Kidney Week

Abstract: FR-OR006

Regulation of PKD2 Channel Function by PKD1

Session Information

• Advances in PKD: From Omics to Therapeutics
  November 08, 2019 | Location: 143, Walter E. Washington Convention Center
  Abstract Time: 05:30 PM - 05:42 PM

Category: Genetic Diseases of the Kidneys

• 1001 Genetic Diseases of the Kidneys: Cystic

Authors

• Padhy, Biswajit, University of Iowa Carver College of Medicine, Iowa City, Iowa, United States

Biswajit Padhy, PhD

• An, Sung Wan, University of Iowa Carver College of Medicine, Iowa City, Iowa, United States

Sung Wan An,

• Xie, Jian, University of Iowa Carver College of Medicine, Iowa City, Iowa, United States

Jian Xie,

• Huang, Chou-Long, University of Iowa Carver College of Medicine, Iowa City, Iowa, United States

Chou-Long Huang,

Background

Mutations of PKD1 and 2 cause autosomal-dominant polycystic kidney disease (ADPKD). PKD1 peptide has a very large extracellular N-terminus (Nt), 11 transmembrane (TM) domains, and intracellular C-terminus (Ct). PKD2 is 6-TM domains channel with intracellular N and C-terminus. The function and relationship of PKD1 and PKD2 and in the pathogenesis of ADPKD remain elusive. Previous studies show PKD1 and PKD2 interact with its C-terminus. Recent cryo-EM structural studies reveal the last 6 TM of PKD1 can interact with PKD2 to form presumably non-functional channel complexes.

Methods

Wild-type (WT) PKD2 and phenylalanine-604 to proline (F604P) mutant PKD2 were expressed in Xenopus oocytes with or without WT and mutant PKD1. PKD2 channel function was studied by using two-electrode voltage-clamp. Extracellular Ca²⁺-inhibitable inward K⁺ currents were measured.

Results

We found that currents in oocytes expressing WT- PKD2 were not different from in control oocytes. WT-PKD1 itself did not produce currents, and had no effects on WT-PKD2 currents when coexpression with it. It is known that F604P mutation on the 5^th TM domain of PKD2 leads to widening of the lower gate and constitutive activation of the channel. We then used F604P-PKD2 to study the regulation by PKD1. Oocytes expressing F604P-PKD2 showed ~3 fold higher currents than background currents in control oocytes, confirming gain-of-function of F604P. Coexpression with WT-PKD1 completely inhibited F604P-PKD2, to the level of control oocytes. To map regions of PKD1 that regulate PKD2, we made five PKD1 mutant constructs with deletion of Nt, deletion of Ct, deletion of the last 6 TM domains, deletion of both Nt and Ct or deletion of Nt as well as the first 5 TM domain. Comparing with WT-PKD1, deletion of Nt and/or the first 5 TM did not alter PKD1's ability to inhibit F604P-PKD2. Deletion of Ct or the last 6 TM resulted in partial reduction in the ability of PKD1 to inhibit F604P-PKD2.

Conclusion

PKD1 inhibits PKD2 channel activity. The C-terminus and last 6 TM domains of PKD1 are involved. The results support the implication of structural studies that PKD1 and PKD2 complexes are non-conducting. Further, they support the hypothesis that ligands or factors target PKD1 to activate channel activities of PKD1/PKD2 complexes.

Funding

• Private Foundation Support