Outer hair cell electromotility is believed to be the result of the concerted action of a large number of independent molecular motors closely associated within the cell's basolateral membrane. Morphological studies show that densely packed IMPs cover the lateral plasma membrane (Gulley and Reese 1977
) where electromotility is generated (Kalinec et al. 1992
). A general hypothesis is that some or the majority of the particles are the molecular motor prestin (Koppl et al. 2004
). Here we provided evidence that the majority of these particles are indeed prestin. We showed that when prestin was absent, the density of IMPs was significantly reduced and the replicas of the lateral membrane showed IMP distribution and density comparable to other membranes that do not contain prestin. Correspondingly, we showed that NLC, motility, and piezoelectric current all disappeared when prestin was absent in the membrane. The correlation between significant reduction of IMPs and loss of NLC and motility strongly suggests that the majority of the IMPs are indeed formed by prestin. The correlation between absence of prestin and loss of motility, voltage-dependent stiffness, and piezoelectricity in prestin-null OHCs further confirms the essential role of prestin for these unique properties of OHCs.
The membrane of WT OHCs is dominated by IMPs with diameter between 9 and 13 nm. The particles with size of less than 8 nm make up less than 5% of the total IMPs seen in the membrane (). It appears that the expression of prestin partially precludes the presence of other proteins due to the high-density packing. In prestin-null OHCs, the majority of the IMPs have a particle diameter of less than 7.5 nm. Although it is hard to determine to what extent the particles in the prestin-null membranes are actually present in the WT, it is likely that many membrane proteins are repopulating the lateral plasma membrane in place of the missing prestin in the prestin-null OHCs. The fact that the OHC lateral membrane can lose ~90 % of its proteins and maintain its structural integrity mirrors the conditions during development of electromotilty when, within a few days, the OHC lateral membrane incorporates and densely packs a remarkable amount of prestin molecules(Souter et al. 1995
In heterozygous OHCs, the prestin density is reduced as demonstrated in NLC measurements and in immunogold labeling. Mechanical stress-induced piezoelectric current is also reduced. All these are consistent with the reduction of OHC motility reported in the previous study (Liberman et al. 2002
). Interestingly, in heterozygous OHCs the distribution of IMPs is not uniform. The membrane exhibits patches of IMP-rich and IMP-poor regions, suggesting that prestin has a natural tendency to aggregate into dense packing. This tendency to form aggregates before it is fully confluent is also observed during development when prestin expression levels increase progressively (data not shown). An example of clustering of a particular type of membrane protein and exclusion of other membrane proteins from large expanses of membrane area occurs during formation of gap junctions (Kachar and Reese 1985
The predicted membrane topology and molecular mass of a single prestin molecule (744 amino acids with a molecular mass of ~81.4 kDa) appear inadequate to account for the size of IMPs. It is well known that ion channels and transporters in the membrane form oligomers and that the nature of oligomerization influences the properties of gating and selectivity. For example, some recent studies using biochemical and electron microscopic techniques suggest that prestin likely is comprised of four homologous subunits (Mio et al. 2008
; Pasqualetto et al. 2008
; Zheng et al. 2000
), while another study suggests that prestin likely forms dimers in the membrane (Detro-Dassen et al., 2008
). Therefore, the oligomeric structure of prestin remained controversial. We examined prestin oligomerization by a different approach. We estimated how many prestin molecules are contained in each IMP by comparing the prestin-related elementary charges per square micron with the average IMP density. The mean charge density (22.1 × 103
) measured from WT OHCs was approximately four times the average density of IMPs (5.6 × 103
), suggesting that each IMP contains four prestin molecules. Santos-Sacchi et al (Santos-Sacchi et al. 1998
) showed that the IMP density is not related with cochlear locations. The average density of IMPs in gerbil OHCs is about 5686 particles/µm2
(Koppl et al. 2004
) similar to the average IMP density found in our study. We used the average IMP density of 5.6 × 103
for the calculation. This requires some explanation since the membrane of prestin-null OHCs still contains the IMPs similar to the membrane of other cells. Although the average density of ~2.9 × 103
is present in the prestin-null OHCs, the size of the majority of particles is ~7.2 nm, which is significantly smaller than the average size of ~11.9-nm particles seen in the WT OHCs (). Although the nature of these small IMPs is unknown, they apparently are not prestin. The membrane of WT OHCs also contains some small IMPs (about 8 nm in diameter); they make up less than 5% of the total IMPs seen in the membrane (). Ninety five percent of the IMPs in the WT OHCs have a diameter larger than 9 nm. It is conceivable that the majority of IMPs seen in the membrane are prestin. Therefore, it is reasonable to use their average density (5.6 × 103
) for the calculation. It is conceivable that reduction in the IMP average diameter in the heterozygous () may reflect a reduction in number of prestin molecules in each IMP. If we use the particle density of 5.5 × 103
for heterozygous OHCs to estimate how many prestin molecules in one particle, the ratio (between mean charge density and the average density of IMPs) is ~3, suggesting that some of the IMPs could be trimers or dimers. If this were indeed the case, it would then suggest that each prestin molecule could function normally and independently, regardless of whether prestin exists as tetramers or other oligomers in the membrane. We recognize that, in the membrane of heterozygous OHCs, the distribution of IMPs is not uniform. The membrane exhibits patches of IMP-rich and IMP-poor regions. This might cause overestimation of the area containing elementary charges and therefore, underestimation of the elementary charge density. Since the size and number of these areas are difficult to measure, it is hard to determine how much they would influence the calculation of charge density. We should point out that dimers, trimers, and tetramers are among all those seen in the prestin-expressing cell lines and in native OHCs using biochemical methods (Detro-Dassen et al. 2008
; Zheng et al. 2006
). It is possible that these high-order oligomers (such as trimer and tetramer) can co-exist and are functional.
