Single Molecule Imaging Reveals The Functional Role Of The Na+/H+ Exchanger-3 Oligomerization

The epithelial Na+/H+ exchanger-3 (NHE3) activity is pivotal in maintaining renal and intestinal sodium homeostasis. NHE3 activity is regulated by a variety of hormonal stimuli and is dysregulated in severe disorders such as hypertension, heart failure, acute kidney injury, diabetic nephropathy and diarrhea. NHE3 exists as monomers and oligomers; though, the requirement of dynamic change in NHE3 stoichiometry for its regulation has not been established. Extra- and intra-cellular tagged NHE3 was expressed on cell membrane of live cells in form of monomers and oligomers (ratio monomer : oligomer, ∼1:1) detected by Total Internal Reflection Fluorescence (TIRF) and Förster Resonance Energy Transfer. The oligomers had slower lateral mobility compared to monomers (determined by TIRF and Fluorescence Recovery After Photobleaching). Activation or inhibition of NHE3 transport by well-characterized stimuli induced a significant shift in the monomer : oligomer ratio toward formation of NHE3 oligomers (∼1:2 and ∼1:1.5, respectively) and reduction in NHE3 lateral mobility. Blocking NHE3 binding to the calcium binding protein, the Calcineurin Homologous Protein-1 (CHP1), by a cell-penetrating peptide from CHP1 binding region (competing for CHP1 binding to NHE3) increased the number of NHE3 oligomers (∼1:2). In summary, a dynamic change in the NHE3 monomer : oligomer ratio toward an increase in NHE3 oligomers is proposed as the molecular signal necessary for stimuli-mediated NHE3 regulation. It is proposed that oligomerization is an essential step in the regulation of NHE3 activity and that changes in NHE3 monomer : oligomer ratio are under control of CHP1 binding to NHE3.