Analysis of the molecular mechanisms of prestin-mediated cochlear amplification via a cysteine accessibility study
Prestin is the protein being responsible for electromotility in mammals. It is a member of the SLC26 (Solute Carrier) family, which function as anion transporters. In contrast to the other members, mammalian prestin does not function as an anion transporter but produces electromotility. Non-mammalia...
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|Summary:||Prestin is the protein being responsible for electromotility in mammals. It is a member of the SLC26 (Solute Carrier) family, which function as anion transporters. In contrast to the other members, mammalian prestin does not function as an anion transporter but produces electromotility. Non-mammalian prestin orthologues such as zebrafish prestin are anion-exchangers with a stoichiometry of 1:1, where a chloride anion is transported in exchange for a divalent anion. Based on the bacterial uracil transporter UraA, a structural homology model of prestin with a 7+7 inverted repeat architecture was predicted.
The mechanism of the SLC26-mediated transport is not fully understood. A potential role of this transport mechanism in the electromotile properties of prestin is elusive, but based on the high sequence similarity of transport-capable and electromotile prestin orthologues the idea arose that both are related and electromotility evolved from transport. This is further supported by the finding that the motor protein prestin interacts with and only undergoes conformational rearrangements in the presence of intracellular monovalent anions. Furthermore, it is not completely understood whether electromotile prestin undergoes structural dynamics that are similar to those of transport-capable members of the SLC26 family.
The aim of the present study is to elucidate whether the function of the SCL26 transporters and voltage-driven motors follows common structural principles. To achieve this aim, two members of the SLC26 family were chosen as model systems: the non-mammalian zebrafish prestin and the mammalian rat prestin. The method mainly used in this study is a cysteine accessibility study. It allows insights into structures that should change their accessibility to the aqueous medium according to their movement during a transport cycle.
The membrane-impermeable MTS reagents were either applied from the intracellular or extracellular side. In rat and zebrafish prestin positions were identified which were sensitive to MTS applications and in consequence accessible.
The presumptive anion-binding site and the anion translocation pathway are mainly located in the transmembrane domains 3 and 10. The data here presented are consistent with the structural models derived from experimental structures. Positions within transmembrane domain 10 are mainly accessibly from the intracellular side and positions within transmembrane domain 3 from the extracellular side.
Furthermore, there are central positions in zebrafish prestin, which are accessible from both sides. This indicates an alternating-access transport consistent with elevator movement of the core domain. The lack of extracellular access of the homologous positions in rat prestin shows that the extracellular-exposed conformation is not obtained in mammalian prestin. This points towards an incomplete transport mechanism performed by mammalian prestin.
The rat prestin mutant V139C is accessible from both sides. This two-sided accessibility suggests an outward movement of the core domain within an incomplete transport transition. This mutant shows that V139 faces the translocation pathway in the inward-open as well as in the occluded conformation and thus stays rigid and does not undergo a conformational rearrangement. An alternating-access transport in elevator mode is characterised by a relatively rigid immobile gate domain, a mobile core domain, which contains the anion-binding site, and a vertical displacement of the bound substrate. The results of the scanning support this mode of transport that was also proposed for a prokaryotic SLC26 family member. While crystal structures record a protein in a certain conformation cysteine labelling provides the opportunity to study the full conformational changes.
The model derived from the scanning results present alternate conformations determined by the extracellular accessibilities of positions of the respective prestin orthologue. To what extent the core domain opens to the extracellular side can be deduced by the positions V139 within transmembrane domain 3 in rat prestin and M400 and S400 within transmembrane domain 10 in zebrafish prestin. There is an obvious difference between the alternate conformation of rat and zebrafish prestin. Zebrafish prestin undergoes a full transport cycle from an inward-open to an outward-open conformation, whereas rat prestin alternates between the inward-open and the intermediate (occluded) conformation. The results of the scanning show that the elevator mode of transport is mainly realised by zebrafish prestin. Rat prestin fails to reach the outward-open conformation, thus it performs an incomplete transport mechanism in an elevator-like manner.
Additionally, rat prestin V139C shows a combination of mechano- and redox-sensitivity. The activity of rat prestin V139C is dependent on the application of fluid-flow. This flow-dependency of activity seems to be correlated with the microenvironment of the mutant - the more inflexible the side chain the more distinct the flow-sensitivity of activity. On the basis of the presented data the applied fluid-flow does not cause membrane tension in V139C. The mechanosensitivity of V139C seems to be provoked by pure shear stress. Furthermore, it is different from the intrinsic tension-sensitivity of prestin wild type. Finally, the phenomenon of the mechano- and redoxsensitivity could not yet be clarified.|
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