Mammalian prestin

Abstract

The invention relates to mammalian prestin protein, which has been discovered to be the mammalian cochlear outer hair cell motor, and to polynucleotides encoding prestin. Full length gerbil prestin and its cDNA are described, full length murine prestin and its cDNA are described, and a partial sequence of human prestin and its chromosomal location are described.

Description

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. §119(c) to U.S. Provisional Application No. 60/183,461, filed on Feb. 19, 2000.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

BACKGROUND OF THE INVENTION

[0003] The mammalian cochlea has two types of hair cells. Cochlear hair cells are non-neuronal epithelial cells that transduce acoustic signals. Outer hair cells (OHCs) are responsible for the exquisite sensitivity and frequency-resolving capacity of the normal mammalian hearing organ, the ear; they provide local mechanical amplification (the “cochlear amplifier”) in the form of feedback (1987, Ashmore, et al., J. Physiol. (London), 388:323-347). In contrast, inner hair cells (IHCs) convey auditory information to the brain (Dallos, P., Overview: Cochlear Neurobiology, pages 1-43, Springer, NY, 1996). OHCs have cylindrical somata of constant diameter and variable length. It is generally believed that the mammalian cochlea owes its remarkable sensitivity, frequency selectivity, and various complex nonlinear properties to a mechanical feedback action by OHCs.

[0004] In response to membrane potential change, the OHC rapidly alters its length and stiffness (1985, Brownell et al., Science, 227:194-196). These mechanical changes, driven by putative molecular motors, are assumed to produce amplification of vibrations in the cochlea that are transduced by IHCs. These somatic shape changes may be up to 5% of the cell length; The cell shortens when depolarized and lengthens when hyperpolarized. Length changes do not depend on either ATP or Ca2+ (1988, Holley et al., Proc. R. Soc. Lond. Ser. B. Biol. Sci., 232:413-429.) and they can be elicited with unchanging amplitude at microsecond rates up to high audio frequencies. Motile responses are accompanied by charge movement, reflected in nonlinear capacitance, akin to the translocation of gating charges of voltage-gated ion channels (1991, Santos-Sacchi et al., J. Neurosci., 11:3096-3110). This nonlinear capacitance is widely used as a “signature” of the electromotile process. Motility is also accompanied by axial stiffness change of the cell. By virtually any test, electromotility and electrically-induced stiffness changes can be correlated with each other and they are collectively described as voltage-dependent mechanical changes of the OHC, heretofore called electromechanics. These observations make it apparent that the fast mechanical changes in OHCs are powered by a novel molecular motor, fundamentally different from other biological force generators, such as the myosin, kinesin or dynein families. The OHC molecular motor performs direct, rapid, reversible electromechanical conversion.

[0005] Despite extensive studies of cellular and biophysical mechanisms of OHC function, very little is known about the genes and molecular events involved in OHC function. OHC electromotility is the likely result of the concerted action of a large number of independent molecular motors that are closely associated with the cell's basolateral membrane, possibly by the densely packed 10 nanometer particles seen therein.

[0006] There have been some suggestions as to the identity of these motor molecules. Based on similarities between the cortical structure of erythrocytes and OHCs, it has been proposed that the motor molecule is a modified anion exchanger. Because their shallow voltage dependence matches that of charge movement in OHCs, transporters have been favored, as opposed to modified voltage-dependent channels, as likely candidates. A recent suggestion is that the motor is related to a fructose transporter, GLUTS. Until the present invention, molecular identification of the motor protein has not been achieved.

SUMMARY OF THE INVENTION

[0007] The invention includes an isolated polynucleotide comprising a portion that anneals with high stringency with at least twenty consecutive nucleotide residues of a coding region of a mammalian pres gene.

[0008] In one aspect, the mammalian pres gene comprises a nucleotide sequence listed in SEQ ID NO: 2 or SEQ ID NO: 4.

[0009] In another aspect, the coding region is one other than a coding region corresponding to any of exons 1-6 of the human pres gene.

[0010] The invention also includes an isolated polynucleotide comprising the coding regions of a mammalian prestin gene, wherein the coding regions of the prestin gene are at least 75% homologous with the coding region of at least one of the gerbil and murine prestin gene.

[0011] In a preferred aspect, the isolated polynucleotide comprises a promoter/regulatory region operably linked with the coding regions.

[0012] An isolated mammalian prestin protein is also encompassed by the invention. In one aspect, the mammalian prestin protein is isolated from a gerbil, a mouse, or a human. In another aspect, the protein has an amino acid sequence listed in SEQ ID NO: 1 or SEQ ID NO: 3. Preferably, the protein is substantially purified.

[0013] Also contemplated by the invention is an isolated antibody which binds specifically with a mammalian prestin protein.

[0014] The invention includes a method of alleviating a hearing disorder in a mammal afflicted with the disorder comprising providing a mammalian prestin protein to cochlear outer hair cells of the mammal, thereby alleviating the disorder. In one aspect, the protein is provided to the outer hair cells by providing a nucleic acid vector comprising a polynucleotide encoding the protein to the outer hair cells.

[0015] A method of rendering the surface area of a lipid bilayer susceptible to modulation by membrane potential is also envisaged in the invention. The method comprises providing a mammalian prestin protein to the bilayer, whereby the bilayer is rendered susceptible to modulation by membrane potential. In an aspect of the invention, the lipid bilayer is the plasma membrane of a cell.

[0016] In another aspect, the protein is provided to the lipid bilayer by providing an expressible nucleic acid vector comprising a polynucleotide encoding the protein to the cell and then expressing the protein in the cell.

[0017] The invention also includes a method of modulating the surface area of a lipid bilayer. The method comprises providing a mammalian prestin protein to the bilayer and modulating the membrane potential, thus modulating the surface area of the bilayer.

[0018] A method for modulating stiffness of a lipid bilyer surrounding a relatively fixed volume is also within the scope of the invention. The method comprises providing a mammalian prestin protein to the bilayer, modulating the membrane potential, and thus, modulating bilayer stiffness.

[0019] The invention also encompasses a method of modulating the volume of a porous bilammelar lipid vesicle. The method comprises providing a mammalian prestin protein to the bilayer and modulating the membrane potential, thereby modulating the volume of the vesicle.

[0020] The invention further includes a method for generating a force between two surfaces. The method comprises interposing a structure having a lipid membrane comprising prestin and enclosing a relatively fixed volume of fluid between the surfaces, restraining the ability of the structure to expand in a direction at least partially parallel to at least one of the surfaces, and altering the membrane potential of the lipid membrane. The structure impacts upon the two surfaces and a force is generated between them.

[0021] The invention also describes a method for generating an electrical impulse by applying a mechanical force to the prestin protein. The method comprises applying a mechanical stress on the prestin protein, such that the protein creates an electrical impulse in response to the mechanical force.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiment(s) which are presently preferred. However, it should be understood that the invention is not limited to the precise arrangements and instrumentalities shown. In the drawings:

[0023] FIG. 1, comprising FIGS. 1A-1D, is a quartet of images depicting the results of subtractive hybridization experiments. Identical cDNA was fixed to each of the 4 blots and separately hybridized with radioactive probes. FIG. 1A represents forward-subtracted probe (OHC-IHC). FIG. 1B represents reverse-subtracted probe (IHC-OHC). FIG. 1C represents OHC probe, and FIG. 1D represents IHC probe. A signal comparison between OHC or OHC-IHC hybridization and IHC or IHC-OHC hybridization is indicated by the arrows.

[0024] FIG. 2 depicts the amino acid sequence of gerbil prestin (upper row), GenBank Accession Number AF230376 and SEQ ID NO: 1, and rat pendrin (lower row), GenBank Accession Number AF167412 (SEQ ID NO: 11). Bolded and underlined residues indicate the differences between the deduced human prestin sequence. Identical residues are indicated with an asterisk (*), while highly similar amino acids are indicated with a solid circle (&Circlesolid;). The sulfate transport motif is boxed with a solid line ( ). The positive charge cluster is outlined by a dotted line ( ), and the negative charge cluster is boxed with a dashed line ( ).

