Pinedo, A. | Search bioRxiv

bioRxiv2023

ADPKD-Causing Missense Variants in Polycystin-1 Disrupt Cell Surface Localization or Polycystin Channel Function

Autosomal dominant polycystic kidney disease (ADPKD) is the leading monogenic cause of kidney failure and affects millions of people worldwide. Despite the prevalence of this monogenic disorder, our limited mechanistic understanding of ADPKD has hindered therapeutic development. Here, we successfully developed bioassays that functionally classify missense variants in polycystin-1 (PC1). Strikingly, ADPKD pathogenic missense variants cluster into two major categories: 1) those that disrupt polycystin cell surface localization or 2) those that attenuate polycystin ion channel activity. We found that polycystin channels with defective surface localization could be rescued with a small molecule. We propose that small-molecule-based strategies to improve polycystin cell surface localization and channel function will be effective therapies for ADPKD patients.

cell biology↗

bioRxiv2022

The cilia enriched oxysterol 7β,27-DHC is required for polycystin activation

PC-1 and PC-2 form a heteromeric ion channel complex (hereafter called the Polycystin complex) that is abundantly expressed on primary cilia of renal epithelial cells. Mutations within the polycystin complex cause Autosomal Dominant Polycystic Kidney Disease (ADPKD). The Polycystin complex forms a non-selective cation channel, yet the spatial and temporal regulation of the polycystin complex within the ciliary membrane remains poorly understood, partially due to technical limitations posed by the tiny ciliary compartment. Here, we employ our novel assays to functionally reconstitute the polycystin complex in the plasma membrane. Using whole-cell and ciliary patch-clamp recordings we identified a ciliary enriched oxysterol, 7{beta},27-DHC, as a critical component required for activation of the polycystin complex. We identified a novel oxysterol binding pocket in PC-2 using molecular docking simulation. We also identified two amino acids within the PC-2 oxysterol binding pocket, E208 and R581, to be critical for 7{beta},27-DHC dependent polycystin activation in both the plasma membrane and ciliary compartment. Further, we can show that the pharmacological and genetic inhibition of oxysterol synthesis by carbenoxolone (CNX) reduces channel activity in primary cilia. Our findings identified a unique second messenger that regulates the polycystin complex. We hypothesize that cilia-enriched lipids license the polycystin complex to be functional only in the ciliary organelle, thus providing novel insights into the spatial regulation of the polycystin complex. Our results also establish a framework to target the same allosteric regulatory site in the polycystin complex to identify activators of the polycystin channels as novel therapeutic strategies for ADPKD.

physiology↗