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

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

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

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

α γ β TRYPSIN FURIN PROSTASIN 1 2 F Pr T

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

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

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 →

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

Video: Oocyte Poking

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

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

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

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

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

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

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

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

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 - =

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

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

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

Thank You Kathi and Ray!

No Questions.