[0025] FIG. 3, comprising FIGS. 3A and 3B, lists the alignment of various sulfate transport motifs in selected proteins. FIG. 3A aligns prestin, pendrin, down-regulated in adenoma (DRA), and distrophic dysplasia (DTD) sequences from human and rodent species. Divergent residues in the prestin sequences are bolded. Amino acid residue numbers refer to gerbil prestin. FIG. 3B depicts sulfate transport motifs in a variety of putative transporters in lower organisms. Invariant residues are indicated with asterisks. The sulfate transport motif is defined by PROSITE PS01130.

[0026] FIG. 4, comprising FIGS. 4A-4D, represents an analysis of pres gene expression. FIG. 4A is a virtual Northern blot analysis of cDNA pools derived from mRNA of gerbil tissues hybridized with a [32P]-labeled pres probe. FIG. 4B depicts PCR results where cDNA pools from different tissues were amplified with pres-specific and cyclophilin-specific primers. In FIGS. 4A and 4B, cochlea was derived from the organ of Corti. FIG. 4C is also a virtual Northern blot analysis. cDNA pools derived from the organ of Corti mRNA at 0, 6, 12, 16, and 20 days after birth (DAB) were hybridized with a [32P]-labeled pres-specific probe, followed by stripping and rehybridization with a [32P]-labeled cyclophilin probe. FIG. 4D indicates PCR results from the cDNA in FIG. 4C, amplified with pres and cyclophilin-specific primers.

[0027] FIG. 5, comprising FIGS. 5A-5D, depicts the voltage-dependent charge movement in TSA201 cells. All recordings were within 24-60 hours of transfection. FIG. 5A illustrates the charge movement measured as transient capacitive currents using a standar subraction technique (P/4). The cell is held at −70 millivolts and voltage steps are 20 millivolts to +50 millivolts. FIG. 5B is a stair-step protocol quantifying voltage dependence of nonlinear capacitance of 20 presentations which were averaged. Voltage was increased from −130 millivolts to +40 millivolts in 10 millivolt increments. FIG. 5C illustrates the membrane capacitance-voltage curve obtained from the stair-step current trace in FIG. 5B. The trace fits data points with a derivative of a Boltzmann function. FIG. 5D demonstrates that localized application of 10 millimolar sodium salicylate reversibly blocks charge movement.

[0028] FIG. 6, comprising FIGS. 6A-6E, depicts examples of voltage-dependent motility expressed in TSA201 cells. FIG. 6A is an image of a TSA201 cell being partially drawn in to a microchamber. FIG. 6B is a trace representing motile responses from control and transfected cells. The top two traces depict motile responses for the transfected cells, while the bottom trace depicts lack of motile response for the control cell. FIG. 6C demonstrates the effect of 10 millimolar sodium salicylate in the external bathing solution on the motile response. FIG. 6D depicts response (top) and stimulus (bottom) waveforms with two different command frequencies. FIG. 6E illustrates Fourier transforms of the response segments in FIG. 6D (top). A 1.2 decibel correction for frequency response was incorporated into the resultant Fourier transforms in FIG. 6E. Response waveforms are the average of 200 presentations.

[0029] FIG. 7, comprising FIGS. 7A and 7B, lists the gerbil cDNA sequence encoding prestin (SEQ ID NO: 2).

[0030] FIG. 8, comprising FIGS. 8A, 8B, and 8C lists a portion of the nucleotide sequence (SEQ ID NO: 2) and the amino acid sequence of gerbil prestin (SEQ ID NO: 1) with the corresponding human amino acid sequence beneath it (SEQ ID NO: 5).

[0031] FIG. 9, comprising FIGS. 9A-9M, lists a portion of the nucleic acid sequence of BAC clone RG107G13 (GenBank Accession Number RG107G13; SEQ ID NO: 6) from nucleotide residue number 90,000 to nucleotide residue number 119, 484.

[0032] FIG. 10, comprising FIGS. 10A and 10B, lists the murine cDNA sequence of prestin (FIG. 10A, SEQ ID NO: 4) and the murine amino acid sequence of prestin (FIG. 10B, SEQ ID NO: 3).

[0033] FIG. 11 is a schematic diagram depicting Stage 1 of the procedure for establishing the OHC subtracted library. After obtaining polyA RNA from each of the OHCs and IHCs, 5′-cap reverse transcription oligo-dT dependent PCR is preformed to obtain the cDNA pools of each of the OHCs and IHCs. After the hybridization screening, the cDNA can be reverse or forward subtracted or subtracted using tester or driver cDNA alone.

[0034] FIG. 12, comprising FIGS. 12A-12D, is a quartet of images depicting IHCs and OHCs isolated from adult gerbil cochlea. Note the differences in stereocilia configuration.

[0035] FIG. 13, comprising FIGS. 13A, 13B, and 13C, is a series of images depicting the typical whole-cell current responses recorded in OHCs (FIG. 13A) and IHCs (FIG. 13B). FIG. 13C depicts the same responses in graph form.

[0036] FIG. 14 is an image of an agarose gel stained with ethidium bromide. The image depicts subracted cDNA pools prior to cloning. Forward-subtracted cDNA is compared to unsubtracted cDNA.

[0037] FIG. 15 is an image depicting amplification and size determination of cloned OHC-subtracted cDNAs. These cDNAs were used for the hybridization screening analysis depicted in FIG. 16.

[0038] FIG. 16, comprising FIGS. 16A-16D, is a series of cDNA hybidization dot-blot assays. These blots demonstrate differential hybridization of the cloned, subtracted OHC cDNAs shown in FIG. 15. FIG. 16A represents a hybridization blot hybridized with radioactive forward subtracted (OHC-IHC) probe. FIG. 16B represents hybridization with radioactive reverse subtracted (IHC-OHC) probe. FIGS. 16C and 16D represent hybridization with radioactive tester (OHC) and driver (IHC) probes, respsectively.

[0039] FIG. 17 is a schematic diagram depicting Stage 2 of the procedure for analyzing an OHC subtracted library.

[0040] FIG. 18 is an image of a virtual Northern dot blot which confirms the differential expression of OHC-subtracted cDNAs.

[0041] FIG. 19, comprising FIGS. 19A and 19B, is a pair of images depicting immunofluorescent expression of C-terminal tagged pres in transfected TSA201 cells. FIG. 19A illustrates cells transfected with control vector and FIG. 19B demonstrates cells transfected with C-terminal tagged prestin.

[0042] FIG. 20 depicts the nonlinear capacitance of TSA201 cells transiently transfected with N-tagged prestin construct.

[0043] FIG. 21, comprising FIGS. 21A-21D, depicts immunofluorescence images of TSA201 cells transiently transfected with C-tagged prestin (V5 epitope; FIGS. 21A and 21C) and N-tagged prestin (Xpress epitope; FIGS. 21B and 21D). FIGS. 21A and 21B depict permeabilized cells and FIGS. 21C and 21D depict nonpermeabilized cells. C-tagged prestin magnification is 100×. N-tagged prestin magnification is 40×.

[0044] FIG. 22, comprising FIGS. 22A-22D, is a set of images depicting immunofluorescence of transiently transfected TSA201 cells. FIGS. 22A and 22B represent the identical cells transfected with C-tagged prestin, with different filters. Magnification at 40×. FIG. 22C and 22D represent identical cells viewed with different filters. Magnification at 20×. FIGS. 22A and 22C depict cells binding with FITC-labeled anti-rabbit IgG, indicating cells expressing native prestin FIGS. 22B and 22D depict cells binding with Cy3-conjugated mouse IgG, indicating cells expressing the V5 tag epitope.

