Effects of Mutation in the Conserved GTSRH Sequence of the Motor Protein Prestin on Its Characteristics

Abstract

Prestin is a motor protein responsible for the outer hair cell (OHC) electromotility which amplifies the vibration of the organ of Corti in the inner ear. Identification of the functional significance of particular amino acids is necessary to characterize prestin. In this study, an attempt was made to clarify the role of the GTSRH sequence at positions 127-131 in prestin conserved in six proteins of the solute carrier (SLC) 26 family of which prestin is a member. To elucidate what role that sequence plays in the characteristics of prestin, mutations were introduced into the sequence and the characteristics of the constructed point mutants were investigated by Western blotting, immunofluorescence experiments and the whole-cell patchclamp technique. The localization of T128A was altered, the anion transport function of H131A and that of S129T were lost and such functions of G127A, T128A, S129A and R130A declined. These results suggest that the GTSRH sequence plays an important role in the localization of prestin, as well as in its anion transport function.

Functional Expression of Prestin, the Outer Hair Cell Motor Protein, Using the Baculovirus/Insect Cell System

“…As the charge density reflects the amount of charge transfer in the unit cell surface, i.e., the expression level of prestin per unit surface area, the small charge density of prestin-expressing Sf9 cells suggests that the prestin expression level is low. α has been reported to be 28.4 ± 2.3, 38.1 ± 4.8 and 30.7 ± 3.7 mV in OHCs, CHO and HEK293 cells, respectively (13,20) . The value of α 45.9 ± 9.8 mV obtained from Sf9 cells is also significantly larger than that from OHC, CHO and HEK293 cells (p < 0.05).…”

“…These significant differences in the values for V p may be attributable to the differences in the characteristics of the plasma membrane (26,27) . The charge density has been reported to be 20.0 ± 9.6 fC/pF in HEK293 cells (20) . The obtained charge density 2.3 ± 2.0 fC/pF in our experiment was significantly smaller than the previously reported value (p < 0.05).…”

“…In those cells, the fitting parameters were obtained as C lin = 27.1 ± 13.8 pF, Q max = 71.5 ± 37.2 fC, V p = -33.5 ± 19.4 mV and α = 45.9 ± 9.8 mV, and the charge density was calculated as being 2.3 ± 2.0 fC/pF. V p is reported to be between -40 and -20 mV in OHCs (17) and -74.8 ± 11.6 and -62.3 ± 9.2 mV in CHO and HEK293 cells, respectively (13,20) . The obtained V p -33.5 ± 19.4 mV in our experiment agrees with these reported values of OHCs and is significantly larger than that from CHO and HEK293 cells (p < 0.05).…”

“…1, in Western blotting, three bands thought to be derived from prestin were detected at around 70 kDa, 38 kDa and 35 kDa in the lane containing the protein from the infected cells. It has been reported that a band showing prestin expressed in mammalian cells appeared at 100 kDa (20) . However, no 100-kDa band was detected in this study.…”

“…The value of α 45.9 ± 9.8 mV obtained from Sf9 cells is also significantly larger than that from OHC, CHO and HEK293 cells (p < 0.05). This large α may be attributable to small charge density (13,20) .…”