Regulatory domain phosphorylation to distinguish the mechanistic basis underlying acute CFTR modulators

Authors

Louise C. Pyle, The University of Alabama at Birmingham
Annette Ehrhardt, The University of Alabama at Birmingham
Lisa High Mitchell, The University of Alabama at Birmingham
Lijuan Fan, The University of Alabama at Birmingham
Aixia Ren, University of Tennessee Health Science Center
Anjaparavanda P. Naren, University of Tennessee Health Science Center
Yao Li, The University of Alabama at Birmingham
J. P. Clancy, The University of Alabama at Birmingham
Graeme B. Bolger, The University of Alabama at Birmingham
Eric J. Sorscher, The University of Alabama at Birmingham
Steven M. Rowe, The University of Alabama at Birmingham

Document Type

Journal Article

Publication Date

10-1-2011

Journal

American Journal of Physiology - Lung Cellular and Molecular Physiology

Volume

301

DOI

10.1152/ajplung.00465.2010

Keywords

Cystic fibrosis; Potentiators; Protein kinase A; Short-circuit current

Abstract

Modulator compounds intended to overcome disease-causing mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) show significant promise in clinical testing for cystic fibrosis. However, the mechanism(s) of action underlying these compounds are not fully understood. Activation of CFTR ion transport requires PKA-regulated phosphorylation of the regulatory domain (R-D) and dimerization of the nucleotide binding domains. Using a newly developed assay, we evaluated nine compounds including both CFTR potentatiators and activators discovered via various high-throughput screening strategies to acutely augment CFTR activity. We found considerable differences in the effects on R-D phosphorylation. Some (including UC CF-152) stimulated robust phosphorylation, and others had little effect (e.g., VRT-532 and VX-770). We then compared CFTR activation by UC CF-152 and VRT-532 in Ussing chamber studies using two epithelial models, CFBE41o - and Fischer rat thyroid cells, expressing various CFTR forms. UC CF-152 activated wild-type-, G551D-, and rescued F508del-CFTR currents but did not potentiate cAMP-mediated CFTR activation. In contrast, VRT-532 moderately activated CFTR short-circuit current and strongly potentiated forskolin-mediated current. Combined with the result that UC CF-152, but not VRT-532 or VX-770, acts by increasing CFTR R-D phosphorylation, these findings indicate that potentiation of endogenous cAMP-mediated activation of mutant CFTR is not due to a pathway involving augmented R-D phosphorylation. This study presents an assay useful to distinguish preclinical compounds by a crucial mechanism underlying CFTR activation, delineates two types of compound able to acutely augment CFTR activity (e.g., activators and potentiators), and demonstrates that a number of different mechanisms can be successfully employed to activate mutant CFTR. © the American Physiological Society.

APA Citation

Pyle, L., Ehrhardt, A., Mitchell, L., Fan, L., Ren, A., Naren, A., Li, Y., Clancy, J., Bolger, G., Sorscher, E., & Rowe, S. (2011). Regulatory domain phosphorylation to distinguish the mechanistic basis underlying acute CFTR modulators. American Journal of Physiology - Lung Cellular and Molecular Physiology, 301 (). http://dx.doi.org/10.1152/ajplung.00465.2010