TY - JOUR
T1 - Rab proteins regulate epithelial sodium channel activity in colonic epithelial HT-29 cells
AU - Saxena, Sunil
AU - Singh, Madhurima
AU - Engisch, Kathrin
AU - Fukuda, Mitsunori
AU - Kaur, Simarna
N1 - Funding Information:
Authors are grateful to Constantine George for help and encouragement during the course of study. Technical support of Amanda Rogers is greatly appreciated. The work was supported in part by the grant from National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) awarded to SS (DK57727).
PY - 2005/12/2
Y1 - 2005/12/2
N2 - ENaC, the sodium-selective amiloride-sensitive epithelial channel, mediates electrogenic sodium re-absorption in tight epithelia and is deeply associated with human hypertension. The ENaC expression at plasma membrane requires the regulated transport, processing, and macromolecular assembly in a defined and highly compartmentalized manner. Ras-related Rab GTPases regulate intracellular trafficking during endocytosis, regulated exocytosis, and secretion. To evaluate the role of these proteins in regulating amiloride-sensitive sodium channel activity, multiple Rab isoforms 3, 5, 6, and Rab27a were expressed in HT-29 cells. Rab3 and Rab27a inhibited ENaC currents, while the expression of other Rab isoforms failed to elicit any statistically significant effect on amiloride-sensitive currents. The immunoprecipitation experiments suggest protein-protein interaction of Rab3 and Rab27a with epithelial sodium channel. Biotinylation studies revealed that modulation of ENaC function is due to the reduced apical expression of channel proteins. Study also indicates that Rabs do not appear to affect the steady-state level of total cellular ENaC. Alternatively, introduction of isoform-specific small inhibitory RNA (SiRNA) reversed the Rab-dependent inhibition of amiloride-sensitive currents. These observations point to the involvement of multiple Rab proteins in ENaC transport through intracellular routes like exocytosis, recycling from ER to plasma membrane or degradation and thus serve as potential target for human hypertension.
AB - ENaC, the sodium-selective amiloride-sensitive epithelial channel, mediates electrogenic sodium re-absorption in tight epithelia and is deeply associated with human hypertension. The ENaC expression at plasma membrane requires the regulated transport, processing, and macromolecular assembly in a defined and highly compartmentalized manner. Ras-related Rab GTPases regulate intracellular trafficking during endocytosis, regulated exocytosis, and secretion. To evaluate the role of these proteins in regulating amiloride-sensitive sodium channel activity, multiple Rab isoforms 3, 5, 6, and Rab27a were expressed in HT-29 cells. Rab3 and Rab27a inhibited ENaC currents, while the expression of other Rab isoforms failed to elicit any statistically significant effect on amiloride-sensitive currents. The immunoprecipitation experiments suggest protein-protein interaction of Rab3 and Rab27a with epithelial sodium channel. Biotinylation studies revealed that modulation of ENaC function is due to the reduced apical expression of channel proteins. Study also indicates that Rabs do not appear to affect the steady-state level of total cellular ENaC. Alternatively, introduction of isoform-specific small inhibitory RNA (SiRNA) reversed the Rab-dependent inhibition of amiloride-sensitive currents. These observations point to the involvement of multiple Rab proteins in ENaC transport through intracellular routes like exocytosis, recycling from ER to plasma membrane or degradation and thus serve as potential target for human hypertension.
KW - Biotinylation
KW - Colonic epithelial cells
KW - ENaC
KW - HT-29 cells
KW - Protein-protein interaction
KW - Rab27a
KW - Rab3
KW - Rab6
KW - Regulation
KW - Trafficking
UR - http://www.scopus.com/inward/record.url?scp=27144473630&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=27144473630&partnerID=8YFLogxK
U2 - 10.1016/j.bbrc.2005.09.186
DO - 10.1016/j.bbrc.2005.09.186
M3 - Article
C2 - 16236259
AN - SCOPUS:27144473630
VL - 337
SP - 1219
EP - 1223
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
SN - 0006-291X
IS - 4
ER -