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. NHERF2 and NHERF3 knockout mice have a strong NHE3 functional defect. It has also been demonstrated that a population of NHE3 resides in lipid rafts of the apical membrane of rabbit ileal epithelia. Similar to the role of the NHERFs, lipid rafts are also thought to be platforms for bringing proteins within close proximity, enabling interaction. The potential role of lipid raft localization of NHE3 towards its function is not properly understood.

Aim: 

We therefore investigated the lipid raft association of NHE3 as well as its interacting NHERF partners in the murine small intestinal brush border membrane (BBM).We also assessed the effect of lipid raft disruption on acid-activated NHE3 activity in microdissected murine jejunal villi by pH_i metry using the pH_i sensitive fluorescent dye BCECF.

Methods and Results: 

Lipid raft disruption by cholesterol depletion and assessment of NHE3 function by pH_i metry showed significant inhibition in activity. Murine BBMs were isolated by the divalent cation precipitation technique, and lipid rafts were 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, NHERF-2 was found to be strongly lipid-raft associated, NHERF-1 partially, and PDZK1 was exclusively in the non-raft fraction.

Conclusions: 

Since NHE3 activity is dependent on the membrane lipid composition and may, like has been shown for other proteins, have a higher basal activity in the lipid raft than in the non-raft fraction, the differential association of NHERFs with lipid-raft and non-raft associated NHE3 may be a component that explains the specific requirement of individual NHERF proteins for different NHE3 regulatory actions.