Correcting Airway dehydration in Cystic Fibrosis BRETT ROLLINS March 5, 2009

Context of Study: Cystic Fibrosis Normal Cell Surface Collapsed Cilia Extended Cilia Airway Surface Liquid (ASL) Mucus Layer Dehydrated ASL Thick/Static Mucus Layer Airways Airways Liver Pancreas Small Intestine Reproductive Tract Cystic Fibrosis Skin

Airway Ion Transport + Proteases CFTR ENaC cAMP - + Na+ Cl- / HCO3-

Airway Ion Transport in CF + Proteases ENaC + cAMP Na+ Cl- / HCO3-

Channel Activating Proteases cleanve and activate ENaC Trypsin CAP Active a g b a

CAP Inhibitors Prevent Cleavage and Activation of ENaC Channel Activating Protease a g b Na+ CAP CAP-Inhibitor Inactive

ENaC is Spontaneously Inhibited over Time in Bronchial Cultures Normal Lung CF Lung Cultured Human Lung Cells V Apical Side Cilia Cell Basolateral Side Tarran et al 2006 ENaC is Active ENaC is Inactive

How To Identify This CAP Inhibitor Add Trypsin-coated beads to the apical side of Normal and CF cells Identify bound proteins by mass spectrometry

Palate, LUng & Nasal epithelial Clone Short PLUNC1 a.k.a. SPLUNC1(256 aa’s) Bingle & Bingle, 2000

SPLUNC1 is typically found in the upper airways & glands Campos et al., 2004

Goals of Project Further Understand Protease Regulation of ENaC Characterize Defective Protease Regulation in CF Use These Findings to Develop Strategies to Treat Overactive ENaC in CF

Experimental Questions If Splunc1 is removed will bronchial cultures be able to properly regulate airway surface liquid levels? Does Splunc1 alone inhibit ENaC conductance?

SPLUNC1 Is Necessary for ASL Regulation in Bronchial Cultures SPLUNC1 knockout SPLUNC1 KO + rSPLUNC1 Liquid Layer Cilia Cells Height of Liquid Layer (mm) ASL height (mm) Control ± rSPLUNC1 time (hours) t (h)

Xenopus Oocyte Injection and Voltage Clamp Recording cRNA Injection Voltage Clamp Voltage Electrode Current Electrode - Reference Electrode + Perfusion Solution Waste 24 hours Xenopus Oocyte Xenopus Oocyte

Voltage Clamp Set-Up Xenopus Oocyte ENaC Inhibitor Wash Buffer ENaC Inhibitor + Trypsin (protease) Wash Buffer ENaC Inhibitor 0nA Baseline Trypisin Activated 2000nA

Splunc-1 is Sufficient To Inhibit ENaC Current Conductance * p< 0.05

Splunc1 Activity is pH Sensitive CF pH Conditions * Percent Inhibition * *

Key Points Channel Activating Proteases Regulate ENaC Activity in Human Bronchial Cultures Splunc1 Is Necessary For ASL Regulation, As Shown By Knockdown Techniques Splunc1 Is Sufficient For ENaC Current Conductance Inhibition, As Shown By Voltage Clamping Techniques

Model of Normal ASL Volume Regulation Na+ CAP (active) ADO CFTR ENaC CAP (inactive) Cl- CFTR ENaC HCO3-/ Cl-