Na+-H+ exchanger 3 (NHE3) is present in lipid rafts in the rabbit ileal brush border: a role for rafts in trafficking and rapid stimulation of NHE3

1. Rabbit ileal Na+-absorbing cell Na+-H+ exchanger 3 (NHE3) was shown to e xist in three pools in the brush border (BB), including a population in lip id rafts. Approximately 50% of BB NHE3 was associated with Triton X-100-sol uble fractions and the other similar to 50% with Triton X-100-insoluble fra ctions; similar to 33% of the detergent-insoluble NTHE3 was present in chol esterol-enriched lipid microdomains (rafts). 2. The raft pool of NHE3 was involved in the stimulation of BB NHE3 activit y with epidermal growth factor (EGF). Both EGF and clonidine treatments wer e associated with a rapid increase in the total amount of BB NHE3. This EGF - and clonidine-induced increase of BB NHE3 was associated with an increase in the raft pool of NHE3 and to a smaller extent with an increase in the t otal detergent-insoluble fraction, but there was no change in the detergent -soluble pool. In agreement with the rapid increase in the amount of NHE3 i n the BB, EGF also caused a rapid stimulation of BB Na+-H+ exchange activit y. 3. Disrupting rafts by removal of cholesterol with methyl-beta -cyclodextri n (M beta CD) or destabilizing the actin cytoskeleton with cytochalasin D d ecreased the amount of NHE3 in early endosomes isolated by OptiPrep gradien t fractionation. Specifically, NTHE3 was shown to associate with endosomal vesicles immunoisolated by anti-EEA1 (early endosomal autoantigen 1) antibo dy-coated magnetic beads and the endosome-associated NHE3 was decreased by cytochalasin D and M beta CD treatment. 4. We conclude that: (i) a pool of ileal BB NHE3 exists in lipid rafts; (ii ) EGF and clonidine increase the amount of BB NHE3; (iii) lipid rafts and t o a lesser extent, the cytoskeleton, but not the detergent-soluble NTHE3 po ol, are involved in the EGF- and clonidine-induced acute increase in amount of BB NTHE3; (iv) lipid rafts and the actin cytoskeleton play important ro les in the basal endocytosis of BB NHE3.