Interaction between CFTR and prestin (SLC26A5)

Abstract

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated chloride channel that is present in a variety of epithelial cell types, and usually expressed in the luminal membrane. In contrast, prestin (SLC26A5) is a voltage-dependent motor protein, which is present in the basolateral membrane of cochlear outer hair cells (OHCs), and plays an important role in the frequency selectivity and sensitivity of mammalian hearing. By using in situ hybridization and immunofluorescence, we found that both mRNA and protein of CFTR are present in OHCs, and that CFTR localizes in both the apical and the lateral membranes. CFTR was not detected in the lateral membrane of inner hair cells (IHCs) or in that of OHCs derived from prestin-knockout mice, i.e., in instances where prestin is not expressed. These results suggest that prestin may interact physically with CFTR in the lateral membrane of OHCs. Immunoprecipitation experiments confirmed a prestin-CFTR interaction. Because chloride is important for prestin function and for the efferent-mediated inhibition of cochlear output, the prestin-directed localization of CFTR to the lateral membrane of OHCs has a potential physiological significance. Aside from its role as a chloride channel, CFTR is known as a regulator of multiple protein functions, including those of the solute carrier family 26 (SLC26). Because prestin is in the SLC26 family, several members of which interact with CFTR, we explored the potential modulatory relationship associated with a direct, physical interaction between prestin and CFTR. Electrophysiological experiments demonstrated that cAMP– activated CFTR is capable of enhancing voltage-dependent charge displacement, a signature of OHC motility, whereas prestin does not affect the chloride conductance of CFTR.

Mechanism and synergism in epithelial fluid and electrolyte secretion

“…The isolated STAS domain was sufficient to fully activate CFTR (66) and when the R domain is targeted to the plasma membrane it activated the SLC26 transporters (127). Similar interaction between CFTR was demonstrated for Slc26a4 (38, 129), Slc26a5 (50), Slc26a8 (118) and Slc26a9 (8, 11). These findings indicate that in the resting state the unphosphorylated CFTR R domain interacts with NBD1 to inhibit CFTR and at the same time to sequester the R domain away from the STAS domain, thus maintain the SLC26 transporters n the inactive state by their STAS domain.…”

STAS Domain Structure and Function

“…This interaction with prestin may direct a proportion of CFTR to the outer hair cell lateral membrane, an unprecedented subcellular localization in mammalian cells. However, prestinmediated non-linear capacitance is not modified in outer hair cells of Cftr -/-mice [67], and cystic fibrosis in humans has not been associated with hearing loss beyond that plausibly attributable to chronic aminoglycoside treatment. MAP1S is a prestin STAS-binding protein discovered through yeast two-hybrid interaction [68].…”

Carcinoembryonic antigen-related cell adhesion molecule 16 interacts with α-tectorin and is mutated in autosomal dominant hearing loss (DFNA4)

“…The procedure for cochlear immunofluorescence is described in Homma et al (35). For subtilisin (Sigma) treatment, cochleae were dissected and incubated with 50 μg/mL subtilisin in Hepes-buffered HBSS solution (21) for 15 min at room temperature before being fixed in 4% formaldehyde.…”

“…Culture medium from Ceacam16-transfected cells was collected and successively centrifuged at 10,000 × g for 20 min and 100,000 × g for 1 h. Proteins in supernatants were precipitated by 5% trichloroacetic acid, resolved by 4-20% gradient gel or 7.5% NEXT-PAGE, and blotted with various antibodies. For coimmunoprecipitation, HEK293T or OK cells cotransfected with various plasmids were harvested, and coimmunoprecipitation experiments were performed as described previously (35). Primary antibody/protein-A or -G beads were saturated with target proteins.…”