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Supplemental material for Furin-Mediated Modification Is Required for Epithelial Sodium Channel-Activating Activity of Soluble (Pro)Renin Receptor in Cultured Collecting Duct Cells

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posted on 2025-01-09, 23:48 authored by Huaqing ZhengHuaqing Zheng

(Pro)renin receptor (PRR) contains overlapping cleavage site for site-1 protease (S1P) and

furin for generation of soluble PRR (sPRR). Although S1P-mediated cleavage mediates the

release of sPRR, the functional implication of furin-mediated cleavage is unclear. Here we tested

whether furin-mediated cleavage was required for the activity of sPRR in activating ENaC in

cultured M-1 cells. M-1 cells were transfected with pcDNA3.4 containing full-length PRR with

(Furin-site Mut) or without (WT) mutagenesis of the furin cleavage site. As compared with empty

vector control (EM), Furin-site Mut showed the attenuation effect on WT-induced α-ENaC

expression and amiloride-sensitive short circuit current. In a separate experiment, M-1 cells were

transfected with pcDNA3.4 containing cDNA for sPRR with S1P cleavage (AA 1-282) (sPRR-S1P)

or with furin cleavage (AA 1-279) (sPRR-furin), indicating overexpression of the two types of

sPRR induced a significant and comparable increase in the release of sPRR, but only sPRR-furin

showed an increase of ENaC activity. Single channel analysis of ENaC activity in Xenopus A6-

2F3 cells confirms sPRR-furin activation of ENaC open probability. Lastly, HEK-293 cells were

pretreated with furin inhibitor α1-antitrypsin Portland (α1-PDX) followed by transfection with EM,

WT PRR. sPRR in the conditioned medium was enriched by using protein centrifugal filter devices

and applied to M-1 cells followed by measurement of ENaC activity, demonstrating that

pretreatment with α1-PDX attenuated ENaC-acting activity induced by overexpression of WT

PRR. In summary, we conclude that furin-mediated modification is required for the activity of

sPRR to increase ENaC-mediated Na+ transport in the CD cells.

Funding

National Institutes of Health Grants HL139689, DK104072, HL135851, HL160020, and Veterans Affairs (VA) Merit Review I01BX004871 and Senior Research Career Scientist award IK6BX005223 from the Department of Veterans Affairs

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