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

描述（申请人提供）：I.项目概述：氨转运蛋白（Amt）和恒河猴蛋白（Rh）是生物体中仅有的两种已鉴定的生物气体通道。Rh蛋白是人体红细胞上的抗原，是二氧化碳的气体通道。它们是绿色莱茵衣藻在高CO2下最佳生长所必需的。Amt蛋白是Rh蛋白的祖先同源物，是NH3的气体通道。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可透过气体物质，因此细胞需要气体通道的原因尚不清楚。对E. coli和革兰氏阳性菌S.鼠伤寒菌显示，在低NH3浓度下，AmtB转运NH3似乎与谷氨酰胺合成酶偶联，该合成酶将NH3同化为谷氨酰胺。因此，在本提案中，我们试图了解AmtB在E. coli中的AmtB与谷氨酰胺合成酶功能性偶联，并假设AmtB与谷氨酰胺合成酶物理结合。AmtB和谷氨酰胺合成酶之间的物理接触可允许NH3从AmtB的孔直接递送到谷氨酰胺合成酶的活性位点，并因此增加NH3的同化速率。该提议的两个具体目的之一是鉴定AmtB中在低NH3下损害生长和铵吸收活性的氨基酸突变。这些amtB突变不应引起AmtB的整体蛋白质错误折叠或AmtB孔功能的丧失，而是局部影响作为其谷氨酰胺合成酶结合位点的蛋白质的胞质区域。第二个具体目的是使用分子生物学和遗传学方法测试AmtB和GS之间的关联。 二.相关性：本研究拟对E. coliAmtB将使我们能够更好地了解气体通道是如何工作以帮助维持健康的细胞生理学。了解气体通道功能将有助于我们确定人类疾病如Rh阴性疾病中功能障碍的基础。

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.