Shun Kumano

Salicylate restores transport function and anion exchanger activity of missense pendrin mutations.

The SLC26A4 gene encodes the transmembrane protein pendrin, which is involved in the homeostasis of the ion concentration of the endolymph of the inner ear, most likely by acting as a chloride/bicarbonate transporter. Mutations in the SLC26A4 gene cause sensorineuronal hearing loss. However, the mechanisms responsible for such loss have remained unknown. Therefore, in this study, we focused on the function of ten missense pendrin mutations (p.P123S (Pendred syndrome), p.M147V (NSEVA), p.K369E (NSEVA), p.A372V (Pendred syndrome/NSEVA), p.N392Y (Pendred syndrome), p.C565Y (NSEVA), p.S657N (NSEVA), p.S666F (NSEVA), p.T721M (NSEVA) and p.H723R (Pendred syndrome/NSEVA)) reported in Japanese patients, and analyzed their cellular localization and anion exchanger activity using HEK293 cells transfected with each mutant gene. Immunofluorescent staining of the cellular localization of the pendrin mutants revealed that p.K369E and p.C565Y, as well as wild-type pendrin, were transported to the plasma membrane, while 8 other mutants were retained in the cytoplasm. Furthermore, we analyzed whether salicylate, as a pharmacological chaperone, restores normal plasma membrane localization of 8 pendrin mutants retained in the cytoplasm to the plasma membrane. Incubation with 10 mM of salicylate of the cells transfected with the mutants induced the transport of 4 pendrin mutants (p.P123S, p.M147V, p.S657Y and p.H723R) from the cytoplasm to the plasma membrane and restored the anion exchanger activity. These findings suggest that salicylate might contribute to development of a new method of medical treatment for sensorineuronal hearing loss caused by the mutation of the deafness-related proteins, including pendrin.

Immune atomic force microscopy of prestin-transfected CHO cells using quantum dots

Prestin, a membrane protein of the outer hair cells (OHCs), is known to be the motor which drives OHC somatic electromotility. Electron microscopic studies showed the lateral membrane of the OHCs to be densely covered with 10-nm particles, they being believed to be a motor protein. Imaging by atomic force microscopy (AFM) of prestin-transfected Chinese hamster ovary (CHO) cells revealed 8- to 12-nm particle-like structures to possibly be prestin. However, since there are many kinds of intrinsic membrane proteins other than prestin in the plasma membranes of OHCs and CHO cells, it was impossible to clarify which structures observed in such membranes were prestin. In the present study, an experimental approach combining AFM with quantum dots (Qdots), used as topographic surface markers, was carried out to detect individual prestin molecules. The inside-out plasma membranes were isolated from the prestin-transfected and untransfected CHO cells. Such membranes were then incubated with antiprestin primary antibodies and Qdot-conjugated secondary antibodies. Fluorescence labeling of the prestin-transfected CHO cells but not of the untransfected CHO cells was confirmed. The membranes were subsequently scanned by AFM, and Qdots were clearly seen in the prestin-transfected CHO cells. Ring-like structures, each with four peaks and one valley at its center, were observed in the vicinity of the Qdots, suggesting that these structures are prestin expressed in the plasma membranes of the prestin-transfected CHO cells.

Atomic force microscopy imaging of the structure of the motor protein prestin reconstituted into an artificial lipid bilayer

Prestin is the motor protein of cochlear outer hair cells and is essential for mammalian hearing. The present study aimed to clarify the structure of prestin by atomic force microscopy (AFM). Prestin was purified from Chinese hamster ovary cells which had been modified to stably express prestin, and then reconstituted into an artificial lipid bilayer. Immunofluorescence staining with anti‐prestin antibody showed that the cytoplasmic side of prestin was possibly face up in the reconstituted lipid bilayer. AFM observation indicated that the cytoplasmic surface of prestin was ring‐like with a diameter of about 11 nm.

