Electrophysiological Characterization of Polycystin-2 Mutants With Disease-Associated Missense Mutations in the Channel's Pore Loop
- 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
- Staudner, Tobias, Friedrich-Alexander-Universitat Erlangen-Nurnberg Institut fur Zellulare und Molekulare Physiologie, Erlangen, Bayern, Germany
- Geiges, Linda, Friedrich-Alexander-Universitat Erlangen-Nurnberg Institut fur Zellulare und Molekulare Physiologie, Erlangen, Bayern, Germany
- Korbmacher, Christoph, Friedrich-Alexander-Universitat Erlangen-Nurnberg Institut fur Zellulare und Molekulare Physiologie, Erlangen, Bayern, Germany
- Ilyaskin, Alexandr V., Friedrich-Alexander-Universitat Erlangen-Nurnberg Institut fur Zellulare und Molekulare Physiologie, Erlangen, Bayern, Germany
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.
Pore mutations F629S, C632R, or R638C were introduced into wild-type PC2 (PC2WT) or two PC2 gain-of-function (GOF) constructs (PC2F604P or PC2L677A/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.
In contrast to PC2WT, 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 PC2F604P. 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 PC2L677A/N681A. Importantly, the R638C mutation significantly altered monovalent cation selectivity of PC2L677A/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.
PC2 pore loop mutations abolished baseline PC2 ion channel function and also the function of the GOF channel PC2F604P. 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).
- Government Support – Non-U.S.