? The Cystic Fibrosis Transmembrane conductance Regulator (CFTR) is a chloride channel that is regulated by phosphorylation and gated by ATP binding and hydrolysis. Mutations in the gene coding for CFTR result in the genetic disease cystic fibrosis (CF). The long term goal of our research is to understand how CFTR works at the molecular level and how mutations in CFTR cause defective functions, to apply the acquired knowledge in therapeutic design for CF.
The first aim of this proposal is to investigate the pathophysiology of the disease-associated CFTR mutant G551D. The result of the glycine-to-aspartate mutation at position 551 is a strongly decreased chloride current. Since G551 is located in the signature sequence of the nucleotide binding domain 1 (NBD1), which together with the Walker A and Walker B regions of nucleotide binding domain 2 (NBD2) form one of the ATP binding pockets, we hypothesize that the mutation of a neutral glycine to a negatively charged aspartate may disrupt the binding of nucleotides and/or the NBD dimerization process due to electrostatic repulsion between the aspartate and the bound ATP molecule. The observed low activity of G551D-CFTR may be spontaneous, ATP-independent openings. Preliminary data show that ATP fails to enhance G551D-CFTR channel activity, whereas the positively charged ions such as Zn and Cd can increase the channel activity of G551D-CFTR. This effect of Cd is not seen in the wild-type channel, suggesting that aspartate 551, together with a yet-to-be identified residue, is involved in coordinating Cd.
The second aim i s to investigate the relationship between the formation of a dimer and the open state in WT-CFTR. Our preliminary data suggest that all that is needed to promote G551D-CFTR channel opening is something that will attract and hold the NBDs together. We will test this hypothesis in WT-CFTR channels with different cysteines pairs engineered in the NBDs and use Cd to promote dimerization.
The third aim i s to study the mechanisms of pharmacological modulation of CFTR. We will test the hypothesis that genistein, the most studied of the CFTR activators, binds at the dimer interface, using a combination of electrophysiological, molecular biological and molecular modeling methodologies. Results from this work will provide invaluable information to help the understanding of one of the most common disease-associated mutations in patients with CF as well as shed light on the mechanisms that enhance CFTR activity. ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Scientist Development Award - Research & Training (K01)
Project #
5K01DK075408-03
Application #
7479166
Study Section
Diabetes, Endocrinology and Metabolic Diseases B Subcommittee (DDK)
Program Officer
Hyde, James F
Project Start
2006-09-01
Project End
2010-08-31
Budget Start
2008-09-01
Budget End
2010-08-31
Support Year
3
Fiscal Year
2008
Total Cost
$109,665
Indirect Cost
Name
University of Missouri-Columbia
Department
Pharmacology
Type
Schools of Medicine
DUNS #
153890272
City
Columbia
State
MO
Country
United States
Zip Code
65211
Yu, Ying-Chun; Miki, Haruna; Nakamura, Yumi et al. (2011) Curcumin and genistein additively potentiate G551D-CFTR. J Cyst Fibros 10:243-52
Jih, Kang-Yang; Li, Min; Hwang, Tzyh-Chang et al. (2011) The most common cystic fibrosis-associated mutation destabilizes the dimeric state of the nucleotide-binding domains of CFTR. J Physiol 589:2719-31
Miki, Haruna; Zhou, Zhen; Li, Min et al. (2010) Potentiation of disease-associated cystic fibrosis transmembrane conductance regulator mutants by hydrolyzable ATP analogs. J Biol Chem 285:19967-75
Wang, Xiaohui; Bompadre, Silvia G; Li, Min et al. (2009) Mutations at the signature sequence of CFTR create a Cd(2+)-gated chloride channel. J Gen Physiol 133:69-77
Bompadre, Silvia G; Li, Min; Hwang, Tzyh-Chang (2008) Mechanism of G551D-CFTR (cystic fibrosis transmembrane conductance regulator) potentiation by a high affinity ATP analog. J Biol Chem 283:5364-9
Bompadre, Silvia G; Sohma, Yoshiro; Li, Min et al. (2007) G551D and G1349D, two CF-associated mutations in the signature sequences of CFTR, exhibit distinct gating defects. J Gen Physiol 129:285-98