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...

To define the topology of the skeletal muscle ryanodine receptor (RyR1), enhanced GFP (EGFP) was fused in-frame to the C terminus of RyR1, replacing a series of C-terminal deletions that started near the beginning or the end of predicted transmembrane helices M1-M10. The constructs were expressed in HEK-293 (human embryonic kidney cell line 293) or mouse embryonic fibroblast (MEF) cells, and confocal microscopy of intact and saponin-permeabilized cells was used to determine the subcellular location of the truncated fusion proteins. The fusion protein truncated after M3 exhibited uniform cytoplasmic fluorescence, which was lost after permeabilization, indicating that proposed M , M , M1, M2, and M3 sequences are not membrane-associated. The fusion protein truncated at the end of the M4 -M5 loop and containing M4 was membrane-associated. All longer truncated fusion proteins were also associated with intracellular membranes. Mapping by protease digestion and extraction of isolated microsomes demonstrated that EGFP positioned after either M5, the N-terminal half of M7 (M7a), or M8 was located in the lumen, and that EGFP positioned after either M4, M6, the C-terminal half of M7 (M7b), or M10 was located in the cytoplasm. These results indicate that RyR1 contains eight transmembrane helices, organized as four hairpin loops. The first hairpin is likely to be made up of M4a-M4b. However, it could be made up from M3-M4, which might form a hairpin loop even though M3 alone is not membrane-associated. The other three hairpin loops are formed from M5-M6, M7a-M7b, and M8 -M10. M9 is not a transmembrane helix, but it might form a selectivity filter between M8 and M10.S keletal muscle contraction is initiated by activation of a Ca 2ϩ release channel (ryanodine receptor isoform 1 or RyR1) located in the junctional terminal cisternae of the sarcoplasmic reticulum. Activation occurs through physical interaction with the dihydropyridine receptor, located in the transverse tubular membrane, where it is directly apposed to the ryanodine receptor (1, 2). Four RyR monomers, each of 565 kDa, assemble as a homotetrameric complex and transmembrane sequences from each of the four monomers interact to form the ion-conducting pore (3). Hydropathy profiles of RyR1 (4, 5), based on criteria defined by Kyte and Doolittle (6), indicate that the bulk of the RyR1 molecule is cytoplasmic and that 4-12 transmembrane sequences lie within the C-terminal one-tenth to one-fifth of the molecule. Among the predicted transmembrane sequences, four potential transmembrane sequences are clearly more hydrophobic than others, with hydropathy indices between 2.0 and 2.9. These four sequences, amino acids Phe-4564-Tyr-4580, Pro-4641-Leu-4664, Gln-4836 -Phe-4859, and Ile-4918 -Ile-4937, were designated M1-M4 and were proposed to form two hairpin loops in the topological model proposed by Takeshima et al. (4). In a second model, proposed by Zorzato et al. Earlier attempts have been made to elucidate the structure and topology of RyR. Proteolytic digestion of RyR1 ...

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 cell's 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 prestin's 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.

Outer hair cells (OHCs) in the mammalian organ of Corti display electromotility, which is thought to provide the local active mechanical amplification of the cochlear response. Prestin is the key molecule responsible for OHC electromotility. Several compounds, including cGMP, have been shown to influence OHC electromotility. There are two potential cAMP/cGMP-dependent protein kinase phosphorylation sites on prestin. Whether these sites are involved in cGMP-dependent reactions is as yet unknown. In this study, prestin cDNA was transiently transfected into TSA 201 cells. Cells that expressed prestin were selected to measure non-linear capacitance (NLC), a signature of outer hair cell motility. We applied cGMP and cAMP analogues and a protein kinase G (PKG) antagonist to the cells. Furthermore, nine mutations at putative phosphorylation sites of prestin were produced. The neutral amino acid alanine replaced serine/threonine at phosphorylation sites to change the conserved phosphorylation motif in order to mimic the dephosphorylated state of prestin, whereas replacement with the negatively charged aspartic acid mimicked the phosphorylated state. The properties of such modified prestin-expressing cells were examined, through measurement of NLC and with confocal microscopy. Our data demonstrate that cGMP is significantly more influential than cAMP in modifying the non-linear, voltage-dependent charge displacement in prestin-transfected cells. The electrical properties of the single and double mutations further indicate a possible interaction between the two PKG target sites. One of these sites may influence the membrane targeting process of prestin. Finally, a new topology map of prestin is proposed.

Prestin is a motor protein of outer hair cells (OHC) that plays a crucial role in mammalian hearing. Prestin is a putative N-glycoprotein with three potential N-linked glycosylation sites. It is not known whether glycosylation affects the function and activity of prestin. Therefore, the effects of N-glycosylation were investigated by producing single-point (N163Q and N166Q) or double-point mutations (NN163/166QQ and NN163/166AA) at putative N-glycosylation sites. Further, treatment with tunicamycin or glycopeptidase-F was used to determine the consequences of removing N-linked glycosylation in wild-type prestin. We determined the effects of these manipulations on prestin's cell surface expression, molecular mass, glycosylation pattern, and electrophysiological properties in different cell-types. Data indicate that prestin is a glycoprotein with N-linked glycosylation sites at N163 and N166. N163 and N166 may have differential programs for synthesis and trimming of the glycans. The N166 site appears to have greater extent of glycosylation than its companion. N-linked glycosylation is not required for plasma membrane targeting of prestin. Both glycosylated and deglycosylated prestin demonstrate non-linear capacitance, a signature of prestin's motor function. Compared to glycosylated prestin, the fully de-glycosylated protein has altered electrophysiological function, with a change in membrane potential at most effective charge transfer to more depolarized values. These data suggest that glycosylation of prestin may quantitatively affect OHC electromotility.