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Abstract: FR-PO266

Lipophilic Methanethiosulfonate (MTS) Reagents Inhibit Ion Channel Function of Polycystin-2 With a Gain-of-Function Mutation (F604P)

Session Information

Category: Genetic Diseases of the Kidneys

  • 1101 Genetic Diseases of the Kidneys: Cystic

Authors

  • Geiges, Linda, Friedrich-Alexander-Universitat Erlangen-Nurnberg, Erlangen, Bayern, Germany
  • Staudner, Tobias, Friedrich-Alexander-Universitat Erlangen-Nurnberg, Erlangen, Bayern, Germany
  • Korbmacher, Christoph, Friedrich-Alexander-Universitat Erlangen-Nurnberg, Erlangen, Bayern, Germany
  • Ilyaskin, Alexandr V., Friedrich-Alexander-Universitat Erlangen-Nurnberg, Erlangen, Bayern, Germany
Background

Polycystin-2 (PC2) mutations account for approximately 15% of reported cases of autosomal-dominant polycystic kidney disease (ADPKD). As a member of the transient receptor potential (TRP) ion channel family, PC2 features a typical structure with 6 membrane-spanning segments (S1–S6). No specific activators or inhibitors of PC2 have been described to date. Interestingly, covalent modification of N-terminal cysteines is a known activation mechanism of another TRP channel, TRPA1. Therefore, we hypothesized that a similar regulatory mechanism may exist in PC2.

Methods

PC2 mutants were generated by site-directed mutagenesis and functionally expressed in Xenopus laevis oocytes. PC2 currents were measured using the two-electrode voltage clamp technique. For covalent cysteine modification, several structurally different methanethiosulfonate (MTS) reagents were applied in the bath. In particular, the effects of positively charged MTSET and MTSEA and lipophilic MTS reagents (methyl-, ethyl-, propyl- and benzyl-MTS) were investigated.

Results

Application of these MTS reagents did not result in an activation of wild-type PC2. To investigate a possible effect of MTS reagents on the channel in an open state, we used two recently discovered gain-of-function (GOF) PC2 mutants (PC2F604P or PC2L677A/N681A). Importantly, we found that lipophilic but not positively charged MTS reagents strongly inhibited PC2F604P. The inhibitory effect could not be reproduced in the structurally related TRPML3 channel containing a corresponding GOF mutation (A419P). The GOF mutant PC2L677A/N681A was not sensitive to any of the tested MTS reagents. The inhibitory effect of methyl-MTS was preserved after mutating all four cysteines in the channel’s N-terminus. Interestingly, replacing the C593 residue in the S4-S5 linker by serine or alanine resulted in a complete loss of PC2F604P function, mimicking the inhibitory effect of lipophilic MTS reagents.

Conclusion

We identified lipophilic MTS reagents as inhibitors of PC2F604P ion channel function. Moreover, C593 in the S4-S5 linker appears to be a crucial residue for PC2 activity. These new findings may lead to a better understanding of PC2 gating mechanisms and promote the development of pharmacological PC2 modulators.
Supported by a grant from IZKF Erlangen (F8)

Funding

  • Government Support – Non-U.S.