Functional characterization of a calcium-sensing receptor-polycystin-2 channel complex in the plasma membrane of LLC-PK1 cells.

SCARINCI N; CANTIELLO HF; PEREZ PL; CANTERO MR

San Francisco, California

Congreso; 62th Annual Meeting of the Biophysical Society; 2018

Polycystin-2 (PC2, TRPP2) is a Ca2+-permeable nonselective cation channel from the TRP superfamily. PC2 is encoded by the PKD2 gene, whose mutations are responsible for autosomal dominant polycystic kidney disease (ADPKD). Recent studies from our laboratory (Dai et al, Exp Cell Res, 2017), determined that the PC2 contribution to the whole cell conductance of wild type LLC-PK1 renal epithelial cells is regulated by changes in external Ca2+ concentration, in a mechanism that implicates the Calcium-Sensing Receptor (CaSR). The present study explored the effect of Ca2+ and CaSR agonists in the regulation of PC2 from isolated LLC-PK1 plasma membranes reconstituted in a lipid bilayer system (BLM). Addition of the CaSR agonist R-568 (5 M) to the ?external? side of the bilayer, activated single channel currents consistent with a functional PC2 by 84 ± 12% (n = 4, p < 0.01). These cation-selective currents were subsequently inhibited (91.3 ± 1.1%, p < 0.0001) by addition to the ?cytosolic? side of the BLM, of a commercially available anti-PC2 C-terminal antibody to the ?cytosolic? or ?internal? side of the BLM. Interestingly, similar findings were observed with the material obtained by co-immunoprecipitation of LLC-PK1 cell lysate with anti-CaSR antibody crosslinked in agarose beads. The data indicate the presence of a membrane locate CaSR-PC2 functional complex in LLC-PK1 renal epithelial cells. The presence of the receptor/channel complex is in agreement with the regulatory role external Ca2+ plays in the modulation of PC2 channel function in the plasma membrane of renal epithelial cells. This receptor-channel complex may be part of a regulatory mechanism connecting Ca2+ signals and PC2 function to onset of ADPKD.