CFTR and interacting proteins from shark rectal gland

鲨鱼直肠腺的 CFTR 和相互作用蛋白

• 批准号: 6440235
• 负责人: JOHN R RIORDAN
• 金额: $ 15.7万
• 依托单位: MAYO CLINIC ARIZONA
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-02-15 至 2004-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6440235
• 关键词: chloride channels; crystallization; mass spectrometry; protein isoforms; protein protein interaction; protein purification; protein structure; proteomics; salt gland; sharks; western blottings

DESCRIPTION (provided by applicant): The cystic fibrosis transmembrane conductance regulator (CFTR) glycoprotein product of the gene mutated in patients with cystic fibrosis is a member of the largest known class of membrane proteins, the ABC (adenine nucleotide binding cassette) superfamily. However, CFTR is a novel ABC protein as the only one known to be an ion channel. Although there has been considerable debate, it is now generally agreed that the ABC protein structural organization which has been so successful evolutionarily was also adapted in metazoa to form an epithelial chloride channel regulated by phosphorylation and by nucleotide interactions at the characteristic ABC nucleotide binding domains (NBDs). A major impediment to more rapid advances in elucidating the structure‑function relationships of the CFTR protein has been the lack of a rich natural or recombinant source of the protein for purification. The levels of expression achievable in heterolgous mammalian cell expression systems is more than an order of magnitude lower than that of other ABC proteins such as P-glycoprotein for which a low resolution 3-dimensional structure has been obtained by electron diffraction of two dimensional crystals. We have previously purified and reconstituted CFTR expressed from insect cells but a strong tendency to aggregate has hindered attempts to obtain crystalline arrays. We have more recently found that native CFTR can be more readily purified from shark rectal gland, the only accessible solid tissue from which significant amounts of the protein can be obtained. Therefore the first specific aim of the present project is to further explore the feasibility of purifying sufficient quantities of functional CFTR to enable crystallization trials and structure determination. The second related specific aim is to determine the molecular identity of the other larger conductance chloride channel present with CFTR in the apical membranes of the rectal gland tubular cells. This objective will be pursued on the basis of two distinct hypotheses: either that this larger conductance channel may be an isoform of a member of the CIC channel family or that, since it is activated by cyclic AMP, it may interact directly with CFTR. It is possible that both of these hypotheses may hold. However, demonstration of the feasibility of employing this highly-specialized non-mammalian model tissue for either of these purposes should serve to advance understanding of the mechanism whereby CFTR functions and controls chloride secretion.

描述（由申请人提供）：