Reference number : GC-2017-Q1-02713, amendment number : , agreement type : G, recipient legal name : Chappe, Valerie (Dalhousie University), recipient country : CA, recipient province : nan, recipient city : Nova Scotia|Nouvelle-Écosse, recipient postal code : nan, program name english : Discovery Grants Program - Individual, program name french : Programme de subventions à la découverte - individuelles, program purpose english : ABC transporters form one of the largest superfamily of membrane proteins which are found in all organisms from bacteria to plants and human. They use the energy generated by ATP binding and hydrolysis to translocate a wide variety of substrates across membranes. Human ABC proteins are mostly exporters involved in secretion, drug detoxification and antigen presentation. Despite the evolutionary divergence and heterogeneity of transported substrates, the basic architecture of ABC transporters is highly conserved. Many questions remain unanswered on ABCs mechanism of transport, especially for eukaryotic transporters for which high resolution 3D structures are limited. Biochemical and functional studies to evaluate the physiological significance of structural prediction based on bacterial transporters are thus needed. ABCC7 or CFTR (Cystic Fibrosis Transmembrane conductance Regulator) is an ATP-dependent, phosphorylation-activated Cl- channel mainly expressed in epithelial cells of the lung, intestine and pancreas. It uses the fundamental mechanism shared by all asymmetric ABC proteins driven by the formation/dissociation of their Nucleotide Binding Domains (NBD1/NBD2). CFTR contains additional domains with physiological significance: a regulatory extension (RE) in NBD1 and a regulatory domain (RD) which plays a key role in the channel activation by Protein kinases A and C. Despite this divergence, CFTR still retains important structural characteristics of asymmetric exporters. The mechanism by which the deferentially phosphorylated RD controls CFTR function remains an important issue to explore. Our main objective is to elucidate the mechanism by which phosphorylation promotes or restrains domains' interactions to regulate chloride secretion. More specifically, we will: 1) Map important interaction sites between the phosphorylated RD and other CFTR domains and identify phosphorylation sites involved. We propose that different phosphorylation profiles of the RD are correlated to changes in binding strength and sites with other domains; 2) Determine the functional significance of phosphorylation-dependent changes in domains interaction. We will study the impact of changes in domains interaction by phosphorylation on CFTR membrane stability and abundance at the cell surface. 3) Study the functional relevance of the current hypothesis that the phosphorylated RD regulates CFTR gating by restraining large movements of NBDs, favoring the dimer stability. The RD is unique to CFTR, and much remains to be elucidated on its role in CFTR gating. 
Our research will help elucidate fundamental aspects of CFTR regulation which are specific to the asymmetric mode of transport. Moreover, we will obtain physiologically relevant models on CFTR structure-function relationship that will complement current structural studies., program purpose french : ABC transporters form one of the largest superfamily of membrane proteins which are found in all organisms from bacteria to plants and human. They use the energy generated by ATP binding and hydrolysis to translocate a wide variety of substrates across membranes. Human ABC proteins are mostly exporters involved in secretion, drug detoxification and antigen presentation. Despite the evolutionary divergence and heterogeneity of transported substrates, the basic architecture of ABC transporters is highly conserved. Many questions remain unanswered on ABCs mechanism of transport, especially for eukaryotic transporters for which high resolution 3D structures are limited. Biochemical and functional studies to evaluate the physiological significance of structural prediction based on bacterial transporters are thus needed. ABCC7 or CFTR (Cystic Fibrosis Transmembrane conductance Regulator) is an ATP-dependent, phosphorylation-activated Cl- channel mainly expressed in epithelial cells of the lung, intestine and pancreas. It uses the fundamental mechanism shared by all asymmetric ABC proteins driven by the formation/dissociation of their Nucleotide Binding Domains (NBD1/NBD2). CFTR contains additional domains with physiological significance: a regulatory extension (RE) in NBD1 and a regulatory domain (RD) which plays a key role in the channel activation by Protein kinases A and C. Despite this divergence, CFTR still retains important structural characteristics of asymmetric exporters. The mechanism by which the deferentially phosphorylated RD controls CFTR function remains an important issue to explore. Our main objective is to elucidate the mechanism by which phosphorylation promotes or restrains domains' interactions to regulate chloride secretion. More specifically, we will: 1) Map important interaction sites between the phosphorylated RD and other CFTR domains and identify phosphorylation sites involved. We propose that different phosphorylation profiles of the RD are correlated to changes in binding strength and sites with other domains; 2) Determine the functional significance of phosphorylation-dependent changes in domains interaction. We will study the impact of changes in domains interaction by phosphorylation on CFTR membrane stability and abundance at the cell surface. 3) Study the functional relevance of the current hypothesis that the phosphorylated RD regulates CFTR gating by restraining large movements of NBDs, favoring the dimer stability. The RD is unique to CFTR, and much remains to be elucidated on its role in CFTR gating. 
Our research will help elucidate fundamental aspects of CFTR regulation which are specific to the asymmetric mode of transport. Moreover, we will obtain physiologically relevant models on CFTR structure-function relationship that will complement current structural studies., agreement title english : Structure/function study of the CFTR (ABCC7) chloride channel, agreement title french : Structure/function study of the CFTR (ABCC7) chloride channel, agreement number : RGPIN, agreement value : 130000.0, agreement start date : 2017-05-10, agreement end date : nan, description english : nan, description french : nan, expected results english : nan, expected results french : nan, additional information english : Grant or Award spanning more than one fiscal year. (2017-2018 to 2022-2023), additional information french : Subvention ou bourse octroyée s'appliquant à plus d'un exercice financier. (2017-2018 à 2022-2023), owner organization : nserc-crsng, owner organization title : Natural Sciences and Engineering Research Council of Canada | Conseil de recherches en sciences naturelles et en génie du Canada