[0045] FIG. 23, comprising FIGS. 23A-23D, is a set of immunofluorescent images of the basilar membrane/organ of Corti complex. FIGS. 23A and 23B depict images of the same cells, viewed through different filters at 20× magnification. FIGS. 23C and 23D depict images of the same cells viewed through different filters at 40× magnification. FIGS. 23A and 23C depict cells binding with FITC-labeled rabbit IgG, indicating cells expressing prestin. FIGS. 23B and 23D depict cells stained with propidium iodide, indicating location of the cell nuclei.

[0046] FIG. 24, comprising FIGS. 24A-24D, is a set of immunofluorescent images of permeabilized (FIGS. 24A and 24B) and nonpermeabilized (FIGS. 24C and 24D) cells binding with FITC-labeled rabbit IgG. FIGS. 24A and 24C are magnified 20× and FIGS. 24B and 24D are magnified 40×.

[0047] FIG. 25 represents a predicted topology of the prestin protein. Charge clusters and sulfate transporter motif are indicated by boxes.

DETAILED DESCRIPTION OF THE INVENTION

[0048] The invention relates to discovery of an integral membrane protein which modulates the shape of mammalian cochlear outer hair cells in response to the membrane potential of the plasma membrane of the cells. In as much as the most distinguishing feature of this novel molecular motor of the invention is its speed, it has been designated as “prestin”, from the musical notation presto. The gene (Prestin) coding for this protein is abbreviated “Pres”, herein.

[0049] The amino acid sequence (SEQ ID NO: 1) of gerbil prestin is listed in FIG. 2, and the nucleotide sequence (SEQ ID NO: 2) of a gerbil EDNA which encodes prestin is listed in FIG. 7. A portion of the human gene (herein designated the pres gene) encoding prestin has been sequenced as a part of the human chromosome 7 sequencing effort. This portion corresponds to at least nucleotide residues 99,895-113,558 of BAC clone RG107G13 (GenBank Accession number RG107G13). The nucleotide sequence of residue numbers 90,000 to 119,484 of this clone (SEQ ID NO: 6) is reproduced in FIG. 9. The portion of the amino acid sequence of human prestin (SEQ ID NO: 5) corresponding to the sequenced region is referred to in FIG. 2 and is listed below the corresponding gerbil prestin amino acid sequence (SEQ ID NO: 1) in FIG. 8. FIG. 19 depicts surface expression of C-tagged prestin in TSA201 cells which were transfected as described in that example. The amino acid sequence (SEQ ID NO: 3) of murine prestin and the nucleotide sequence (SEQ ID NO: 4) of a cDNA encoding it have been determined, and are listed in FIGS. 10B and 10A, respectively. Portions of the murine pres gene were reported by others in a BAC clone derived from murine chromosome 5. The nucleotide sequence of this BAC clone is listed in GenBank accession number AC023284.

[0050] The invention includes an isolated polynucleotide which anneals with high stringency with at least twenty consecutive nucleotide residues of at least one strand of a mammalian pres gene, (e.g. the human, murine, or gerbil pres gene), or with one strand having a nucleotide sequence comprising SEQ ID NO: 2 having naturally-occurring introns interposed therewithin, or a coding region thereof. Preferably, the isolated polynucleotide of the invention anneals with high stringency with at least 25, more preferably with at least 30, and even more preferably with at least 40, at least 50, or at least 100 consecutive nucleotide residues. In certain embodiments, the isolated polynucleotide of the invention has a length not greater than about 200 nucleotide residues, more preferably not greater than about 100 nucleotide residues, and even more preferably not greater than about 50, 40, or even 35 nucleotide residues.

[0051] The isolated polynucleotide of the invention preferably has a sequence that is substantially homologous with at least 20, 25, or 35 consecutive nucleotide residues of at least one strand of the mammalian pres gene. More preferably, the isolated polynucleotide of has a sequence completely homologous with at least 20, 25, or 35 consecutive nucleotide residues of at least one strand of the mammalian pres gene, and even more preferably with at least 20, 25, or 35 consecutive nucleotide residues of at least one strand of a DNA having the nucleotide sequence SEQ ID NO: 2 or SEQ ID NO: 4.

[0052] The isolated polynucleotide can be inserted into a gene vector in order to facilitate cloning, fusion protein production, or delivery of the polynucleotide to a cell (i.e. in vitro or in vivo) for the purpose of inducing or enhancing expression of pres in the cell or for the purpose of inhibiting or preventing expression of pres in the cell (e.g. using an antisense oligonucleotide which binds specifically with at least one strand of a portion of the pres gene). When the vector is intended for use to induce or enhance expression of pres in a cell, the vector includes one or more promoter/regulatory sequences which cause the gene to be expressed in the presence of the normal transcription and/or translation mechanism of the cell.

[0053] When the isolated polynucleotide of the invention is to be hybridized or annealed with a nucleic acid having a sequence wherein at least a portion is complementary to the isolated polynucleotide, the necessary degree of homology between the isolated polynucleotide and the at least one strand of pres is dependent on the length of the polynucleotide. It is well known in the art that, as the length of a polynucleotide increases, the degree of complementarity necessary to anneal the polynucleotide with another polynucleotide with high stringency decreases. Numerous methods, algorithms, computer programs, and the like are known whereby the skilled artisan may predict the stringency of binding between two polynucleotides (e.g. Suhai, Ed., 1992, Computational Methods in Genome Research, Plenum Press, New York; Swindell, Ed., 1997, Sequence Data Analysis Guidebook, Humana Press, New Jersey; Bishop, Ed., 1998, Guide to Human Genome Computing, Academic Press, New York). Any of these methods, etc., may be used by the skilled artisan, in light of the present disclosure, to design or select isolated polynucleotides of various lengths which will anneal with at least one strand of a human pres gene with high stringency. All such isolated polynucleotides are included within the invention.

[0054] When the isolated polynucleotide of the invention is to be used to express all or a portion of a mammalian (e.g. human, murine, or gerbil) prestin protein, either in vitro or in vivo, it is important that (i) the homology of the isolated polynucleotide of the pres gene (e.g. SEQ ID NO: 2) is such that the amino acid sequence encoded by the isolated polynucleotide is identical to the corresponding region of pres, (ii) the differences between the sequence of the isolated polynucleotide and the corresponding region of pres does not result in differences in the encoded amino acid sequence (i.e. any sequence difference in a coding region merely substitutes a codon encoding an amino acid in place of another codon encoding the same amino acid), or (iii) any differences in the encoded amino acid sequence between the isolated polynucleotide and the corresponding region of pres results only in one or more conservative amino acid substitutions, as described in greater detail elsewhere herein. The following Human Codon Table may be used to select or identify alternate codons which encode the same amino acid. 1 Human Codon Table Amino Acid Codons Encoding the Amino Acid Alanine GCA GCC GCG GCU Cysteine UGC UGU Aspartic acid GAC GAU Glutamic acid GAA GAG Phenylalanine UUC UUU Glycine GGA GGC GGG GGU Histidine CAC CAU Isoleucine AUA AUC AUU Lysine AAA AAG Leucine UUA UUG CUA CUC CUG CUU Methionine AUG Asparagine AAC AAU Proline CCA CCC CCG CCU Glutamine CAA CAG Arginine AGA AGG CGA CGC CGG CGU Serine AGC AGU UCA UCC UCG UCU Threonine ACA ACC ACG ACU Valine GUA GUC GUG GUU Tryptophan UGG Tyrosine UAC UAU

