On the Modulation of CFTR Channel Function by Sphingomyelinase and VX-770 Público

Abstract

Cystic Fibrosis (CF) is a debilitating disease that results from loss-of-function mutations in the Cystic Fibrosis Transmembrane Conductance Regulator chloride channel (CFTR). CF is associated with widespread exocrine dysfunction and patients most often succumb to pulmonary failure following the accumulation of mucus, inflammatory cells, and bacteria in the small airways. The discovery of small molecules that directly improve CFTR function, for example the potentiator VX-770, has ushered is a new era of CF therapeutics in which the goal is to directly improve protein ion channel function. The advent of CFTR pharmacotherapy provokes new questions about what kind of cellular and biophysical factors influence the efficacy of these novel potentiators. Sphingolipids are of particular interest because their metabolism is disrupted in CF and the breakdown of plasma membrane sphingomyelin by sphingomyelinase (SMase) has been shown to directly inhibit CFTR channel function. Despite potentially acting as a hurdle to successful treatment, the full extent of this interaction remains unknown. The Background section will provide an overview of CF pathophysiology, abnormalities in sphingolipid metabolism that have been associated with CF, and briefly discuss evidence suggesting that these two pathways interact. The first Results section will discuss experiments performed to characterize the functional interaction of sphingomyelin metabolism on CFTR channel function. Results herein provide evidence that SMase is a state-dependent gating modifier of CFTR. The second Results section will focus on our efforts to characterize the effect of VX-770 on CFTR channel function. Given that these CFTR potentiators are relatively new, much has yet to be determined about their mechanism of action and ideal screening parameters. Our data provide evidence that current small molecule screening approaches have likely underestimated the value of these drugs, and we provide rationale for how to improve screening approaches for CFTR potentiators. Finally, the Discussion will provide a broader context for our results regarding these two CFTR modulators and pose new questions for future studies.

Table of Contents

1) Introduction

1.1. Cystic Fibrosis: CFTR Dysfunction, Inflammation, and Infection

1.2. CFTR: An overview of Structure and Function

1.2.1. CFTR Function in Epithelia

1.2.2. CFTR Structure and Gating

1.2.3. CFTR Inhibitors

1.3. Cystic Fibrosis-Associated Mutations and Therapeutics

1.4. Sphingolipids in Epithelia and Cystic Fibrosis

1.4.1. General Lipid Considerations

1.4.2. Sphingolipid Handling in Epithelial Cells

1.4.3. Evidence for a Functional Interaction Between CFTR and Sphingolipid Metabolism

1.4.4. Consequences of Abnormal Sphingolipid Metabolism in CF - Infection

1.4.5. Consequences of Abnormal Sphingolipid Metabolism in CF - Inflammation

1.4.6. Evidence for Direct Interactions Between CFTR and Sphingolipids

1.4.7. Sphingomyelinase Activity Inhibits CFTR Channel Function in Some Cell Systems

2. Summary

3. Methods

3.1. Protein Purification

3.2. Amplex Red Sphingomyelinase Assay

3.3. Preparation of Oocytes and cRNA

3.4. Xenopus Oocyte Electrophysiology (TEVC and Inside-Out Patch Clamp)

3.5. Voltage Clamp Fluorometry

3.6. Xenopus Oocyte cell-ELISA

3.7. Xenopus Oocyte Biotinylation

3.8. Ussing Chamber Electrophysiology

3.9. Statistics

4. Results

4.1. Primary Research Project: Inhibition of CFTR Currents by SMase in Xenopus Oocytes and Human Bronchial Epithelial Cells

4.1.1. Introduction

4.1.2. Results

4.1.3. Discussion

4.2. Collaborative Research Project: Phosphorylation Dependence of CFTR Potentiation by VX-770

4.2.1. Introduction

4.2.2. Results

5. General Discussion and Future Directions

5.1. On the inhibition of CFTR channel activity by SMase

5.2. On the potentiation of CFTR by VX-770

6. Concluding Remarks

7. Appendices - Synopses of Coauthored Works

7.1. Potentiators exert distinct effects on human, murine, and Xenopus CFTR. Cui G, Khazanov N, Stauffer BB, Infield DT, Imhoff BR, Senderowitz H, McCarty NA. AJP: Lung Cellular and Molecular Physiology 2016 June:311(2):L192-207

7.2. Junctional Abnormalities In Human Airway Epithelial Cells Expressing F508del CFTR. Molina SA, Stauffer BB, Moriarty HK, Kim AH, McCarty NA, Koval M. AJP: Lung Cellular and Molecular Physiology 2015 Sep.;309(5):L475-L487.

7.3. Inflammation and ER stress downregulate BDH2 expression and dysregulate intracellular iron in macrophages. Zughaier SM, Stauffer BB, McCarty NA. J Immunol Res. 2014;2014(11):1-16.

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Molecular & Systems Pharmacology
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Health Sciences, Pharmacology
Palabra Clave	Ceramide Sphingomyelinase Cystic Fibrosis Cystic Fibrosis Transmembrane Conductance Regulator CFTR
Committee Chair / Thesis Advisor	McCarty, Nael, Emory University
Committee Members	Hall, Randy A, Emory University Hartzell Jr., Criss, Emory University Jenkins, Andrew, Emory University Balog, Edward, GA Tech

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