Prestin forms oligomer with four mechanically independent subunits

Abstract

Prestin is the motor protein of cochlear outer hair cells (OHCs) with the unique capability of performing direct, rapid and reciprocal electromechanical conversion. Prestin consists of 744 amino acids with a molecular mass of ~81.4 kDa. The predicted membrane topology and molecular mass of a single prestin molecule appear inadequate to account for the size of intramembrane particles (IMPs) expressed in the OHC membrane. Although recent biochemical evidence suggests that prestin forms homo-oligomers, most likely as a tetramer, the oligomeric structure of prestin in OHCs remains unclear. We obtained the charge density of prestin in the gerbil OHCs by measuring their nonlinear capacitance (NLC). The average charge density (22,608 μm−2) measured was four times the average IMP density (5,686 μm−2) reported in the freeze-fracture study. This suggests that each IMP contains four prestin molecules, based on the general notion that each prestin transfers a single elementary charge. We subsequently compared the voltage dependency and the values of slope factor of NLC and somatic motility simultaneously measured from the same OHCs to determine whether NLC and motility are fully coupled and how prestin subunits function within the tetramer. We showed that the voltage dependency and slope factors of NLC and motility were not statistically different, suggesting that NLC and motility are fully coupled. The fact that the slope factor is the same between NLC and motility suggests that each prestin monomer in the tetramer is in parallel, each interacting independently with cytoplasmic or other partners to facilitate the mechanical response.

Disparities in voltage-sensor charge and electromotility imply slow chloride-driven state transitions in the solute carrier SLC26a5

“…3C). Finally, there is a decrease in disparity with shorter ramp durations in the hyperpolarizing direction, and with 140 mM chloride, little disparity exists, which accounts for assertions of good electromechanical coupling during the last couple of decades (8)(9)(10)(11)(12).…”

“…OHC eM is voltage dependent and necessarily coupled to its charged voltage sensor (8,9,12,26,27,33). Thus, it is expected that sensor-charge movement, measured as displacement currents or NLC, should correspond to mechanical movements of the cell.…”

“…Following the molecular identification of prestin (3), an SLC26 family member (a5) that recapitulates the electromechanical properties of the native OHC's voltage sensor/motor (4,5), a preponderance of evidence pointed to prestin-based electromotility (eM) as the basis of this boost (6,7). During the last couple of decades, measures of nonlinear charge movement or capacitance (NLC), an electrical correlate of voltage-dependent conformational changes within the motor protein prestin, have served as surrogate, indeed as signature, of voltage-evoked eM of the OHC (8)(9)(10)(11)(12). Intracellular chloride plays an important role in prestin function (13), and recent evidence indicates that anions serve a modulatory, allosteric-like role (14)(15)(16)(17).…”

Low Resolution Structure of a Bacterial SLC26 Transporter Reveals Dimeric Stoichiometry and Mobile Intracellular Domains

“…Initial studies of gerbil prestin using Native PAGE and yeast two-hybrid systems suggested a tetrameric state (23), a hypothesis subsequently supported by single particle analysis (5). Recently however, Wang et al (7) used the charge density of prestin in gerbil outer hair cells to conclude that a tetrameric state was possible, but that their data would also be consistent with a dimer. In contradiction to the original hypothesis, Detro-Dassen et al, (6) used copurification, blue native PAGE and chemical crosslinking to show that human SLC26A3, rat, and zebrafish prestin, as well as a SLC26 homologue from Pseudomonas aeruginosa, form dimers with the monomers working in parallel.…”

Glutamate Transporter Homolog-based Model Predicts That Anion-π Interaction Is the Mechanism for the Voltage-dependent Response of Prestin

“…Furthermore, although the Glt Ph protein is a trimer and prestin is known to function in homooligomeric form (57, 58), our model does not address the issue of oligomerization. Previous studies have shown that each prestin monomer is mechanically independent in the oligomer (57,58) and interacts with cytoplasmic partners to facilitate the mechanical response.…”