Carbachol-mediated endocytosis of NHE3 involves a clathrin-independent mechanism requiring lipid rafts and Cdc42.
BACKGROUND
In intestinal epithelial cells, acute regulation of the brush border Na(+)/H(+) exchanger, NHE3, usually occurs by changes in endocytosis and/or exocytosis. Constitutive NHE3 endocytosis involves clathrin. Carbachol (CCH), which elevates intracellular Ca(2+) ([Ca(2+)]i), decreases NHE3 activity and stimulates endocytosis; however, the mechanism involved in calcium-mediated endocytosis of NHE3 is unclear. A pool of NHE3 resides in lipid rafts, which contributes to basal, but not cAMP-mediated, NHE3 trafficking, suggesting that an alternative mechanism exists for NHE3 endocytosis. Cdc42 was demonstrated to play an integral role in some cases of cholesterol-sensitive, clathrin-independent endocytosis. Therefore, the current study was designed to test the hypotheses that (1) clathrin-mediated endocytosis (CME) is involved in constitutive, but not CCH-mediated, endocytosis of NHE3, and (2) CCH-mediated endocytosis of NHE3 occurs through a lipid raft, activated Cdc42-dependent pathway that does not involve clathrin.


METHODS
The role of Cdc42 and lipid rafts on NHE3 activity and endocytosis were investigated in polarized Caco-2/BBe cells using pharmacological and shRNA knockdown approaches.


RESULTS
Basal NHE3 activity was increased in the presence of CME blockers (chlorpromazine; K(+) depletion) supporting previous reports that constitutive NHE3 endocytosis is clathrin dependent. In contrast, CCH-inhibition of NHE3 activity was abolished in Caco-2/BBe cells treated with MβCD (to disrupt lipid rafts) as well as in Cdc42 knockdown cells but was unaffected by CME blockers.


CONCLUSIONS
CCH-mediated inhibition of NHE3 activity is not dependent on clathrin and involves lipid rafts and requires Cdc42.
Department of Medicine/Gastroenterology and Hepatology, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
Nicholas C
NC
Zachos
Bharath
B
Alamelumangpuram
Luke J
LJ
Lee
Peng
P
Wang
Olga
O
Kovbasnjuk
D018938:Caco-2 Cells; D002217:Carbachol / Q000494:pharmacology*; D002966:Clathrin / Q000378:metabolism*; D004705:Endocytosis / Q000187:drug effects*; D006801:Humans; D021962:Membrane Microdomains / Q000378:metabolism*; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism*; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism*
D002966:Clathrin; C000613385:SLC9A3 protein, human; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers; D002217:Carbachol; D020764:cdc42 GTP-Binding Protein