[0055] In situations in which it is necessary or desirable to introduce nucleotide residue changes into a polynucleotide, or into a prestin protein or a portion thereof, a variety of well-known techniques may be used, such as site-specific mutagenesis. Site-specific mutagenesis, for example, allows production of mutants through the use of specific oligonucleotides which encode the sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complementarity to form a stable duplex on both sides of the nucleotide sequence to be altered (e.g. a codon). Typically, a primer of about 17 to 25 nucleotides in length is preferred, with about 5 to 10 residues on both sides of the junction of the sequence being altered. This technique typically employs a phage vector which exists in both a single stranded and double stranded form. Typical vectors useful in site-directed mutagenesis include vectors such as M13 phage. Such vectors are commercially available, and their use is well known in the art. Double stranded plasmids are also routinely employed in site-directed mutagenesis protocols, to eliminate the need to transfer the gene of interest from a plasmid to a phage vector. Site-directed mutagenesis is performed by first obtaining a single-stranded vector or dissociating the two strands of a double stranded vector which includes within its sequence a DNA sequence which comprises the desired site of mutagenesis. The oligonucleotide primer described above is annealed with the single-stranded vector, and subjected to DNA polymerization, in order to generate a mutation-bearing strand. A heteroduplex is formed between the mutation-bearing strand and either the original non-mutated strand of the double-stranded vector or an added or synthesized strand which is substantially complementary to the mutation-bearing strand. This heteroduplex is then used to transform appropriate cells, such as E. coli or cultured human cells, and clones are selected which comprise recombinant vectors bearing the mutated sequence arrangement. Preparation of sequence variants of the isolated polynucleotide of the invention using site-directed mutagenesis is provided merely as an example of a method of producing potentially such variants, and is not intended to be limiting, as there are other well-known methods for producing such variants. By way of example, recombinant vectors comprising or encoding the desired isolated polynucleotide may be treated with mutagenic agents, such as hydroxylamine, to obtain sequence variants.

[0056] The isolated polynucleotide of the invention may be single stranded or double-stranded, it being understood that a single-stranded form is the form referred to herein when annealing of the isolated polynucleotide of the invention with another nucleic acid is described.

[0057] The determination of percent identity between two nucleotide or amino acid sequences can be accomplished using a mathematical algorithm. For example, a mathematical algorithm useful for comparing two sequences is the algorithm of Karlin and Altschul (1990, Proc. Natl. Acad. Sci. USA 87:2264-2268), modified as in Karlin and Altschul (1993, Proc. Natl. Acad. Sci. USA 90:5873-5877). This algorithm is incorporated into the NBLAST and XBLAST programs of Altschul, et al. (1990, J. Mol. Biol. 215:403-410), and can be accessed, for example at the National Center for Biotechnology Information (NCBI) world wide web site having the universal resource locator “http://www.ncbi.nlm.nih.gov/BLAST/”. BLAST nucleotide searches can be performed with the NBLAST program (designated “blastn” at the NCBI web site), using the following parameters: gap penalty=5; gap extension penalty=2; mismatch penalty=3; match reward=1; expectation value 10.0; and word size=11 to obtain nucleotide sequences homologous to a nucleic acid described herein. BLAST protein searches can be performed with the XBLAST program (designated “blastn” at the NCBI web site) or the NCBI “blastp” program, using the following parameters: expectation value 10.0, BLOSUM62 scoring matrix to obtain amino acid sequences homologous to a protein molecule described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997, Nucleic Acids Res. 25:3389-3402). Alternatively, PSI-Blast or PHI-Blast can be used to perform an iterated search which detects distant relationships between molecules (Id.) and relationships between molecules which share a common pattern. When utilizing BLAST, Gapped BLAST, PSI-Blast, and PHI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.

[0058] The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically exact matches are counted.

[0059] The invention also relates to an isolated prestin protein which has a sequence which is identical or highly homologous (e.g. 70%, 80%, 85%, 90%, 95%, 98%, or 99% or more homologous) with the amino acid sequence of gerbil prestin (SEQ ID NO: 1) or murine prestin (SEQ ID NO: 3). Preferably, the isolated prestin protein is substantially purified. The isolated prestin protein may be in the form of a suspension of the native or denatured protein in a liquid such as water, a buffer, or the like, aIyophilized powder, an immunogenic composition comprising the protein and one or more adjuvants or immunogenicity enhancers such as are known in the art, or a pharmaceutical composition.

[0060] The isolated prestin protein of the invention may be made by a variety of techniques. For example, the protein may be expressed in an in vitro expression mixture using an isolated polynucleotide of the invention. The isolated polynucleotide of the invention may also be operably linked with a constitutive or other promoter, and the prestin protein is then over-expressed in a human or non-human cell, and subsequently purified therefrom. Alternately, the prestin protein may be purified using, for example, standard chromatographic techniques from a naturally occurring source of prestin protein (e.g. mammalian cochlear outer hair cells).

[0061] The isolated prestin protein can have an amino acid sequence completely homologous with SEQ ID NO: 1 or SEQ ID NO: 2, or it can comprise one or more conservative amino acid substitutions relative to either of these two sequences). For example, the protein can have an amino acid sequence which incorporates the differences between any two of the gerbil, murine, and human prestin amino acid sequences described herein. Furthermore, it is within the level of skill of the ordinary worker to determine the remainder of the sequence of the human pres gene (e.g. by constructing an oligonucleotide primer that is complementary to the end of BAC clone RG107G13 corresponding to pres, isolating a genomic fragment that hybridizes with the primer, and determining the sequence of the genomic fragment beyond that provided in BAC clone RG107G13). Thus, the present invention includes isolated human prestin as well.

[0062] Certain amino acids of prestin may be substituted for other amino acids without appreciably affecting the biological activity of the protein. Preferably, the amino acid sequence of the isolated prestin protein is substantially homologous with SEQ ID NO: 1 or the sequence of human prestin. The hydropathic index of naturally occurring prestin amino acid residues may be compared with those of potential substitute amino acid residues. The significance of amino acid hydropathic index similarity between naturally occurring and potential substitute amino acid residues, as it relates to retention of biologic function of a protein is generally understood in the art. It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.

[0063] Each naturally occurring amino acid residue has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics, as described (Kyte et al., 1982, J. Mol. Biol. 157:105). These hydropathic index values are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5);glutamine (−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine (−4.5). Amino acid residues may be substituted in place of other amino acid residues which having a similar hydropathic index without significantly affecting biological activity of the protein. Preferably, the substitute amino acid residue has a hydropathic index which differs from the hydropathic index of the naturally occurring amino acid residue by less than 2.0, preferably by less than 1.0, and more preferably by less than 0.5. For example, if the hydropathic index of a naturally occurring amino acid residue is 1.8, then a substitute amino acid residue should have a hydropathic index in the range from 3.8 to −0.2, preferably in the range from 2.8 to 0.8, and more preferably in the range from 2.3 to 1.3.

[0064] An alternate method may be used to predict amino acid residues which may be substituted in place of naturally occurring prestin amino acid residues in regions of prestin which are predicted to interact with other molecules, such as regions predicted to interact with the plasma membrane of cells. This method has been described in the art (Hoop et al., 1981, Proc. Natl. Acad. Sci. USA 78:3824), and involves assigning the following hydrophilicity values to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0); glutamate (+3.0); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); proline (0.0); threonine (−0.4); alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4). Amino acid residues may be substituted in place of other amino acid residues having a similar hydrophilicity value without significantly affecting biological activity of the protein. Preferably, the substitute amino acid residue has a hydrophilicity value which differs from the hydrophilicity value of the naturally occurring amino acid residue by less than 2.0, preferably by less than 1.0, and more preferably by less than 0.5. For example, if the hydrophilicity value of a naturally occurring amino acid residue is 1.8, then a substitute amino acid residue should have a hydrophilicity value in the range from 3.8 to −0.2, preferably in the range from 2.8 to 0.8, and more preferably in the range from 2.3 to 1.3.

[0065] As outlined above, amino acid substitutions may be based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. For example, conservative amino acid substitutions may include substitutions within the following groups:

[0066] glycine, alanine;

[0067] valine, isoleucine, leucine;

[0068] aspartic acid, glutamic acid;

[0069] asparagine, glutamine;

[0070] scrine, threonine;

[0071] lysine, arginine;

[0072] phenylalanine, tyrosine.

