Studies of the structure, regulation, and pharmacology of the cystic fibrosis transmembrane conductance regulator Open Access

Infield, Danny (2016)

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Cystic Fibrosis (CF) is a devastating, life-shortening disease affecting approximately 70,000 patients worldwide. The research in this dissertation concerns the structure, function, and regulation of the product of the gene responsible for CF, an anion channel called the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). The Introduction (Chapter 1) primarily serves to provide information relevant to our understanding of CF pathogenesis and of CFTR. The Methods (Chapter 2) detail the techniques I learned and modified to perform experiments testing CFTR function. The Results section (Chapter 3) is comprised of three related sections detailing discoveries on the conformational dynamics, regulation, and pharmacology of CFTR. Finally, the Discussion (Chapter 4) interprets the Results in light of existing knowledge on CFTR.

Table of Contents

Chapter 1: Introduction. 1

1.1 A brief history of research on CF pathophysiology. 2

1.2 Cloning and characterization of the CF gene. 5

1.3 Physiological Roles of CFTR. 21

1.4 Discovery of a clinically efficacious potentiator of CFTR. 27

1.5 CFTR: the evolution of structure and function. 31

1.6 Statement of research questions. 62

References. 63

Chapter 2: Methods. 80

2.1 Xenopus laevis husbandry and optimization of oocyte quality. 81

2.2 Electrophysiological methods to study CFTR. 96

2.3 Development of an assay to report on CFTR localization in oocytes. 101

References. 106

Chapter 3: Results. 109

3.1 The position of ECL1 affects the pore gating of CFTR. 109

3.2 Conformational restriction between ECL1 and ECL4 alters the sensitivity of CFTR to activation by phosphorylation. 125

3.3 CFTR lacking the regulatory R-domain is highly potentiated by Ivacaftor, an FDA-approved CFTR potentiator. 130

References. 133

Chapter 4: Discussion. 136

4.1 A structural model of CFTR pore gating that relates it to the transport cycle of ABC exporters. 137

4.2 Integrating the present studies into an energetic model of CFTR pore gating. 145

4.3 Evidence for allosteric modulation of CFTR activity by phosphorylation Status of the R-domain. 153

4.4 Insights into the mechanism of action of Ivacaftor enabled by WT-ΔR-CFTR. 154

4.5 Conclusions. 158

References. 163

Appendix A: Localization of the Insulin receptor in primary airway cells. 167

Appendix B: Multiple sequence alignments used for analysis in section 1.5. 172

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