Aberrant ion transport caused by either hypo- or hyper-functioning of epithelial transporters, can be detrimental, and may result in life-threatening diseases, such as cystic fibrosis or secretory diarrhea. Recently, we reported biochemical and functional associations between the PDZ domain-containing protein Shank2 and a couple of epithelial transporters, the cystic fibrosis transmembrane conductance regulator (CFTR) and Na+/H+ exchanger 3 (NHE3) (1, 2). Interestingly, Shank2 attenuated the cAMP-dependent regulation of CFTR and NHE3. On the other hand, it has been previously identified that another PDZ-based scaffold EBP50 can enhance the effects of cAMP on these transporters by recruiting cAMP-dependent protein kinase anchoring protein (AKAP) (3). Shank2 and EBP50 have very similar PDZ structures that can bind to the carboxy terminus of CFTR and NHE3 (4). Therefore, Shank2 and EBP50 may compete with each other in associating with the epithelial transporters. In the present study, we determined the dynamic properties and physiological significance of these competitive interactions between PDZ-based scaffolds and epithelial transporters. In the surface plasmon resonance study, the dissociation constants (KD) of Shank2-CFTR and Shank2-NHE3 bindings were within a range similar to those of EBP50-CFTR and EBP50-NHE3, respectively, supporting the possibility of Shank2 vs. EBP50 competition in the physiological tissues. In addition, patch clamp studies revealed that the cAMP-activated CFTR chloride channel activity was dynamically regulated by the Shank2 and EBP50 competition. Lastly, in contrast to AKAP-recruiting activity of EBP50, Shank2 was found to be associated with proteins that attenuate cAMP-PKA signals. These results strongly suggest that the competitive balance between Shank2-transporter binding and EBP50-transporter binding may maintain the homeostatic regulation of epithelial ion and fluid transport.