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The Calcineurin Homologous Protein‐1 Facilitates the Na + /H + exchanger‐3 Traffic through the Golgi Complex and Impedes Traffic to the Early Endosomes
Author(s) -
Crosby Garrett S,
Babich Victor,
Di Sole Francesca
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.lb860
Subject(s) - endosome , endoplasmic reticulum , transfection , microbiology and biotechnology , golgi apparatus , chemistry , hek 293 cells , transport protein , cell , biology , biochemistry , receptor , gene
Hypertension is a leading risk factor for cardiovascular and renal diseases, and a major burden for the health care system. It is characterized by disturbance in kidney function that increases sodium (Na + ) re‐absorption and decreases Na + output below Na + intake. The Na + /H + exchanger‐3 (NHE3) is a major regulator of body Na + , and blood volume and pressure. Physiological functions of NHE3 are dependent on NHE3 trafficking and precise localization to the cell surface. The Calcineurin Homologous Protein‐1 (CHP1) is a calcium‐binding protein and binding partner of NHE3. Defect in CHP1 action leads to hypotension and reduced cell surface NHE3 activity and protein expression. The goal of this study was to determine whether CHP1 effect on NHE3 function is via a control on NHE3 trafficking to and from the cell surface. NHE3‐mGFP alone or in combination with CHP1‐mRFP was expressed in opossum kidney (OK) cells and NHE3 localization to specific cellular compartments (determined using biomarkers) was analyzed by immunofluorescence. NHE3 co‐localized with biomarkers of the endoplasmic reticulum (PDI), Golgi Complex (GM130) and early endosomes (EEA1) after 24hrs, 48hrs, 72hrs and 96hrs of transfection (measured by the Mander's co‐localization coefficient). NHE3 and PDI co‐localization was steady for 48hrs after transfection but was rapidly reduced after 72hrs and 96hrs of transfection. NHE3 and GM130 co‐localization was abruptly increased after 48 hrs of transfection and reversed back to the 24hrs value after 96hrs of transfection. NHE3 co‐localization with EEA1 was steady through the time points under study. When NHE3 was co‐expressed with CHP1, NHE3 co‐localization with PDI did not change, while NHE3 co‐localization with GM130 or EEA1 was consistently reduced starting from 48hrs (30% reduction, NHE3 co‐localized with GM130) or 24hrs (20% reduction, NHE3 co‐localized with EEA1) of transfection. NHE3 and CHP1 co‐localization was constant through the time points under study. These findings suggest that CHP1: 1. co‐localized with NHE3 during its early steps in the biosynthetic pathway 2. facilitated NHE3 traffic through the Golgi Complex, while it impeded NHE3 traffic to the early endosomes. This study advances our understanding on the role of CHP1 in the control of NHE3 function and blood pressure.