Supplemental material for Furin-Mediated Modification Is Required for Epithelial Sodium Channel-Activating Activity of Soluble (Pro)Renin Receptor in Cultured Collecting Duct Cells
<p dir="ltr">(Pro)renin receptor (PRR) contains overlapping cleavage site for site-1 protease (S1P) and</p><p dir="ltr">furin for generation of soluble PRR (sPRR). Although S1P-mediated cleavage mediates the</p><p dir="ltr">release of sPRR, the functional implication of furin-mediated cleavage is unclear. Here we tested</p><p dir="ltr">whether furin-mediated cleavage was required for the activity of sPRR in activating ENaC in</p><p dir="ltr">cultured M-1 cells. M-1 cells were transfected with pcDNA3.4 containing full-length PRR with</p><p dir="ltr">(Furin-site Mut) or without (WT) mutagenesis of the furin cleavage site. As compared with empty</p><p dir="ltr">vector control (EM), Furin-site Mut showed the attenuation effect on WT-induced α-ENaC</p><p dir="ltr">expression and amiloride-sensitive short circuit current. In a separate experiment, M-1 cells were</p><p dir="ltr">transfected with pcDNA3.4 containing cDNA for sPRR with S1P cleavage (AA 1-282) (sPRR-S1P)</p><p dir="ltr">or with furin cleavage (AA 1-279) (sPRR-furin), indicating overexpression of the two types of</p><p dir="ltr">sPRR induced a significant and comparable increase in the release of sPRR, but only sPRR-furin</p><p dir="ltr">showed an increase of ENaC activity. Single channel analysis of ENaC activity in Xenopus A6-</p><p dir="ltr">2F3 cells confirms sPRR-furin activation of ENaC open probability. Lastly, HEK-293 cells were</p><p dir="ltr">pretreated with furin inhibitor α1-antitrypsin Portland (α1-PDX) followed by transfection with EM,</p><p dir="ltr">WT PRR. sPRR in the conditioned medium was enriched by using protein centrifugal filter devices</p><p dir="ltr">and applied to M-1 cells followed by measurement of ENaC activity, demonstrating that</p><p dir="ltr">pretreatment with α1-PDX attenuated ENaC-acting activity induced by overexpression of WT</p><p dir="ltr">PRR. In summary, we conclude that furin-mediated modification is required for the activity of</p><p dir="ltr">sPRR to increase ENaC-mediated Na+ transport in the CD cells.</p>
