Analysis of the Oligomeric Structure of the Motor Protein Prestin

Abstract

Prestin, a member of the solute carrier family 26, is expressed in the basolateral membrane of outer hair cells. This protein provides the molecular basis for outer hair cell somatic electromotility, which is crucial for the frequency selectivity and sensitivity of mammalian hearing. It has long been known that there are abundantly expressed ϳ11-nM protein particles present in the basolateral membrane. These particles were hypothesized to be the motor proteins that drive electromotility. Because the calculated size of a prestin monomer is too small to form an ϳ11-nM particle, the possibility of prestin oligomerization was examined. We investigated possible quaternary structures of prestin by lithium dodecyl sulfate-PAGE, perfluoro-octanoate-PAGE, a membrane-based yeast two-hybrid system, and chemical cross-linking experiments. Prestin, obtained from different host or native cells, is resistant to dissociation by lithium dodecyl sulfate and behaves as a stable oligomer on lithium dodecyl sulfate-PAGE. In the membrane-based yeast two-hybrid system, homo-oligomeric interactions between prestin-bait/prestinprey suggest that prestin molecules can associate with each other. Chemical cross-linking experiments, perfluoro-octanoate-PAGE/Western blot, and affinity purification experiments all indicate that prestin exists as a higher order oligomer, such as a tetramer, in prestin-expressing yeast, mammalian cell lines and native outer hair cells. Our data from experiments using hydrophobic and hydrophilic reducing reagents suggest that the prestin dimer is connected by a disulfide bond embedded in the prestin hydrophobic core. This stable dimer may act as the building block for producing the higher order oligomers that form the ϳ11-nM particles in the outer hair cell's basolateral membrane.Hearing impairment, the most common congenital sensory defect, affects millions of people from newborns to senior citizens, resulting in large hearing-related health care costs (1). Causes of hearing impairment are often associated with damage to outer hair cells (OHCs). 2 These sensory receptor cells, located in the mammalian organ of Corti, rapidly change their length (2) and stiffness (3) at acoustic frequencies when their transmembrane voltage is altered. Corresponding to this somatic cell-length change, OHCs exhibit voltage-dependent non-linear capacitance (4). This unique somatic electromotility is thought to provide active mechanical amplification of the cochlear response to sound (5). It has long been known that large (ϳ11-nM diameter) membrane protein particles constitute a substantial portion of the lateral membrane in OHCs (6). It is suspected that these abundantly expressed particles are the "motor proteins" responsible for somatic electromotility (7).Prestin, the OHC motor protein (8), is located at the same location where the 11-nM protein particles are found, i.e. in the lateral membrane of OHCs (9 -11). When prestin is heterologously expressed in several mammalian cell lines, the prestinexpressing cells demonstrate all of...

Evidence That Prestin Has at Least Two Voltage-dependent Steps

“…On the other hand, the density of the intramembrane particles found in the lateral membrane of OHCs is reported to be ϳ5600/m 2 (28). Thus, a prestin oligomer is expected to be composed of at least three prestin molecules and in conformity with the recent estimates, likely four (27,28).…”

“…OHC displacement, d, was measured directly. (26,27)) is independently functional. The most intriguing and surprising feature of the q-d relation is the positive d intercepts at q ϭ 0, which were statistically significant (p Ͻ 0.001) for all the q-d relations irrespective of the models used for estimating basal capacitances (C lin or C sa ) for determining q values.…”

Conserved Dimeric Subunit Stoichiometry of SLC26 Multifunctional Anion Exchangers

“…Our conclusions are in conflict with a recent report that postulated that gerbil prestin exists as a tetramer formed by association of two disulfide-linked dimers (34). To identify possible reasons for these differences in experimental results, we performed three experiments.…”

“…We can thus only speculate about the reasons for the different results of our group and those by Zheng et al (34). We solubilized prestin with digitonin, whereas Zheng et al used PFO (34).…”

“…Whereas we employed digitonin as detergent and BN-PAGE, Zheng et al used PFO and PFO-PAGE (34). To assess the consequences of this difference, we purified WT and glycosylation-deficient mutant rat prestin from oocytes using digitonin and PFO as detergents.…”