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Prestin Contributes to Membrane Compartmentalization and Is Required for Normal Innervation of Outer Hair Cells.

ABSTRACT: Outer hair cells (OHC) act as amplifiers and their function is modified by medial olivocochlear (MOC) efferents. The unique OHC motor protein, prestin, provides the molecular basis for somatic electromotility, which is required for sensitivity and frequency selectivity, the hallmarks of mammalian hearing. Prestin proteins are the major component of the lateral membrane of mature OHCs, which separates apical and basal domains. To investigate the contribution of prestin to this unique arrangement, we compared the distribution of membrane proteins in OHCs of wildtype (WT) and prestin-knockout (KO) mice. In WT, the apical protein PMCA2 was exclusively localized to the hair bundles, while it was also found at the lateral membrane in KOs. Similarly, a basal protein KCNQ4 did not coalesce at the base of OHCs but was widely dispersed in mice lacking prestin. Since the expression levels of PMCA2 and KCNQ4 remained unchanged in KOs, the data indicate that prestin is required for the normal distribution of apical and basal membrane proteins in OHCs. Since OHC synapses predominate in the basal subnuclear region, we also examined the synaptic architecture in prestin-KO mice. Although neurite densities were not affected, MOC efferent terminals in prestin-KO mice were no longer constrained to the basal pole as in WT. This trend was evident as early as at postnatal day 12. Furthermore, terminals were often enlarged and frequently appeared as singlets when compared to the multiple clusters of individual terminals in WT. This abnormality in MOC synaptic morphology in prestin-KO mice is similar to defects in mice lacking MOC pathway proteins such as ?9/?10 nicotinic acetylcholine receptors and BK channels, indicating a role for prestin in the proper establishment of MOC synapses. To investigate the contribution of prestin's electromotility, we also examined OHCs from a mouse model that expresses non-functional prestin (499-prestin). We found no changes in PMCA2 localization and MOC synaptic morphology in OHCs from 499-prestin mice. Taken together, these results indicate that prestin, independent of its motile function, plays an important structural role in membrane compartmentalization, which is required for the formation of normal efferent-OHC synapses in mature OHCs.

SUBMITTER: Takahashi S

PROVIDER: S-EPMC6062617 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Prestin Contributes to Membrane Compartmentalization and Is Required for Normal Innervation of Outer Hair Cells.

Takahashi Satoe S Sun Willy W Zhou Yingjie Y Homma Kazuaki K Kachar Bechara B Cheatham Mary Ann MA Zheng Jing J

Frontiers in cellular neuroscience 20180720

Outer hair cells (OHC) act as amplifiers and their function is modified by medial olivocochlear (MOC) efferents. The unique OHC motor protein, prestin, provides the molecular basis for somatic electromotility, which is required for sensitivity and frequency selectivity, the hallmarks of mammalian hearing. Prestin proteins are the major component of the lateral membrane of mature OHCs, which separates apical and basal domains. To investigate the contribution of prestin to this unique arrangement, ...[more]

PMID: 30079013

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Project description:Prestin is a membrane protein in the outer hair cell (OHC) that has been shown to be essential for electromotility. OHCs from prestin-null mice do not express prestin, do not have a nonlinear capacitance (the electrical signature of electromotility), and are smaller in size than wild-type OHCs. We sought to determine whether prestin-null OHCs can be transduced to incorporate functional prestin protein in a normal fashion. A recombinant helper-dependent adenovirus expressing prestin and green fluorescent protein (HDAd-prestin-GFP) was created and tested in human embryonic kidney cells (HEK cells). Transduced HEK cells demonstrated membrane expression of prestin and nonlinear capacitance. HDAd-prestin-GFP was then applied to cochlear sensory epithelium explants harvested from wild-type and prestin-null mice at postnatal days 2-3, the age at which native prestin is just beginning to become functional in wild-type mice. At postnatal days 4-5, we investigated transduced OHCs for (1) their prestin expression pattern as revealed by immunofluorescence; (2) their cell surface area as measured by linear capacitance; and (3) their prestin function as indicated by nonlinear capacitance. HDAd-prestin-GFP efficiently transduced OHCs of both genotypes and prestin protein localized to the plasma membrane. Whole-cell voltage clamp studies revealed a nonlinear capacitance in transduced wild-type and prestin-null OHCs, but not in non-transduced cells of either genotype. Prestin transduction did not increase the linear capacitance (cell surface area) for either genotype. In peak nonlinear capacitance, voltage at peak nonlinear capacitance, charge density of the nonlinear capacitance, and shape of the voltage-capacitance curves, the transduced cells of the two genotypes resembled each other and previously reported data from adult wild-type mouse OHCs. Thus, prestin introduced into prestin-deficient OHCs segregates normally to the cell membrane and generates a normal nonlinear capacitance, indicative of normal prestin function.

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Prestin Contributes to Membrane Compartmentalization and Is Required for Normal Innervation of Outer Hair Cells.

Publications

Prestin Contributes to Membrane Compartmentalization and Is Required for Normal Innervation of Outer Hair Cells.

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