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

描述（申请人提供）： 一、项目概要：氨转运（Amt）蛋白和恒河猴（Rh）蛋白是生物体中唯一已确定的两种生物气体通道。 Rh 蛋白是众所周知的人类红细胞抗原，是二氧化碳的气体通道。它们是绿藻莱茵衣藻在高CO 2 下最佳生长所必需的。 Amt 蛋白被称为 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渗透气体种类，因此细胞需要气体通道的原因尚不清楚。对大肠杆菌和鼠伤寒沙门氏菌的生理研究表明，在低 NH3 浓度下，AmtB 的 NH3 转运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，在本提案中，我们试图了解大肠杆菌中的 AmtB 如何与谷氨酰胺合成酶功能性偶联，并假设 AmtB 和谷氨酰胺合成酶发生物理关联。 AmtB 和谷氨酰胺合成酶之间的物理接触可以使 NH3 从 AmtB 孔直接输送到谷氨酰胺合成酶的活性位点，从而提高 NH3 的同化速率。该提案的两个具体目标之一是识别 AmtB 中的氨基酸突变，这些突变会损害低 NH3 条件下的生长和铵吸收活性。这些 amtB 突变不应导致 AmtB 整体蛋白质错误折叠或 AmtB 孔功能丧失，而是局部影响蛋白质的细胞质区域，即其与谷氨酰胺合成酶的结合位点。第二个具体目标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：这项针对大肠杆菌 AmtB 的拟议研究将使我们能够更好地了解气体通道如何发挥作用，帮助维持健康的细胞生理机能。了解气体通道功能将有助于我们确定人类疾病（如 Rh 缺失病）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.