[0073] Modifications (which do not normally alter primary sequence) include in vivo, or in vitro chemical derivatization of polypeptides, e.g., acetylation, or carboxylation. Also included are modifications of glycosylation, e.g., those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in further processing steps; e.g., by exposing the polypeptide to enzymes which affect glycosylation, e.g., mammalian glycosylating or deglycosylating enzymes. Also embraced are sequences which have phosphorylated amino acid residues, e.g., phosphotyrosine, phosphoserine, or phosphothreonine.

[0074] Isolated prestin protein, or a fragment thereof, may be used to generate polyclonal or monoclonal antibodies using known methods. As is well known, administration of prestin protein to an animal can induce a soluble immune response against the protein or fragment in the animal. Preferably, the protein or fragment is mixed with an adjuvant or other immune system enhancer. Such adjuvants include, but are not limited to, mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, and polyanions, other peptides, and oil emulsions. Antibodies which bind specifically with the prestin protein or fragment may be identified and isolated using well known methods (see, e.g. Harlow et al., 1988, Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.). Likewise, immortal hybridomas may be generated using known methods to provide a supply of such antibodies.

[0075] The amino acid and nucleotide sequences of prestin and pres, respectively, which are provided herein can be used to diagnose a disorder associated with aberrant expression of pres. Aberrant expression of pres includes, for example, expression of prestin protein having an amino acid sequence that differs from the normal (i.e. wild type) sequence of prestin, over- or under-expression of pres relative to normal expression levels, expression of pres in tissues in which it is not normally expressed, and non-expression of pres in tissues in which it normally is expressed. By way of examples, disorders which are associated with aberrant expression of pres include various forms of deafness, other hearing impairment, and other hearing disorders, including disorders linked with the autosomal recessive locus designated DFN B14, such as a congenital, sensorineural, autosomal recessive form of non-syndromic deafness associated with this locus.

[0076] The amino acid and nucleotide sequences of prestin and pres, respectively, which are provided herein can also be used to treat a disorder associated with aberrant expression of pres. For example, wild type prestin protein or wild type pres can be provided to a cell which expresses less than a normal amount of the wild type protein (e.g. one which expresses a non-functional form of prestin or one which expresses no prestin). Alternatively, an antisense oligonucleotide which binds specifically with at least one strand of pres can be provided to a cell which expresses more than a normal or desirable amount of wild type or altered prestin protein, in order to inhibit or prevent such expression.

[0077] The ability of prestin to alter the surface area of a lipid bilayer, as described herein, can be used to affect the properties of either a naturally occurring lipid bilayer or a synthetic lipid bilayer. By way of example, providing prestin (i.e. in the form of the protein or in the form of a gene vector which will express the protein) to a cell renders the surface area of the plasma membrane of the cell susceptible to modulation by prestin. In the presence of an increased membrane potential (i.e. a membrane potential having a more negative value), a lipid bilayer comprising prestin is expanded relative to the surface area of the bilayer in the presence of a lower (i.e. less negative; more positive) membrane potential. If the lipid bilayer encloses a fixed volume (or a volume which cannot change as quickly as the surface area changes), then the tension within the bilayer is altered as the membrane potential changes. Thus, in the presence of a low membrane potential, the surface area of a lipid bilayer comprising prestin tends to decrease, so the surface tension of the bilayer increases if the bilayer surrounds a fixed volume, yielding a stiffer structure. Upon increasing membrane potential, the surface area of the bilayer increases, so the surface tension of a bilayer surrounding a fixed volume decreases yielding a more pliant structure.

[0078] The ability of prestin to modulate the surface area of a lipid bilayer can be used to modulate the volume of structure which is enclosed by a lipid bilayer if fluid can pass across the bilayer upon expansion or contraction of the bilayer mediated by prestin. Thus, for example, the volume (i.e. and diameter) of a ‘leaky’ (i.e. porous) bilammelar lipid membrane vesicle which comprises prestin can be modulated by changing the potential across the membrane. Similarly, the stiffness or shape of a structure which comprises a lipid bilayer surrounding a fluid can be modulated if the bilayer comprises prestin and if the rate at which the volume of the structure can change is less than the rate at which prestin can change the surface area of the bilayer (i.e. taking into account the geometric relationship between volume and surface area). Thus, a structure which comprises a lipid membrane comprising prestin enclosing a fixed (or relatively fixed) volume of fluid can be used as a force generator by interposing the structure between two surfaces upon which force can be exerted, restraining the ability of the structure to expand in a direction at least partially parallel to (i.e. absolutely parallel to or oblique with respect to) at least one surface, and modulating the membrane potential of the lipid membrane. Upon increasing the membrane potential (i.e., the electrical aspect), the structure expands in a direction normal to at least one surface, and outward force (relative to the structure) is exerted upon the surfaces, thereby urging the surfaces apart (i.e., the mechanical aspect). Upon decreasing the membrane potential, the structure contracts in a direction normal to at least one surface, and inward force (relative to the structure) is exerted upon the surfaces, thereby urging the surfaces together. Because structures comprising a lipid bilayer can be made on a microscopic (or even sub-microscopic) scale, these force generators can be exceedingly small, and are suitable for incorporation into very small (e.g. microscopic or nanometer-scale) apparatus.

[0079] It is also contemplated by the invention that by applying a mechanical force to prestin, an electrical impulse is created in response to the mechanical force. While the electrical impulse generated may be small, this aspect of the invention has use in electrical circuitry, and more specifically, in nanocircuitry. Thus, for example, prestin has the ability to modulate the electrical characteristics associated with the functions of the cochlea and, more generally, electrical functions associated with hearing.

[0080] Definitions

[0081] As used herein, each of the following terms has the meaning associated with it in this section.

[0082] The articles “a” and “an” are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0083] “Antisense” refers particularly to the nucleic acid sequence of the non-coding strand of a double stranded DNA molecule encoding a protein, or to a sequence which is substantially homologous to the non-coding strand. As defined herein, an antisense sequence is complementary to the sequence of a double stranded DNA molecule encoding a protein. It is not necessary that the antisense sequence be complementary solely to the coding portion of the coding strand of the DNA molecule. The antisense sequence may be complementary to regulatory sequences specified on the coding strand of a DNA molecule encoding a protein, which regulatory sequences control expression of the coding sequences.

[0084] “Complementary” refers to the broad concept of sequence complementarity between regions of two nucleic acid strands or between two regions of the same nucleic acid strand. It is known that an adenine residue of a first nucleic acid region is capable of forming specific hydrogen bonds (“base pairing”) with a residue of a second nucleic acid region which is anti-parallel to the first region if the residue is thymine or uracil. Similarly, it is known that a cytosine residue of a first nucleic acid strand is capable of base pairing with a residue of a second nucleic acid strand which is anti-parallel to the first strand if the residue is guanine. A first region of a nucleic acid is complementary to a second region of the same or a different nucleic acid if, when the two regions are arranged in an anti-parallel fashion, at least one nucleotide residue of the first region is capable of base pairing with a residue of the second region. Preferably, the first region comprises a first portion and the second region comprises a second portion, whereby, when the first and second portions are arranged in an anti-parallel fashion, at least about 50%, and preferably at least about 75%, at least about 90%, or at least about 95% of the nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion. More preferably, all nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion.

[0085] “Homologous” as used herein, refers to nucleotide sequence similarity between two regions of the same nucleic acid strand or between regions of two different nucleic acid strands. When a nucleotide residue position in both regions is occupied by the same nucleotide residue, then the regions are homologous at that position. A first region is homologous to a second region if at least one nucleotide residue position of each region is occupied by the same residue. Homology between two regions is expressed in terms of the proportion of nucleotide residue positions of the two regions that are occupied by the same nucleotide residue. By way of example, a region having the nucleotide sequence 5′-ATTGCC-3′ and a region having the nucleotide sequence 5′-TATGGC-3′ share 50% homology. Preferably, the first region comprises a first portion and the second region comprises a second portion, whereby, at least about 50%, and preferably at least about 75%, at least about 90%, or at least about 95% of the nucleotide residue positions of each of the portions are occupied by the same nucleotide residue. More preferably, all nucleotide residue positions of each of the portions are occupied by the same nucleotide residue.

