Charakterisierung der Lokalisation von Prestin (SLC26A5) in Membran-Mikrodomänen

Prestin (SLC26A5; Pres) nimmt eine Schlüsselstellung für die Funktion des SäugetierInnenohres ein. Es ist das Motorprotein der ÄHZ (Zheng et al. 2000), welches in Antwort auf Änderungen des Membranpotenzials ultraschnelle Längenänderungen der ÄHZ generiert (Brownell et al. 1985). Es wird angenommen,...

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Bibliographic Details
Main Author: Feuer, Anja
Contributors: Oliver, Dominik (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2016
Online Access:PDF Full Text
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Prestin (SLC26A5; Pres) takes a key position for the function of the mammalian inner ear. It is the motor protein of the outer hair cells (Zheng et al. 2000) which generates as an answer to changes in membrane potential ultrafast changes in length of the overall OHC (Brownell et al. 1985). It is supposed that this as electromotility designated process is the base for the electromechanical amplification performed from OHC and that this mechanism is accountable for the remarkable sensitivity of hearing (Liberman et al. 2002). Loss of the OHC amplification is the most frequent reason for hearing loss in humans. Immunolocalisation of native Pres presents a subcellular localisation in the lateral plasma membrane of the OHC (Winter et al. 2006). The present study characterises the distinctive membrane localisation of the three different Pres orthologs from rat (Rattus norvegicus, rPres), zebrafish (Danio rerio, zPres) and chicken (Gallus gallus, cPres) in heterologous expression systems using high-resolution fluorescence microscopy techniques. All analysed Pres orthologs showed a membrane staining, but differences in their distribution within the membrane. Pres proteins from cPres displayed a homogenous distribution. Orthologs from rat and zebrafish expressed in a demonstrative manner, they localised as punctuated clusters. The internal transmembrane domain (TMD) has been identified to act as a molecular determinant for the cluster formation. For this reason the distribution of Pres was accountable to intrinsic features of the TMD portion. Moreover, biophysical properties of the plasma membrane influenced the clustering: The distribution of Pres depended on cellular cholesterol content and temperature. Additionally, specific proteins co-determined the distribution of Pres. Microdomain-associated Cav-1 co-localised with Pres and induced cPres clustering, but was not essential for localisation as clusters. Pres clustering was strongly cytoskeleton associated as Actin and Map1S spots co-localised with Pres clusters. Depolymerisation of Actin cytoskeleton partially dissolved the Pres clusters, but heterologously expressed Map1S prevented the dissolution. Finally, it has been shown biochemically that Actin and Cav-1 interact with Pres in a cholesterol-dependent manner. In conclusion, Pres localises in cholesterol- and cytoskeleton-dependent lipid raft-like membrane domains as a protein complex with Cav-1, Actin and Map1S. This study identified molecular characteristics of Pres membran domains and reveals candidates that could organise the motor protein of the OHC along the lateral membrane. These candidates could be involved in regulating the electromotility.