Structure-based electrophysiological analysis of polycystin-2 ion channel function as homomer or in complex with polycystin-1

• Applicant: Alexandr Ilyaskin, Ph.D.
• Subject Area: Anatomy and Physiology ; Nephrology
• Term: since 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 517598260

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the development of fluid-filled renal cysts ultimately leading to kidney failure. It is caused by mutations in the PKD1 or PKD2 gene coding for polycystin-1 (PC1) or polycystin-2 (PC2), respectively. No clear consensus exists regarding the (patho-)physiological roles of PC1 and PC2. Recent cryo-EM data support the concept that PC2 functions as cation channel in a homotetrameric configuration or as heterotetramer with 3 PC2 monomers and 1 PC1 protein. Altered PC2 ion channel function may contribute to the pathophysiology of ADPKD. However, conflicting findings have been reported regarding the ion channel properties of PC2 and its functional interaction with PC1. This justifies the need to develop novel approaches to explore the ion channel function of PC2. In the proposed project we will benefit from recently published cryo-EM data and newly developed PC2 constructs, with improved expression at the cell surface or gain-of-function mutations, to study ion channel properties of PC2 alone or in complex with PC1. We have three major aims, which will be addressed using a combination of electrophysiological techniques, computational methods and site-directed mutagenesis: 1.) Investigate the functional importance of the pore loop, TOP domain finger 2 and S4-S5-linker domains for PC2 function. 2.) Investigate mechanisms of PC2 modulation by plasma membrane lipids, amphiphilic or lipophilic substances. 3.) Investigate the functional interaction of PC2 and PC1. To pursue aim 1, we will address the following questions: Do ADPKD-associated PC2 mutations in the pore loop domain (F629S, C632R, R638C) alter ion channel properties of PC2? Is an intersubunit cation-π interaction formed by the TOP domain finger 2 residue (W380) and the pore domain residue (R654) critical for PC2 ion channel function? Do ADPKD-associated PC2 mutations in the TOP domain finger 2 (A384P, G390S) alter ion channel properties of PC2? How does the ADPKD-associated N580K mutation within the S4-S5-linker domain affect ion channel function of PC2? For aim 2 we will explore the following: Is the PC2 homomeric channel modulated by cholesterol and/or phosphatidylinositol phosphates (PIP2)? Is the putative PIP2 binding site identified in the PC2 cryo-EM structure functionally important? Can we identify novel lipophilic or amphiphilic PC2 modulators acting via the PIP2 binding site? For aim 3 we will focus on the following aspects: How does coexpression of PC1 affect ion channel properties and cell surface expression of PC2? Which PC2 and PC1 domains are critical for heteromeric PC2-PC1 ion channel function? In summary, using our novel experimental strategy we expect to gain new insights into PC2 ion channel function as homomer or in complex with PC1. This may promote the development of pharmacological modulators of these channels and lead to novel (patho)physiological and therapeutic concepts for ADPKD treatment.

• DFG Programme: Research Grants
• Co-Investigator: Professor Dr. Christoph Korbmacher