Mutation-induced reinforcement of prestin-expressing cells

The motor protein prestin in cochlear outer hair cells is a member of the solute carrier 26 family, but among the proteins of that family, only prestin can confer the cells with nonlinear capacitance (NLC) and motility. In the present study, to clarify contributions of unique amino acids of prestin, namely, Met-122, Met-225 and Thr-428, to the characteristics of prestin, mutations were introduced into those amino acids. As a result, NLC remained unchanged by both replacement of Met-122 by isoleucine and that of Thr-428 by leucine, suggesting that those amino acids were not important for the generation of NLC. Surprisingly, the replacement of Met-225 by glutamine statistically increased NLC as well as the motility of prestin-expressing cells without an increase in the amount of prestin expression in the plasma membrane. This indicates that Met-225 in prestin somehow adjusts NLC and the motility of prestin-expressing cells.

Salicylate-induced translocation of prestin having mutation in the GTSRH sequence to the plasma membrane

Prestin is a key molecule for mammalian hearing. The present study investigated changes in characteristics of prestin by culturing prestin‐transfected cells with salicylate, an antagonist of prestin. As a result, the plasma membrane localization of prestin bearing a mutation in the GTSRH sequence, which normally accumulates in the cytoplasm, was recovered. Moreover, the nonlinear capacitance of the majority of the mutants, which is a signature of prestin activity, was also recovered. Thus, the present study discovered a new effect of salicylate on prestin, namely, the promotion of the plasma membrane expression of prestin mutants in an active state.

Analysis of Membrane Topology of Prestin Expressing in CHO Cells

Outer hair cell (OHC) motility is thought to be based on the voltage‐dependent conformational changes of the motor protein prestin. However, little is known about its structure and function. In this study, the membrane topology of prestin was investigated by single molecule force spectroscopy using an atomic force microscope (AFM). The C‐terminus of prestin was tagged with an Avi‐tag and biotinylated. Prestin was then connected with a streptavidin‐coated AFM cantilever via biotin‐streptavidin binding. The prestin was pulled out from the plasma membrane by retracting the cantilever and force curves were obtained. Obtained force curves suggested the existence of 12 transmembrane domains of prestin.

High‐Resolution AFM Imaging of Prestin Purified and Reconstituted into an Artificial Lipid Bilayer

In the present study, purified prestin was observed by atomic force microscopy (AFM). First, the lipid bilayer was formed on a freshly cleaved mica disk by the deposition of small unilamellar lipid vesicles. Such bilayer was destabilized by incubation with a detergent. Afterward, purified prestin was added to the destabilized lipid bilayer. Finally, a high resolution image of the prestin‐reconstituted lipid bilayer was acquired by AFM. As a result, densely embedded prestin with a diameter of 11.0±1.3 nm was visualized. From the obtained image, cytoplasmic side of prestin was found to form a ring‐like structure with four peaks and one valley at its center.

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.

Effects of Mutations in Unique Amino Acids of Prestin on Its Characteristics

Prestin is believed to be the motor protein ex- pressed in the plasma membrane of outer hair cells (OHCs). In this study, characterization of prestin was carried out by site- directed mutagenesis focusing on the unique amino acids in prestin. Five prestin mutants, namely, M122I, C192A, M225Q, C415A and T428L, were engineered to be separately expressed in HEK293 cells. The characteristics of those mutants were then investigated. As a result, it was found that the charge density of M225Q, which reflects the charge transfer of prestin in the plasma membrane, was 1.7 times larger than that of wild-type prestin (WT), although the charge density of the other mutants were similar to that of WT. On the other hand, the amount of M225Q molecules in the plasma membrane was 1.2 times greater than that of WT molecules. The increase in the amount of prestin molecules due to the mutation of M225 probably indicates that prestin became more stable, but such increase does not correspond to the increase of the charge density. Hence, the mutation of M225 was considered toincrease the stability of prestin, leading to an increase in the ratio of active prestin molecules to the total amount of prestin molecules in the plasma membrane.