Proceedings of The Physiological Society
University College London (2011) Proc Physiol Soc 24, C18 and PC18
The apical Na+/H+ exchanger NHE3 interacting PDZ protein NHERF2 is strongly lipid raft-associated and determines the raft association of NHE3 in murine small intestine
A. Sultan1, M. Chen1, B. Riederer1, C. C. Yun2, H. deJonge3, M. Donowitz4, U. Seidler1
1. Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Lower Saxony, Germany. 2. Emory University, Atlanta, Georgia, United States. 3. Erasmus MC, Rotterdam, Netherlands. 4. John Hopkins Medical School, Baltimore, Maryland, United States.
Backround: Na+/H+ exchangers play a major role in the regulation of intracellular and intravascular volume and maintain it within a physiological range. Na+/H+ exchanger 3 (NHE3) is expressed on the apical membrane of intestinal epithelia and is scaffolded by Na+/H+ exchanger regulatory factor (NHERF) family of PDZ-adapter proteins which are also involved in its regulation and membrane trafficking. It has been demonstrated that a population of NHE3 resides in lipid rafts of the Brush Border Membrane (BBM). Similar to the role of the NHERFs, lipid rafts are also considered to be platforms enabling protein-protein interaction and signal transduction. Aim: In this study, we asked the question whether the NHERFs are differentially associated with the raft and non-raft fraction of NHE3, and if the presence or absence of the NHERFs influences the raft vs. non-raft distribution of NHE3, and its transport activity. Methods and Results: Murine BBMs were isolated by the divalent cation precipitation technique, and lipid rafts were subsequently separated by Triton X-100 membrane solubilisation and flotation of detergent-resistant membranes in an OptiPrep density gradient. NHE3 was found to be partially lipid raft associated. Interestingly, NHERF2 was found to be strongly lipid-raft associated, NHERF1 partially, and NHERF3 was exclusively in the non-raft fraction. In the absence of NHERF2 expression, the percentage of raft-associated NHE3 was strongly decreased, and NHE3 function, measured both in isolated BCECF-loaded small intestinal villi by pHi-fluorometry and in luminally perfused mice in vivo, was altered, as was NHE3 trafficking within the Brush border membrane. Conclusions: The data demonstrate that in the Brush border membrane of murine small intestine, NHE3 differentially associates with the different NHERFs in the raft and non-raft fraction, with NHERF2 being strongly associated with raft-associated NHE3. NHERF2 deletion resulted in loss of raft-associated NHE3 as well as alteration of NHE3 transport function. This suggests that PDZ-scaffolding proteins are involved in the retention of membrane proteins within lipid raft platforms.
Where applicable, experiments conform with Society ethical requirements