Functional Characterization of a Polycystin-2-Calcium Sensing Receptor Complex Present in LLC-PK1 Renal Epithelial Cells

CANTIELLO HF; SCARINCI MN; CANTERO MR; PEREZ PL

Mar del Plata

Congreso; Reunión Anual de la Sociedad Argentina de Fisiología; 2018

Polycystin-2 (PC2, TRPP2) is a Ca2+-permeable nonselective cation channel encoded by the PKD2 gene whose mutations cause autosomal dominant polycystic kidney disease (ADPKD). Recent studies from our laboratory (Dai et al, Exp Cell Res, 2017), determined that the expression of plasma membrane PC2 in LLC-PK1 renal epithelial cells is regulated by changes in external Ca2+ concentration through the Calcium-Sensing Receptor (CaSR). The present study explored the existence of a structural coupling between PC2 and CaSR in LLC-PK1 cells. The presence of a PC2-CaSR complex was explored as follows. Cell lysates with either anti-CaSR (CaSR elution) or PC2 antibody (PC2 elution), were added to Pierce Protein A/G coupled to magnetic beads (Thermo Scientific) for elution of the complexes. CaSR elution co-immunoprecipitated with PC2 as indicated by dot-blot labeling with anti?PC2 antibody. Conversely, PC2 elution co-immunoprecipitated CaSR. In the presence of high Ca2+ (6 mM), CaSR-PC2 colabeling was at least 60% lower for either elution sample. A BLM reconstitution system was used to test for the presence of ion channel activity in the Co-IP materials. Reconstitution was carried out in a KCl gradient (150:15 mM). Both Co-IP materials showed spontaneous cation-selective ion channel activity whose I/V relationship rendered a single channel conductance of 128 ± 11 pS (n = 3) and 104 ± 28 pS (n = 3) for the CaSR and PC2 materials, respectively. The data indicate that the Co-IP technique using either anti-PC2 or anti-CaSR antibodies, results in the elution of a functional and structural PC2-CaSR complex. Further studies will be required to assess the interaction between the CaSR and PC2, including physical interactions with cytoskeletal connections.