Kidney Week

Abstract: TH-PO597

Disruption of Ca2+-Binding in the EF-Hand Domain of Polycystin-2 Does Not Result in a Cystic Phenotype

Session Information

• Cystic Kidney Diseases - I
  November 02, 2017 | Location: Hall H, Morial Convention Center
  Abstract Time: 10:00 AM - 10:00 AM

Category: Genetic Diseases of the Kidney

• 801 Cystic Kidney Diseases

Authors

• Krappitz, Matteus, Yale University School of Medicine, New Haven, Connecticut, United States

Matteus Krappitz,

• Dong, Ke, Yale University School of Medicine, New Haven, Connecticut, United States

Ke Dong,

• Fedeles, Sorin V., Yale University School of Medicine, New Haven, Connecticut, United States

Sorin V. Fedeles,

• Gallagher, Rachel, Yale University School of Medicine, New Haven, Connecticut, United States

Rachel Gallagher,

• Cai, Yiqiang, Yale University School of Medicine, New Haven, Connecticut, United States

Yiqiang Cai,

• Somlo, Stefan, Yale University School of Medicine, New Haven, Connecticut, United States

Stefan Somlo,

Background

Polycystin-2 (PC2/TRPP2), the product of one of the genes mutated in ADPKD, is expressed in the endoplasmic reticulum (ER) and ciliary membranes. The COOH-terminal tail of PC2 contains 2 potential Ca²⁺ binding EF hand motifs, an ER retention domain and a coiled-coiled domain. Only the second EF hand motif can bind Ca²⁺. It has been proposed that a mutation in this EF hand abrogates Ca²⁺ binding which leads to a substantial reduction of channel function resulting in decreased inward Ca²⁺ currents conducted by PC2. This study investigates the relevance of abolishing the Ca²⁺ binding properties of the EF-hand domain in PC2 by generating a mouse model mutated at critical residues.

Methods

CRISPR/Cas9 methodology was used to generate a mouse model termed Pkd2^TEAA, in which critical residues at the EF hand –z, –x coordination vertices were substituted with alanine (T769A, E772A) to eliminate Ca²⁺ binding. Mutant alleles were further modified by insertion of a V5 epitope tag in-frame at the C-terminus.

Results

The animals were aged up to 18 months. All organs were extracted and analyzed. The kidney and livers were examined by measuring kidney weight (KW) and liver weight (LW) as a fraction of body weight (BW) and by detailed histological examination. Expression of the mutated protein in the kidney was confirmed by immunoblot analysis with anti-V5. Homozygous Pkd2^TEAA/TEAA animals survive up to and past 18 months of age with no histological signs of cystic disease in either kidney or liver. There was no significant difference in BW, KW or LW between wild type, Pkd2^TEAA/TEAA or Pkd2^+/- animals at the same age. The Pkd2^TEAA/+ mice were crossed with Pkd2^+/- animals to generate Pkd2^TEAA/- mice. These mice also did not display a kidney or liver cystic phenotype at 9 months of age.

Conclusion

Inactivation of the Ca²⁺-binding properties of PC2-EF hand does not result in a significant loss of PC2 function as evidenced by the lack of cystic disease phenotype in the kidney and livers of the Pkd2^TEAA/TEAA and Pkd2^TEAA/- animals. This suggests that the PC2 channel does not require EF-hand Ca²⁺ binding in order to function in vivo.

Funding

• NIDDK Support