Rab proteins regulate epithelial sodium channel activity in colonic epithelial HT-29 cells.
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.
Saxena
Sunil
S
Center for Cell and Molecular Biology, Department of Chemistry and Chemical Biology, Stevens Institute of Technology, Hoboken, NJ 07030, USA. ssaxena@stevens.edu
Singh
Madhurima
M
Engisch
Kathrin
K
Fukuda
Mitsunori
M
Kaur
Simarna
S
D000584:Amiloride / Q000494:pharmacology; D019921:Biotinylation; D003110:Colonic Neoplasms / Q000378:metabolism* / Q000473:pathology; D053503:Epithelial Sodium Channels; D005786:Gene Expression Regulation; D019073:HT29 Cells; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects; D011485:Protein Binding; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D015222:Sodium Channels / Q000378:metabolism*; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*
D053503:Epithelial Sodium Channels; D020033:Protein Isoforms; D034741:RNA, Small Interfering; D015222:Sodium Channels; D000584:Amiloride; D020691:rab GTP-Binding Proteins