[0086] An “isolated” polynucleotide or protein refers to a molecule which has been separated from sequences which flank it in a naturally occurring state, e.g., a DNA fragment which has been removed from the sequences which are normally adjacent to the fragment, e.g., the sequences adjacent to the fragment in a genome in which it naturally occurs. The term also applies to molecules which have been substantially purified from other components which naturally accompany the nucleic acid, e.g., RNA or DNA or proteins, which naturally accompany it in the cell.

[0087] As used herein, the term “promoter/regulatory sequence” means a nucleic acid sequence which is required for expression of a gene product operably linked to the promoter/regulatory sequence. In some instances, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product. The promoter/regulatory sequence may, for example, be one which expresses the gene product in a tissue specific manner.

[0088] By describing two polynucleotides as “operably linked” is meant that a single-stranded or double-stranded nucleic acid moiety comprises the two polynucleotides arranged within the nucleic acid moiety in such a manner that at least one of the two polynucleotides is able to exert a physiological effect by which it is characterized upon the other. By way of example, a promoter operably linked with the coding region of a gene is able to promote transcription of the coding region.

[0089] A first oligonucleotide anneals with a second oligonucleotide “with high stringency” if the two oligonucleotides anneal under high stringency hybridization conditions.

[0090] By “high stringency hybridization conditions” is meant those hybridizing conditions that (1) employ low ionic strength and high temperature for washing, for example, but not limited to 0.015 molar NaCl, 1.5 millimolar sodium citrate, and 0.1% (w/v) sodium dodecyl sulfate (SDS) at 50° C.; (2) employ during hybridization a denaturing agent such as formamide, for example, but not limited to 50% (v/v) formamide, 0.1% (w/v) bovine serum albumin, 0.1% (w/v) Ficoll, 0.1% (w/v) polyvinylpyrrolidone, and 50 millimolar sodium phosphate buffer at pH 6.5 with 750 millimolar NaCI, 75 millimolar sodium citrate at 42° C.; or (3) employ 50% (v/v) formamide, 5×SSC (0.75 molar NaCl, 75 millimolar sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50 micrograms per milliliter), 0.1% (w/v) SDS, and 10% (w/v) dextran sulfate at 42° C, with washes at 42° C. in 0.2×SSC and 0.1% (w/v) SDS. Under stringent hybridization conditions, only highly complementary nucleic acids hybridize. Preferably, such conditions prevent hybridization of nucleic acids having 1 or 2 mismatches out of 20 contiguous nucleotides.

[0091] The term “substantially pure” describes a compound, e.g., a protein, polynucleotide, or polypeptide which has been separated from components which naturally accompany it. Typically, a compound is substantially pure when at least 10%, more preferably at least 20%, more preferably at least 50%, more preferably at least 60%, more preferably at least 75%, more preferably at least 90%, and most preferably at least 99% of the total material (by volume, by wet or dry weight, or by mole percent or mole fraction) in a sample is the compound of interest. Purity can be measured by any appropriate method, e.g., in the case of polypeptides by column chromatography, gel electrophoresis or HPLC analysis. A compound, e.g., a protein, is also substantially purified when it is essentially free of naturally associated components or when it is separated from the native contaminants which accompany it in its natural state.

[0092] By the term “specifically binds,” as used herein, is meant a protein (e.g., an antibody) which recognizes and binds a prestin protein but does not substantially recognize or bind other molecules in a sample.

[0093] By the term “lipid bilayer”, as used herein, is meant a membrane formed by phospholipids in a bimolecular layer. This term should be construed to include naturally-occurring, as well as artificially prepared membranes.

[0094] The term “modulation” describes regulation of some quantity or measure of a product or process. For example, a protein is said to be modulated when expression is either increased or decreased.

[0095] The invention is now described with reference to the following Examples. These Examples are provided for the purpose of illustration only and the invention is not limited to these Examples, but rather encompasses all variations which are evident as a result of the teaching provided herein.

EXAMPLES

Example 1

[0096] Prestin is the Motor Protein of Cochlear Outer Hair Cells

[0097] Tissue Preparation and Pres cDNA Isolation by Subtractive PCR Hybridization

[0098] Outer hair cells and inner hair cells are distinct sensory receptor cells in the mammalian organ of Corti. They both have hair bundles at the cell apex, though their bundle lengths, numbers and configuration are quite different. They both are rich in mitochondria in order to maintain their active physiologic function. It is likely that a vast array of housekeeping genes as well as genes that code for metabolism, the transducer apparatus and a variety of common ion channels are duplicated in IHC and OHC. These and other similarities between OHC and IHC make IHC an ideal “driver” in order to “subtract-away” common hair cell genes, allowing isolation of OHC specific genes. The use of inner and outer hair cells as sources of cell-type specific mRNA should reduce the number of candidate genes obtained after the subtraction-hybridization process and thus enhance the likelihood of successful isolation of OHC specific genes.

[0099] A unique and abundant OHC-specific cDNA from a gene, designated Prestin (Pres), has been identified herein via OHC minus IHC subtractive PCR hybridization and differential screening. The protein prestin is novel, but some of its regions exhibit moderate homology to pendrin and sulfate/anion transport proteins. Voltage-induced shape change can be elicited in human kidney (TSA201) cells that express prestin. The OHC's mechanical response to voltage change is accompanied by a “gating current,” manifested as nonlinear capacitance. This nonlinear capacitance was also demonstrated in transfected TSA201 cells.

[0100] The materials and methods used in the experiments presented herein are now described.

[0101] Mature gerbils were euthanized and the organ of Corti and associated basilar membrane were isolated by dissection as described in He et al.(1997, J. Neurosci., 15:3634-3643). The tissue was placed in calcium-free S-MEM medium, followed by brief trypsin treatment (1 milligram per milliliter) and trituration (20 minutes at 37 degrees Celsius). OHCs and IHCs were isolated in a low-calcium solution. Single OHCs and IHCs were identified and separated based on the presence of particular stereocilia configuration, depicted in FIG. 12. Using a glass pipette, the cells were separately isolated based on their morphological appearance and were collected in lithium chloride buffer. Messenger RNA was isolated from tissue using 20 microliters of oligo-dT magnetic beads (Dynal, Oslo, Norway). cDNA pools were created with Superscript II RNAase H reverse transcriptase (Gibco BRL, Gaithersburg, Md.) followed by amplification using a 5′-cap and oligo dT-dependent PCR technique according to manufacturer's specifications (SMART PCR™, Clontech, Palo Alto, Calif.). These cDNA pools were used for PCR subtractive hybridization, qualitative PCR, and virtual Northern (cDNA) dot-blot hybridization experiments.

[0102] The PCR subtractive hybridization procedures, control reactions, and subcloning were according to the manufacturer's instructions (PCR Select cDNA Subtraction Kit, Clontech). Clones were subjected to a differential screening procedure using a PCR-Select Differential Screening Kit (Clontech). PCR amplified cDNA inserts from these clones were denatured and vacuum filtered onto four identical nylon membranes. Each cDNA sample was separately hybridized with one of the following probes: forward subtracted (OHC-IHC) cDNA; reverse subtracted (IHC-OHC) cDNA; un-subtracted OHC cDNA; and un-subtracted IHC cDNA. These cDNA pools were random primed labeled with [32P]-dCTP and hybridized to the blots. Positively hybridizing clones were scored for differential hybridization with forward (OHC-IHC) and reverse (IHC-OHC) subtracted probes. This differential expression was confirmed by correlation with simultaneous differential hybridization with the original un-subtracted OHC and IHC cDNA pools. Sequencing was performed using dRhodamine terminator cycle sequencing ready reaction kit (ABI Prism, Foster City, Calif.) and an automated DNA sequencer (Model 377, Perkin Elmer, Norwalk, Conn.). DNA and amino acid sequences were compared and analyzed using sequence analysis software of GCG and TMHMM (1998, Sonnhammer, et al., Proc. 6th Intern. Conf. On Intelligent Systems for Mol. Biol., 175-182) and Tmpred (1993, Hoffman, et al., Biol. Chem., 347:166).

