Chloride channels play crucial roles in many aspects of cell physiology. The genes encoding these channels are the loci for several human diseases relevant to the NIDDK. Understanding the structure and function of these proteins, and development of pharmaceutical agents targeting them, relies upon the availability of specific, high-affinity probes. The goal of this proposal is to characterize a novel peptide inhibitor which interacts with high affinity with the CFTR chloride channel. CFTR is defective in the lethal genetic disease, Cystic Fibrosis (CF), and also plays an important role in polycystic kidney disease (PKD) and secretory diarrhea. Peptide toxins from animal venom are among the most selective and useful tools for the study of ion channels; however, until now, no peptide toxins have been found that interact with anion channels of known molecular identity. This laboratory recently isolated a peptide toxin that inhibits CFTR. The novel toxin, """"""""GaTx1"""""""", inhibits CFTR in a state-dependent manner by locking channels into a long closed state. Hence, GaTx1 represents a quantum advance in how we can approach structure/function studies in CFTR, compared to the structural probes currently available. The present application proposes a series of objectives to characterize GaTx1, in three aims as follows.
Aim 1 is to characterize the wildtype toxin by: determining kinetics of inhibition using single-channel patch clamp and macropatch recording, determining effects on ATP binding and hydrolysis by purified CFTR cytosolic domain polypeptides, and asking whether GaTx1 inhibits the conformational change underlying gating of the channel pore itself.
Aim 2 is to localize the toxin's binding site by a series of independent studies using electrophysiological and biochemical approaches followed by site-directed mutagenesis.
Aim 3 is to identify determinants of activity by mutating the toxin itself, leading to identification of the interacting surfaces. The approach takes advantage of a list of highly qualified collaborators with expertise complementary to that of the PI's lab. This work will provide the unique opportunity to use the GaTx1 toxin as a research tool, and also will likely aid in the design of novel therapeutics for CF, PKD, secretory diarrhea, and other pathologies that involve CFTR.

Public Health Relevance

TO DISEASE The gene defective in the lethal genetic disease cystic fibrosis encodes the CFTR protein, which forms a chloride ion channel expressed in many epithelial cell types. Studies of the structure and function of this clinically-relevant protein have been hampered by the lack of pharmacologically useful tools. This laboratory has isolated a peptide toxin inhibitor of CFTR, which locks the channel closed with high affinity and high specificity;the present proposal aims to characterize the toxin, GaTx1, and to use it to understand how the CFTR channel protein is regulated.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK075016-05
Application #
8460503
Study Section
Cellular and Molecular Biology of the Kidney Study Section (CMBK)
Program Officer
Mckeon, Catherine T
Project Start
2009-07-01
Project End
2014-04-30
Budget Start
2013-05-01
Budget End
2014-04-30
Support Year
5
Fiscal Year
2013
Total Cost
$316,251
Indirect Cost
$110,540
Name
Emory University
Department
Pediatrics
Type
Schools of Medicine
DUNS #
066469933
City
Atlanta
State
GA
Country
United States
Zip Code
30322
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Infield, Daniel T; Cui, Guiying; Kuang, Christopher et al. (2016) Positioning of extracellular loop 1 affects pore gating of the cystic fibrosis transmembrane conductance regulator. Am J Physiol Lung Cell Mol Physiol 310:L403-14
Cui, Guiying; McCarty, Nael A (2015) Murine and human CFTR exhibit different sensitivities to CFTR potentiators. Am J Physiol Lung Cell Mol Physiol 309:L687-99
Cui, Guiying; Freeman, Cody S; Knotts, Taylor et al. (2013) Two salt bridges differentially contribute to the maintenance of cystic fibrosis transmembrane conductance regulator (CFTR) channel function. J Biol Chem 288:20758-67
Rishishwar, Lavanya; Varghese, Neha; Tyagi, Eishita et al. (2012) Relating the disease mutation spectrum to the evolution of the cystic fibrosis transmembrane conductance regulator (CFTR). PLoS One 7:e42336