Recovery by Salicylate of the Plasma Membrane Expression of Prestin Mutants

The motor protein prestin in the plasma membrane of cochlear outer hair cells is believed to be the origin of their electromotility. Several characteristics of prestin have been clarified by introduction of mutations into prestin. In the present study, the aim was to investigate whether or not salicylate has the ability to promote the plasma membrane expression of prestin mutants accumulated in the cytoplasm. Six prestin mutants created in our previous study, namely, G127A, T128A, S129A, R130A, H131A and S129T, were used. These mutants were engineered to be expressed in HEK293 cells by transfection and effects of salicylate on prestin were then investigated by immunofluorescence staining and the whole-cell patch-clamp. When the cells were cultured without salicylate, immunofluorescence staining showed that all prestin mutants were accumulated in the cytoplasm. The patch-clamp recording indicated that H131A and S129T did not show nonlinear capacitance (NLC), which reflects the amount of functional prestin in the plasma membrane, and that the other four mutants showed NLC significantly smaller than that of wild-type (WT) prestin. On the other hand, when 10 mM salicylate was used, immunofluorescence staining suggested that the plasma membrane expression of all prestin mutants was recovered. Especially, the plasma membrane expression of G127A and R130A was recovered to the WT prestin level. By the patch-clamp method, NLC in G127A, T127A, S129A and R130A were shown to statistically increase, although H131A and S129T did not exhibit NLC. As in the plasma membrane expression, NLC of G127A and that of R130A recovered to the WT prestin level by salicylate. The results suggest that the prestin mutants were misfolded in the cytoplasm and that salicylate has the ability to induce mutants’ correct folding, promoting their transport to the plasma membrane, which led to the recovery of NLC.