Kidney Week

Abstract: FR-OR13

Electrophysiological Analysis of a Gain-of-Function Polycystin-1/Polycystin-2 Ion Channel

Session Information

• Cystic Kidney Diseases: Basic and Translational Research
  October 25, 2024 | Location: Room 23, Convention Center
  Abstract Time: 05:00 PM - 05:10 PM

Category: Genetic Diseases of the Kidneys

• 1201 Genetic Diseases of the Kidneys: Cystic

Authors

• Staudner, Tobias, Friedrich-Alexander-Universitat Erlangen-Nurnberg Institut fur Zellulare und Molekulare Physiologie, Erlangen, Bayern, Germany

Tobias Staudner, DrMed

• Khamseekaew, Juthamas, Friedrich-Alexander-Universitat Erlangen-Nurnberg Institut fur Zellulare und Molekulare Physiologie, Erlangen, Bayern, Germany

Juthamas Khamseekaew, PhD

• Madej, M. Gregor, Universitat Regensburg Fakultat fur Biologie und Vorklinische Medizin, Regensburg, Bayern, Germany

M. Gregor Madej, PhD

• Geiges, Linda, Friedrich-Alexander-Universitat Erlangen-Nurnberg Institut fur Zellulare und Molekulare Physiologie, Erlangen, Bayern, Germany

Linda Geiges

• Azemi, Bardha, Friedrich-Alexander-Universitat Erlangen-Nurnberg Institut fur Zellulare und Molekulare Physiologie, Erlangen, Bayern, Germany

Bardha Azemi

• Ziegler, Christine Maria, Universitat Regensburg Fakultat fur Biologie und Vorklinische Medizin, Regensburg, Bayern, Germany

Christine Maria Ziegler

• Korbmacher, Christoph, Friedrich-Alexander-Universitat Erlangen-Nurnberg Institut fur Zellulare und Molekulare Physiologie, Erlangen, Bayern, Germany

Christoph Korbmacher

• Ilyaskin, Alexandr V., Friedrich-Alexander-Universitat Erlangen-Nurnberg Institut fur Zellulare und Molekulare Physiologie, Erlangen, Bayern, Germany

Alexandr V. Ilyaskin, PhD

Background

Autosomal-dominant polycystic kidney disease (ADPKD) is caused by mutations in the polycystin-1 (PC1) or polycystin-2 (PC2) coding gene. Structural data imply that PC1 and PC2 form a heterotetrameric channel with a 1:3 stoichiometry. This study investigates the assembly and ion channel properties of PC1/PC2 complexes using a novel gain-of-function (GOF) PC1 construct.

Methods

Amino acid residues identified as critical for ion permeation in the cryo-electron microscopy (cryo-EM) model of the PC1/PC2 complex [PDB ID: 6A70] were mutated to alanine in PC2 and the C-terminal fragment of PC1 (aa: 3049-4303) to obtain the known PC2^GOF (L677A/N681A)) and a putative PC1^GOF (R4100A/R4107A/H4111A) construct. Wildtype (WT) and mutant PC1 and PC2 were expressed in Xenopus laevis oocytes and analysed using the two-electrode voltage clamp technique. Cell surface expression of PC1 and PC2 was detected using a biotinylation approach. The formation of PC1/PC2 complexes was confirmed by co-immunoprecipitation experiments.

Results

Co-expression of PC1^WTand PC2^GOF decreased Na⁺ inward currents compared to PC2^GOF alone. Importantly, Na⁺ inward currents were ~5-fold larger in oocytes co-expressing PC1^GOF/PC2^GOF than in those co-expressing PC2^GOF/PC1^WT. A mutagenesis approach identified the residues R4107 in PC1 and L677 in PC2 as critical for the GOF effect. Homomeric PC2^GOF channels exhibit a preference for K⁺ over Na⁺ and Li⁺ and are inhibited by DMA⁺ and DEA⁺. In contrast, PC1^GOF/PC2^GOF heteromers conduct Na⁺, K⁺, Li⁺, and DMA⁺ equally well. Intriguingly, PC2^GOF channels exhibit a permeability for Ca²⁺, whereas PC1^GOF/PC2^GOF complexes do not. By varying the expression ratio of PC1^GOF and PC2^GOF, we demonstrate that PC2 preferentially forms heteromeric complexes with PC1. A re-interpretation of the published cryo-EM map of PC1 implies that its pore-forming domain exhibits a TRP-like conformation. This interpretation is supported by cysteine-modification experiments.

Conclusion

Taken together, these results support the concept that PC2 homomers and PC1/PC2-heteromers are ion channels with distinct physiological functions. The novel PC1^GOF construct described here may serve as a tool to investigate the effect of pathogenic ADPKD mutations on PC1/PC2 ion channel function.

Funding

• Government Support – Non-U.S.