Kidney Week

Abstract: FR-PO265

Electrophysiological Characterization of Polycystin-2 Mutants With Disease-Associated Missense Mutations in the Channel's Pore Loop

Session Information

• Genetic Diseases of the Kidneys: Cystic - II
  November 04, 2022 | Location: Exhibit Hall, Orange County Convention Center‚ West Building
  Abstract Time: 10:00 AM - 12:00 PM

Category: Genetic Diseases of the Kidneys

• 1101 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,

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

Linda Geiges,

• 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,

Background

In ~15% of patients with autosomal dominant polycystic kidney disease (ADPKD) mutations of polycystin-2 (PC2) are causative for the disease. PC2 belongs to the family of transient receptor potential (TRP) channels characterised by six transmembrane segments (S1 - S6) and a pore loop between S5 and S6. Here we investigated the effects of three disease-associated pore loop mutations (F629S, C632R, and R638C) on PC2 ion channel properties.

Methods

Pore mutations F629S, C632R, or R638C were introduced into wild-type PC2 (PC2_WT) or two PC2 gain-of-function (GOF) constructs (PC2_F604P or PC2_L677A/N681A) using site-directed mutagenesis. PC2 constructs with or without pore mutations were heterologously expressed in Xenopus laevis oocytes for functional analysis using the two-electrode voltage clamp technique. PC2-mediated Na⁺ inward currents were elicited by divalent cation removal. Monovalent cation selectivity and Ca²⁺ permeability were assessed.

Results

In contrast to PC2_WT, expression of PC2 with F629S, C632R or R638C pore mutations produced no detectable baseline Na⁺ inward currents. These findings are consistent with a loss-of-function effect of the pore loop mutations on baseline PC2 activity. Importantly, F629S, C632R and R638C also completely abolished the ion channel function of the GOF construct PC2_F604P. This finding suggests that pore loop mutations disturb the gating mechanism associated with the F604P GOF mutation. Interestingly, the R638C mutation reduced but did not abolish Na⁺ inward currents mediated by the GOF construct PC2_L677A/N681A. Importantly, the R638C mutation significantly altered monovalent cation selectivity of PC2_L677A/N681A and completely abolished its Ca²⁺ permeability. Interestingly, introducing the corresponding mutation R518C in the closely related polycystin-2L1 channel rendered it impermeable to Ca²⁺ without affecting its Na⁺ permeability.

Conclusion

PC2 pore loop mutations abolished baseline PC2 ion channel function and also the function of the GOF channel PC2_F604P. This suggests that pore mutations disturb PC2 channel gating. Moreover, our findings highlight an important role of R638 in monovalent cation selectivity and Ca²⁺ permeability of PC2.
Supported by a grant from IZKF Erlangen (F8).

Funding

• Government Support – Non-U.S.