas channels for CO2. They are required for optimal growth of the green alga Chlamydomonas reinhardtii at high C02. Amt proteins, which are known as the-ancestral homolog to Rh proteins, are gas channels for NH3. They are required for optimal growth of enteric bacteria Escherichia coli and Salmonella typhimurium at low NH3. Since phospholipid bilayers of the cell membrane are permeable to the gas species, the reason why gas channels are needed by cells is not known. Physiological studies in E. coli and S. typhimurium show that at low NH3 concentrations NH3 transport by AmtB appears to be coupled to glutamine synthetase which assimilates NH3 into glutamine. Therefore, in this proposal we seek to understand how AmtB in E. coli is functionally coupled to glutamine synthetase and hypothesize that AmtB and glutamine synthetase associate physically. The physical contact between AmtB and glutamine synthetase may allow the direct delivery of NH3 from the pore of AmtB to the active site of glutamine synthetase and hence increase the rate of assimilation of NH3. One of the two specific aims of this proposal is to identify amino acid mutations in AmtB that impair growth and ammonium uptake activity at low NH3. These amtB mutations should not cause global protein misfolding of AmtB or loss of function of the pore of AmtB but rather locally affect cytoplasmic regions of the protein that are its binding site for glutamine synthetase. The second specific aim is to test the association between AmtB and GS using molecular biology and genetics approaches. II. Relevance: This proposed study of E. coli AmtB will enable us to gain a better understanding of how gas channels work to help maintain healthy cell physiology. Knowing gas channel function will help us identify the basis of dysfunction in human disease such as Rh null disease.