[0103] Virtual Northern (cDNA) Dot-Blot Hybridization

[0104] 0.5 Micrograms of SMART™ cDNA from gerbil thyroid, IHC, OHC, and 0, 6, 12, 16, and 20 day old organs of Corti were denatured and vacuum filtered onto membranes. A 1067 base pair NcoI restriction fragment of Pres cDNA was radioactively labeled with [32P]-dATP and used as a probe (Strip-EZ Kit™ and UltraHyb™ buffer, Ambion, Austin Tex.).

[0105] PCR

[0106] PCR reactions used templates of 200 nanograms of SMART™ cDNA from gerbil thyroid, newborn and adult organs of Corti, isolated IHC, and organ of Corti from various post-natal ages (0 to 20 days after birth). Pres-specific primers (sense: 5′-TACCTCACGGAGCCGCTGGT-3′ (SEQ ID NO: 7), and antisense: 5′-GCAGTAATCAGTCCGTAGTCC-3′ (SEQ ID NO: 8)) were used to amplify an 863-base pair fragment from the cDNA. Cyclophilin primers (sense: 5′-TGGCACAGGAGGAAAGAGCATC-3′ (SEQ ID NO: 9), and antisense: 5′-AAAGGGCTTCTCCACCTCGATC-3′ (SEQ ID NO: 10)) that amplify a 301-base pair DNA fragment were used as an internal control. Cycle parameters were 3 minutes at 94 degrees Celsius followed by 25-30 cycles of 94 degrees Celsius for 45 seconds, 56 degrees Celsius for 45 seconds, and 72 degrees Celsius for 1 minute, with a final extension at 72 degrees Celsius for 10 minutes.

[0107] Transient Transfection

[0108] TSA201 cells, clones of human embryonic kidney 293 cells that express the simian virus 40 (SV40) large T-antigen in a stable manner, were cultured in DMEM with 5% fetal calf serum, 100 units of penicillin per millilter, and 100 micrograms of streptomycin per milliliters. Cells were plated for 24 hours before calcium phosphate transfection. The transfection reaction mixture contained:

[0109] a) control plasmid, pcDNA3.1; b) 2 micrograms of Pres cDNA plasmid; c) 2 micrograms of Pres cDNA plasmid plus 0.2 micrograms of green fluorescent protein (GFP) plasmid as transfection marker; or d) 2 micrograms of pendrin cDNA plasmid plus 0.2 micrograms GFP plasmid.

[0110] After 24 hours the transfected cells were used for electrophysiological and physical measurements. Large, rounded, non-clustered TSA201 cells exhibiting considerable granulation were selected for electrophysiological and motility experiments.

[0111] Capacitance Measurements

[0112] Whole-cell voltage-clamp recordings were made with an Axopatch™ 200B amplifier (Axon Instruments, Foster City, Calif.) Recording pipettes had open tip resistances of 2-3 megaohms and were filled with internal solution containing 140 millimolar CsCl, 2 millimolar MgCl2, 10 millimolar EGTA, and 10 millimolar HEPES at pH 7.2. The external solution contained: 120 millimolar NaCl, 20 millimolar TEA-Cl, 2 millimolar CoCl2, 2 millimolar MgCl2, 10 millimolar HEPES, and 5 millimolar glucose at pH 7.2. Osmolarity was adjusted to 300 milliosmolar per liter with glucose. Capacitive currents were filtered at 5 kilohertz and digitized at 50 kilohertz using pClamp 7.0 software (Axon Instruments).

[0113] Voltage-dependent capacitance was measured using two methods. First, a standard P/4 linear subtraction procedure was employed to detect the presence of nonlinear charge movement. After nonlinear transient current was detected, a stair-step voltage protocol was used to obtain the parameters of charge movement. For each voltage step, the measured membrane capacitance (Cm) was plotted as a function of membrane voltage (Vm) and fitted with the derivative of a Boltzmann function: 1 C m = Q max ⁢   ⁢ α Exp ⁢   [ α ⁡ ( V m - V 1 / 2 ) ] ⁢ ( 1 + exp [ - α ( V m - V 1 / 2 ] ) 2 + C lin ( 1 )

[0114] where Qmax is maximum charge transfer, V1/2 is the voltage at which the maximum charge is equally distributed across the membrane, Clin is linear capacitance, and &agr;=ze/kT is the slope factor of the voltage dependence of charge transfer where k is Boltzmann's constant, T is absolute temperature, z is valence, and e is electron charge. From the stair-step analysis we have obtained the membrane resistance: Rm=130.45±20.14 megaohms.

[0115] Motility Measurements

[0116] TSA201 cells were drawn into a microchamber with gentle suction (FIG. 6A). The microchamber was mounted on a three-dimensional micromanipulator attached to the stage of a Zeiss inverted microscope. The most distal part of the excluded segment of the cell was imaged onto a photodiode with a rectangular slit interposed into the light path. A change of photocurrent signified cell extension or contraction. The signal was current-to-voltage transformed, amplified, A/D converted, and averaged. The command voltage, delivered between the electrolyte solutions (L15, Gibco BRL) surrounding and filling the microchamber, produces different voltage drops on the included and excluded cell membrane segments, determined by the electrical voltage-divider effect of these membranes. During motility measurements, the location of the cell within the slit was monitored via a video camera placed behind the slit. The appearance of the whole cell in the microchamber was also continuously displayed with the aid of a second video camera. This permitted an independent verification of a lack of movement of the microchamber itself.

[0117] The results of these experiments are now described.

[0118] Cloning of Pres and the Properties of Prestin

[0119] The molecular approach to identify the OHC sensor-motor protein faced two initial challenges: first, isolation of cDNA from limited amounts of biological material, and second, recognition of the candidate motor protein cDNA among the isolated unknown genes. Isolation of sufficient number of OHCs is a fairly routine task.

[0120] Obtaining comparable numbers of IHCs requires some modification of techniques. mRNA was isolated from approximately 1000 OHCs and 1000 IHCs and reverse transcribed followed by 5′-mRNA cap and oligo dT-dependent PCR amplification to obtain OHC and IHC cDNA pools.

[0121] Recognition of the cDNAs that encoded the candidate motor protein was based on the following assumptions: (1) the protein is expressed in OHC and not in the non-motile IHC, (2) ontogenic expression should relate to the known development of electromechanical responses, (3) the protein should be relatively abundant in OHC, (4) the protein should be a transmembrane protein, and (5) electromechanical responses should be demonstrable when expressed in a heterologous system. It was reasonable to expect that gene expression related to mechano-electric transduction and genomic, metabolic, and structural functions would be shared by these two types of hair cells. Therefore suppression subtractive hybridization PCR procedure was used to amplify and enrich the OHC cDNA pool for uniquely expressed gene products. This procedure uses two rounds of cDNA hybridization to subtract out common OHC cDNAs using a vast excess of IHC cDNA. The unique fragments are amplified by PCR, RsaI-digested, and cloned to create a library of candidate clones. This technique is useful because it simultaneously enriches for differentially expressed cDNA fragments and suppresses non-target DNA amplification.