Since prestin was discovered, a great deal of effort has been made to understand whether somatic motility is responsible for cochlear amplification (Dallos et al. 2008
; Gao et al. 2007
; Liberman et al. 2002
; Mellado Lagarde et al. 2008
). What is still not well understood is how OHC stiffness and length changes emerge from prestin function and whether stiffness and length changes are related at the structural or molecular level. Ultimately, what is still missing is a clear structural framework for the lateral wall and information on how prestin molecules interact with each other and with the underlying cortical cytoskeleton. The electromotility mechanism is unique in its basic organization and is fundamentally different from other well-studied motile systems (Frolenkov et al. 1998a
). The mechanisms of force generation are membrane-based but the underlying cytoskeleton is an integral part of the force translation into the cell shape and stiffness changes that characterize the electromotility. The detailed mechanism of force production and propagation depends on a detailed structural framework, in particular the identity of the pillar elements that connect the underlying cytoskeleton to the membrane. The fact that prestin is required to form or retain the organized actin cytoskeletal lattice suggests that the membrane and the cortical lattice are structurally and functionally interdependent.
The present study shows that the average axial stiffness of the prestin-null OHCs is reduced by as much as 62% of that of the WT OHCs. The reduction is consistent with that reported in a previous study (Dallos et al. 2008
). This reduction is greater than the stiffness reduction seen in the previous study (He et al. 2003
), which showed that the average axial stiffness is reduced by about ~50% when the prestin-based motility is blocked by removal of intracellular Cl−
. There is also a significant reduction of stiffness in the heterozygous OHCs (), consistent with the reduction of prestin charge density in the membrane ( and ). It is conceivable that a significant portion of the stiffness reduction of the prestin-null OHCs is the result of loss of prestin in the lateral wall. The disruption of organization and orientation of actin filaments and pillar proteins in the cortical lattice may also contribute to the reduction of overall stiffness. Our results suggest that the plasma membrane, which contains the closely packed motor protein, is a significant contributor to the stiffness of the OHC’s composite lateral wall. The possibility that the plasma membrane is a significant contributor to the OHC lateral wall’s longitudinal or axial stiffness is quite intriguing. In thin section electron microscopy, the plasma membrane appears as just a very slim line contributing to the overall structure of the lateral wall, which also contains the cortical lattice and in some hair cells also contains multiple parallel cisternae. However, this apparent “thin-shell structure” is consistent with the conclusions of Holley and Ashmore (Holley and Ashmore 1988
), who suggest that most of the lateral wall stiffness is inherent to the plasma membrane.
By measuring the amplitude of driven vibrations of a fiber that was loaded by an isolated OHC while the cell was electrically stimulated under whole-cell voltage-clamp, it was shown that the amplitude of fiber motion was significantly modulated during the contraction—elongation cycle of the cell. The finding is interpreted as a voltage-dependent axial stiffness change of OHCs (Frolenkov et al. 1998b
; He and Dallos 1999
). It is noted that Hallworth (2007)
did not see any voltage-dependent stiffness in the basal turn OHCs from the guinea pig. However, the voltage-dependent stiffness and voltage-dependent cell length are clearly related; several such relationships were revealed in a previous study (He and Dallos 2000
). The most suggestive relationship is the same dependence on prestin, as shown in this study: absence of prestin abolishes both motility and voltage-dependent stiffness (). The general covariance of stiffness and length changes indicates that they may arise from a common mechanism, the motor protein.
It is interesting to note that NLC, pizeoelectrical current, and passive stiffness were reduced in heterozygous OHCs. However, the differences in all these measurements did not reach the 50% reduction that one would expect from one functional allele in the heterozygous as opposed to two functional alleles in the WT mice. Cheatham et al (2005)
(Cheatham et al. 2005
) also showed that while one copy of prestin generates about half the mRNA as revealed by real-time PCR, the level of prestin protein (determined by western blot) in the heterozygous OHC is reduced by only 10–12 %. This is consistent with our functional measurements of prestin. The nature of this discrepancy is unknown. It is possible that prestin expression is regulated post-transcription and OHCs compensate for the deleted prestin gene in the heterozygous mice, thereby minimizing deleterious effects on peripheral auditory function.