MEMBRANE TOPOLOGY OF NHE3 - EPITOPES WITHIN THE CARBOXYL-TERMINAL HYDROPHILIC DOMAIN ARE EXOPLASMIC

Experimental data indicate that the relatively hydrophilic carboxyl-te rminal domains of Na+-H+ exchangers mediate the regulation of transpor ter activity through interactions with cytoskeletal effecters. It has therefore been assumed that this entire domain lies on the cytoplasmic surface of the plasma membrane. The purpose of the present study was to determine the membrane orientation of the COOH-terminal 131 amino a cids of Na+-H+ exchanger isoform NHE3 by use of three monoclonal-antib odies that recognize at least two distinct epitopes within this region . Enzyme-linked immunosorbent assay studies demonstrated binding of th ese monoclonal antibodies (mAbs) to intact right-side-out renal brush border membrane vesicles in the absence of detergent, Moreover, when c oupled to an affinity matrix to isolate membrane vesicles, the anti-NH E3 mAbs bound structures that were morphologically identical to intact microvilli, To confirm the identity of the exoplasmic antigen bound b y the antibodies, immunoprecipitation studies were performed. Intact r ight-side-out brush border membrane vesicles were incubated with the m Abs in the absence of detergent. The membranes were pelleted, supernat ant with unbound antibody was removed, the pellet was solubilized, and then immunoprecipitation with secondary antibody was performed. Immun oblot analysis indicated that NHE3 was precipitated after binding of t he mAbs to intact membranes, Finally, the localization of the mAb epit opes was determined using high resolution immunocytochemistry, Ultrath in cryosections of rat kidney were labeled with the mAbs and bound ant ibody detected with the colloidal gold technique. Labeling was restric ted to the exoplasmic surface of microvilli of the proximal tubule, Ta ken together, these findings indicate that epitopes within the carboxy l terminus of the Na+-H+ exchanger isoform NHE3 are exposed to the out side of the plasma membrane.