Carbachol-Mediated Endocytosis of NHE3 Involves a Clathrin-Independent Mechanism Requiring Lipid Rafts and Cdc42

• Published in: Cellular physiology and biochemistry Vol. 33; no. 3; pp. 869 - 881
• Main Authors: Zachos, Nicholas C., Alamelumangpuram, Bharath, Lee, Luke J., Wang, Peng, Kovbasnjuk, Olga
• Format: Journal Article
• Language: English
• Published: Basel, Switzerland Cell Physiol Biochem Press GmbH & Co KG 01.01.2014
• Subjects: Caco-2 Cells; Carbachol - pharmacology; cdc42 GTP-Binding Protein - metabolism; Clathrin - metabolism; Endocytosis - drug effects; Humans; Intestinal epithelial cells; Intracellular calcium; Membrane Microdomains - metabolism; Original Paper; Protein trafficking; Sodium-Hydrogen Exchanger 3; Sodium-Hydrogen Exchangers - metabolism; Intracellular calcium; Intestinal epithelial cells; Protein trafficking
• ISSN: 1015-8987; 1421-9778
• DOI: 10.1159/000358659

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. Conclusion: CCH-mediated inhibition of NHE3 activity is not dependent on clathrin and involves lipid rafts and requires Cdc42.