1. Regulators: Protease and kinase regulation of epithelial transport Wednesday June 5, 2013 Mentor: Ray A-team: Kathi Jessica Buckley, Ricky Kim, Lucas Butler, Evan Levy, Amit Mittal, Azim Munivar, Lorenzo Senawan

2. Outline Protease regulation of ENaC – Cell biology of protease activation of ENaC – Experiment 1: Effect of cleavage site mutations – Experiment 2: Effect of protease inhibitors WNK regulation of ROMK – Cell biology of WNK inhibition of ROMK – Familial Hyperkalemic Hypertension – Experiment 3: WNK mutants in oocytes

3. Outline Protease regulation of ENaC – Cell biology of protease activation of ENaC – Experiment 1: Effect of cleavage site mutations – Experiment 2: Effect of protease inhibitors WNK regulation of ROMK – Cell biology of WNK inhibition of ROMK – Familial Hyperkalemic Hypertension – Experiment 3: WNK mutants in oocytes

4. Furin Cleaves 2 α and 1 γ on ECF side Golgi Membrane RXXR to RXXA Prostasin Cleaves γ on ECF Anchored on Apical PM Airway Epithelial Trypsin Non-specific on ECF Open Probability Proteases cleave ENaC and increase Pо

5. α γ β TRYPSIN FURIN PROSTASIN 1 2 F Pr T

6. Outline Protease regulation of ENaC – Cell biology of protease activation of ENaC – Experiment 1: Effect of cleavage site mutations – Experiment 2: Effect of protease inhibitors WNK regulation of ROMK – Cell biology of WNK inhibition of ROMK – Familial Hyperkalemic Hypertension – Experiment 3: WNK mutants in oocytes

7. ENaC protease regulation experiment Hypothesis: (a)Cleavage site mutations in ENaC subunits will decrease open probability and thus decrease current through the ENaC channel (b)Trypsin will “rescue” cleavage site mutants and restore wild-type open probability in the ENaC channel

8. Wild Type: F 1 F 2 Cleavage Site = RXXR vs. Mutant: F 1 F 2 Cleavage Site = RXXA I ringer – I ameloride = I ENaC 1.Wt → Ringer → → Amiloride → 2.αF 1 F 2 → Ringer → → Amiloride →

9. Wild Type: F 1 F 2 Cleavage Site = RXXR vs. Mutant: F 1 F 2 Cleavage Site = RXXA I ringer – I ameloride = I ENaC 1.Wt Baseline →...…→ Trypsin →...… → Amiloride 2.αF 1 F 2 Baseline →...…→ Trypsin →…… → Amiloride Trypsin is expected to cleave indiscriminately and restore normal function

10. Video: Oocyte Poking

11. Mutation in F 1 F 2 cleavage site decreases ENaC current. *

12. Trypsin affects ENaC current more for F 1 F 2 mutant than for WT.

13. Conclusion P o of ENaC increases with cleavage. Trypsin increases Na + current @ nonspecific sites. Trypsin has greater effect on ENaC F 1 F 2 mutant— 3 to 4 times more effective!

14. Outline Protease regulation of ENaC – Cell biology of protease activation of ENaC – Experiment 1: Effect of cleavage site mutations – Experiment 2: Effect of protease inhibitors WNK regulation of ROMK – Cell biology of WNK inhibition of ROMK – Familial Hyperkalemic Hypertension – Experiment 3: WNK mutants in oocytes

15. Aprotinin, 16hr Untreated Control t (min) 12 15 Current (μA) Inhibition of prostasin decreases ENaC activity Forskalin Trypsin Amiloride

16. Outline Protease regulation of ENaC – Cell biology of protease activation of ENaC – Experiment 1: Effect of cleavage site mutations – Experiment 2: Effect of protease inhibitors WNK regulation of ROMK – Cell biology of WNK inhibition of ROMK – Familial Hyperkalemic Hypertension – Experiment 3: WNK mutants in oocytes

17. WNK Regulation of ROMK ROMK – ATP Dependent Potassium Channel – Potassium Secretion – Found in Thick Ascending Limb and Cortical Collecting Duct WNK Kinase 1 – Removes ROMK from Cell Surface – Activated WNK 1 binds ROMK – Initiates clathrin-mediated endocytosis & removal of ROMK from plasma membrane

18. Outline Protease regulation of ENaC – Cell biology of protease activation of ENaC – Experiment 1: Effect of cleavage site mutations – Experiment 2: Effect of protease inhibitors WNK regulation of ROMK – Cell biology of WNK inhibition of ROMK – Experiment 3: WNK mutants in oocytes – Familial Hyperkalemic Hypertension

19. ROMK Experiment: How does WNK regulate ROMK? Xenopus Oocyte ROMK ROMK w/WNK ROMK w/ WNK Q562E ROMK cDNA ROMK mRNA WNK cDNA ROMK mRNA Primers RT-PCR Primers Directed mutagenesis RT-PCR WNK cDNA ROMK Q562E mRNA ND96Barium Chloride I_total I_total – I_K I_K - =

20. WNK1 tends to decrease ROMK current. WNK1mutation tends to restore ROMK current.

21. Outline Protease regulation of ENaC – Cell biology of protease activation of ENaC – Experiment 1: Effect of cleavage site mutations – Experiment 2: Effect of protease inhibitors WNK regulation of ROMK – Cell biology of WNK inhibition of ROMK – Experiment 3: WNK mutants in oocytes – Familial Hyperkalemic Hypertension

22. Familial Hyperkalemic Hypertension -WNK mutation leads to increase in WNK1 isoform -Decoupling of ENaC and ROMK -More ENaC expressed on surface (decreased inhibition of ENaC) -Reduced ROMK on surface due to ROMK endocytosis

23. Thank You Kathi and Ray!

25. No Questions.