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 ∼11-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 ∼11-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/prestin-prey 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 ∼11-nm particles in the outer hair cell's basolateral membrane.

Prestin is a unique molecular-motor protein expressed in the lateral plasma membrane of outer hair cells (OHC) in the organ of Corti of the mammalian cochlea. It is thought that prestin undergoes conformational changes driven by the cell9s membrane potential. The resulting alterations in OHC-length are assumed to constitute the cochlear amplifier. Prestin is a member of the anion solute carrier family 26 (SCL26A), but it is different from other family members in its unique function of voltage-driven motility. Because the C-terminus is the least conserved region in the family, we investigated its influence with a series of deletion, point and chimeric mutants. The function and cellular expression of mutants were examined in a heterologous expression system by measurement of nonlinear capacitance (NLC) and immunofluorescence. Each mutant produced a unique mixture of patterns of cell morphologies, which were classified as to the location of prestin within the cell. The data from deletion mutants (Del516, Del525, Del630, Del590, Del709, Del719) revealed that nearly the full length (>708 amino acids) of the protein was required for normal prestin expression and function. Since most deletion mutations eliminated plasma membrane targeting, chimeric proteins were constructed by fusing prestin, at amino acid 515 or 644, with the homologous portion of the C-terminus from the two most closely related SLC26A members, pendrin and putative anion exchanger 1. These chimeric proteins were again improperly (but differently) targeted than simple truncation mutants, and all lacked functional phenotype. When two of the potential basolateral membrane-targeting motifs were mutated (Y520A/Y526A), incomplete plasma membrane expression was seen. We also show that some double point mutations (V499G/Y501H) fully express in the plasma membrane but lack NLC. These non-charged amino acids may have unrevealed important roles in prestin9s function. Together, these data suggest that certain specific sequences and individual amino acids in the C-terminus are necessary for correct cellular distribution and function.

Prestin is a gene recently cloned from mammalian cochlear outer hair cells (OHC) using a single cell type, outer minus inner hair cell, specific suppressive subtractive hybridization procedure. The localization and gene expression profile of the prestin protein fits the pattern of OHC’s development of electromotility. When prestin is abundantly expressed in normally nonmotile kidney cells, nonlinear capacitance and motility that are normally only seen in OHCs can be recorded. Furthermore, both nonlinear capacitance and motility can be reduced by salicylate, a well-known inhibitor of electromotility. These data suggest that prestin is the motor protein of OHCs. Amino acid sequence and gene structure analysis indicate that prestin is the fifth member of a newly discovered anion transport family (SLC26) that includes PDS, DRA and DTDST, which are chloride-iodide transporters, Cl^–/HCO^–₃ exchangers or sulfate transporters. Prestin shares overall structure similarity with this anion transporter family. Recently, intracellular anions (chloride or bicarbonate) were found to be essential for OHC electromotility and prestin’s function.

Niemann-Pick type C1 disease (NPC1) is a fatal genetic disorder caused by impaired intracellular cholesterol trafficking. Recent studies reported ototoxicity of 2-hydroxypropyl- β-cyclodextrin (HPβCD), a cholesterol chelator and the only promising treatment for NPC1. Because outer hair cells (OHCs) are the only cochlear cells affected by HPβCD, we investigated whether prestin, an OHC-specific motor protein, might be involved. Single, high-dose administration of HPβCD resulted in OHC death in prestin wildtype (WT) mice whereas OHCs were largely spared in prestin knockout (KO) mice in the basal region, implicating prestin’s involvement in ototoxicity of HPβCD. We found that prestin can interact with cholesterol in vitro, suggesting that HPβCD-induced ototoxicity may involve disruption of this interaction. Time-lapse analysis revealed that OHCs isolated from WT animals rapidly deteriorated upon HPβCD treatment while those from prestin-KOs tolerated the same regimen. These results suggest that a prestin-dependent mechanism contributes to HPβCD ototoxicity.

Synchronized beating of cilia on multiciliated cells (MCCs) generates a directional flow of mucus across epithelia. This motility requires a “9 + 2” microtubule (MT) configuration in axonemes and the unidirectional array of basal bodies of cilia on the MCCs. However, it is not fully understood what components are needed for central MT-pair assembly as they are not continuous with basal bodies in contrast to the nine outer MT doublets. In this study, we discovered that a homozygous knockdown mouse model for MT minus-end regulator calmodulin-regulated spectrin-associated protein 3 (CAMSAP3), Camsap3^tm1a/tm1a, exhibited multiple phenotypes, some of which are typical of primary ciliary dyskinesia (PCD), a condition caused by motile cilia defects. Anatomical examination of Camsap3^tm1a/tm1a mice revealed severe nasal airway blockage and abnormal ciliary morphologies in nasal MCCs. MCCs from different tissues exhibited defective synchronized beating and ineffective generation of directional flow likely underlying the PCD-like phenotypes. In normal mice, CAMSAP3 localized to the base of axonemes and at the basal bodies in MCCs. However, in Camsap3^tm1a/tm1a, MCCs lacked CAMSAP3 at the ciliary base. Importantly, the central MT pairs were missing in the majority of cilia, and the polarity of the basal bodies was disorganized. These phenotypes were further confirmed in MCCs of Xenopus embryos when CAMSAP3 expression was knocked down by morpholino injection. Taken together, we identified CAMSAP3 as being important for the formation of central MT pairs, proper orientation of basal bodies, and synchronized beating of motile cilia. Significance Cilia are composed of hundreds of proteins whose identities and functions are far from being completely understood. In this study, we determined that calmodulin-regulated spectrin-associated protein 3 (CAMSAP3) plays an important role for the function of motile cilia in multiciliated cells (MCCs). Global knockdown of CAMSAP3 protein expression in mice resulted in defects in ciliary structures, polarity, and synchronized beating in MCCs. These animals also displayed signs and symptoms reminiscent of primary ciliary dyskinesia (PCD), including a mild form of hydrocephalus, subfertility, and impaired mucociliary clearance that leads to hyposmia, anosmia, rhinosinusitis, and otitis media. Functional characterization of CAMSAP3 enriches our understanding of the molecular mechanisms underlying the generation and function of motile cilia in MCCs.