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

2. 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

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

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

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

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

7. 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

8. 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

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

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

11. 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

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

13. 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)

14. 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

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

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

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

18. 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

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