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ADP Ribosylation Factor 6 (Arf6) Modulates the Cell Surface Expression of the Cardiac Acetylcholine‐Activated Inward Rectifier Potassium Current (I KACh ) Proteins
Author(s) -
Alhadidy Mohammed M,
Reiser Michelle,
Szekeres Charles,
Cha Byeong,
Noujaim Sami F.
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.991.8
Subject(s) - flow cytometry , g protein coupled inwardly rectifying potassium channel , chemistry , potassium channel , hek 293 cells , g protein , microbiology and biotechnology , adp ribosylation factor , biophysics , biochemistry , cell , biology , signal transduction , golgi apparatus , receptor
It has been suggested that the ADP ribosylation factor 6 (Arf6) plays a role in the trafficking of Kir3.1 and Kir3.4 proteins ‐ the molecular correlates of the cardiac acetylcholine‐activated inward rectifier potassium current (I KACh ). However, further details need to be elucidated. Therefore, we investigated the effects of Arf6 activity on the localization of Kir3.1 and Kir3.4 proteins using flow cytometry, Arf6 activation pull‐down assay, and live confocal microscopy. We tested the hypothesis that Arf6 cycling between the GDP‐ and GTP‐bound forms modulates the cell surface expression of I KACh proteins. Methods Studies were performed in a human embryonic kidney (HEK293) cell line stably expressing Kir3.1 and Kir3.4 (GIRK cells). The I KACh proteins at the cell surface were labelled with TertiapinQ‐ATTO‐488 and quantified using flow cytometry in GIRK cells transiently transfected with Arf6WT (wild type), Arf6T27N (dominant negative, GDP‐bound form), or Arf6Q67L (constitutively active, GTP‐bound form). Additionally, GIRK cells were treated for 30 minutes with 10 μM Chloroquine or Primaquine, then cell surface I KACh proteins were quantified using flow cytometry, and their internalization was studied using live confocal microscopy. Arf6 activation by Chloroquine was assayed using purified Arf6 protein and human atrial tissue samples in pull‐down experiments. Results are reported as average ± standard error. Results In flow cytometry experiments, both Arf6T27N (dominant negative) and Arf6Q67L (constitutively active) showed significant increase in I KACh proteins expression at the cell surface compared to Arf6WT (Arf6T27N, 14% ± 3, p<0.01; Arf6Q67L, 28.5% ± 4, p<0.01). This suggested that interference with Arf6 cycling between the GDP‐ and GTP‐bound forms leads to cell surface accumulation of the I KACh proteins. Moreover, treatment of GIRK cells with 10 μM Chloroquine for 30 minutes significantly decreased cell surface I KACh proteins (8% ± 1.7, p<0.01). Conversely, treatment with Primaquine, a structurally similar 4‐aminoquinoline, did not show the same effect. In live confocal microscopy studies, Chloroquine promoted the internalization of I KACh proteins compared to control. Arf6 activation pull‐down experiments using purified Arf6 protein and human atrial tissue samples suggested that Chloroquine, but not Primaquine, increased Arf6 activity. Conclusion Expression of the I KACh proteins at the cell surface is, in part, modulated by an Arf6‐dependent pathway. Constitutive interference with Arf6 cycling between GDP‐ and GTP‐bound forms increases the cell surface density of I KACh proteins, while transient pharmacological activation of Arf6 could lead to their internalization. Support or Funding Information Funded in part by NIH Grant R01HL129136 to SFN