[0122] Approximately 1300 fragment clones were screened using four cDNA pools (OHC-IHC, IHC-OHC, OHC unsubtracted and IHC unsubtracted) to identify differentially expressing clones (FIG. 1). Once false positive clones were excluded, 487 clones were identified as differentially expressing genes and of these, 108 clones were sequenced. These 108 sequences contained 50 unique cDNA fragments. Eighteen of the clones contained sequences which were highly similar to known proteins including Type I collagen pro-alpha 2, serine kinase, oncomodulin, ribonucleotide reductase, ATP synthase, etc., while 32 were believed to be unique in that there were no obvious homologous sequences to them in the genetic database searched. Eleven of the 32 apparently unique clones contained DNA having open reading frames. These were examined in greater detail by dot-blot hybridization using radioactively labeled clone cDNA against immobilized OHC and IHC cDNA pools. Pres was one of the eleven candidate clones identified. Pres fragments in the PCR subtracted library were abundant (>10% of 487 differentially expressed clones) and demonstrated consistent differential hybridization with OHC and IHC derived probes (FIG. 16).

[0123] Using one of these Pres cDNA RsaI fragments as a probe, three cDNA clones were subsequently isolated from a gamma-gt11 gerbil adult cochlea library. The largest of these clones was 4.1 kilobases in length having an open reading frame of 2232 base pairs, encoding a 744 amino acid protein (FIG. 2). The sequence around the putative translation start site contains a consensus Kozak sequence and an in-frame stop codon is present 12 bases upstream of this predicted start site. There is a short 223 base pair untranslated 5′ end and a large 1654 base pair, 3′ untranslated region. A computer search of the Pres sequence revealed that about one-third of the human PRES gene has been fortuitously sequenced as part of the human chromosome 7 effort (BAC clone RG107G13, bases 99,895 to >113,558). The amino acid homology between human and gerbil prestin, deduced from the genomic sequence of the first 6 exons, is 98% (FIG. 2).

[0124] Analysis of the prestin sequence revealed that its highest homology was to members of a family of anion/sulfate transport proteins including pendrin and DRA. The pendrin homology (40%) is of particular interest because this chloride-iodide transport protein is known to be a cause of the genetically inherited deafness observed in Pendred's syndrome. This homology, although modest overall, is similar to the homology among other members of the sulfate/anion transport family. Because gerbil cDNA was used and only rat, mouse, and human pendrin sequences were known at the time, it was necessary to demonstrate that prestin was not the gerbil homolog of pendrin.

[0125] It has been shown by others that mouse pendrin is expressed in the endolymphatic duct and sac, the utricle and saccule, and within the cochlea only in the external sulcus, but not in the organ of Corti. Confirmation that prestin was not a gerbil homolog of pendrin was achieved by cloning an 824 base pair fragment of gerbil pendrin cDNA, and demonstrating that the sequence was distinct from prestin. The gerbil pendrin amino acid sequence derived from the above cDNA fragment was 88% homologous to the amino acid sequence of human pendrin, but only 38% homologous to the amino acid sequence of gerbil prestin.

[0126] The protein prestin (744 amino acids; 81.4 kiloDaltons) is hydrophobic, containing approximately 50% non-polar residues; 27.8% of the protein is composed of the amino acids valine, leucine and isoleucine. Computer modeling of the amino acid sequence produces ambiguous results using multiple modeling programs. Specifically, the models TMHMM and TMPred report ambiguous results as to the number and location of transmembrane regions and are unable to predict the topology of the amino and carboxy termini in relation to the membrane. By comparison, when pendrin is subjected to the same modeling programs, 11 transmembrane regions, and an intracellular amino terminus are unambiguously predicted. The consistent ambiguity of the modeling programs with regard to prestin is potentially significant, and suggests that the mechanism by which prestin produces electromechanical action is by altering the protein/membrane interface as a result of voltage induced changes in its structure.

[0127] Two distinctive charged regions are located in the carboxy terminal region of prestin. A positive charge cluster of residues is located at 557-580; adjacent to the positive cluster, a negative charge cluster is at residues 596 to 613 (FIG. 2). Prestin's overall predicted hydropathy profile is similar to pendrin, DRA, and other members of the sulfate/anion transport family. The homology to pendrin is highest in the 50 amino acid region (97-146) that encompasses the sulfate transport motif (109-130), but does not have long regions of amino acid identity elsewhere. Instead, there is a pattern of moderate homology using both amino acid identity and similarity throughout the protein with multiple, discrete 8-12 residue segments of amino acid identity. The charged cluster regions of prestin are conserved in their net charges, but are otherwise not remarkably homologous. The proteins differ most in the region between the two charge clusters and at the termini. Although prestin exhibits high homology in the sulfate transport region, a highly conserved domain, found in homologs in organisms as distant as yeast, C. elegans, and plants, does not conform to the sulfate transporter signature as currently defined. Specifically, both human and gerbil prestin differ from the consensus sequence at three positions (FIG. 3). Consequently, while prestin appears related to sulfate transporters, these differences in the conserved region suggest that the protein may have distinct properties.

[0128] The tissue-specific and developmental expression pattern of Pres in the gerbil was also determined. Northern blot analysis using a Pres probe did not detect expression of prestin in liver, lung, brain, spleen, ovary, kidney, muscle, and heart (total RNA 20 micrograms). Virtual Northern dot-blot analysis using cDNA prepared from IHC, OHC, mature and newborn organs of Corti, and thyroid revealed prestin expression only in mature OHC and 20 day old organ of Corti (FIG. 4A). Similar results were obtained using Pres-specific primers and a qualitative PCR assay (FIG. 4B). Both virtual Northern and PCR exhibited progressively increasing Pres expression in isolated organ of Corti from birth up to 20 days after birth (FIGS. 4C and 4D), by which time electromotility is fully developed in the altricial gerbil. In OHCs, the motors are usually congruent with abundant approximately 10 nanometer membrane particles. The density of these particles in gerbil OHCs increases with development which density then decelerates toward adult values 16-18 days after birth. Thus the ontogenic expression of Pres is similar to that of membrane particles, electromotility, and the onset of high sensitivity hearing.

[0129] Functional Tests of Prestin

[0130] In order to investigate the function of the prestin cDNA, the functional properties of prestin in eukaryotic cells were examined. To accomplish this, Pres was subcloned into the eukaryotic expression vector pcDNA3.1. A carboxy-terminal epitope-tagged (V5) version (pcDNA6/V5-HisA vector) of the expression vector was created to facilitate detection of expression of the full-length protein in transfected cells. The expression of the V5 version was examined by indirect immunofluorescence to assure its presence in transfected cells. The epitope-modified prestin was found to be located in the cell membrane, with a punctate distribution in permeabilized cells. In addition, Western blot analysis using an anti-V5 antibody against transfected cell extracts demonstrated production of the appropriate sized prestin protein.

[0131] To test the sensor-motor function of prestin, voltage-dependent charge movement was measured in TSA201 cells after transient transfection of unmodified, native Pres cDNA. A second plasmid (pEGFP-N2) containing GFP cDNA was used as an independent marker for successful transfection of the cells. Transfection efficiency was 30-40% as judged from the fluorescence of GFP in transfected cells. Consistent with this, 36.8% of cells transfected with Pres alone displayed transient capacitive currents under voltage clamp using a standard subtraction protocol after blocking ionic currents (FIG. 5A). In experiments conducted on cells co-transfected with prestin and GFP cDNA, transient capacitive currents were obtained from 91.8% of the GFP positive cells. In contrast, untransfected cells or cells transfected solely with the control plasmid exhibited no measurable nonlinear capacitance. The capacitive currents were directed towards the outside as the membrane potential was depolarized and toward the inside as it was hyperpolarized. Charges transferred at command onset and offset were approximately equal. The decay of transient currents was rapid (100-200 microseconds) and could be well-fitted with a single exponential. The voltage protocol used to estimate membrane capacitance and charge transfer across the membrane was an ascending stair-step voltage waveform (FIG. 5B). The membrane capacitance was fitted to the derivative of a first-order Boltzmann equation (FIG. 5C). In a group of seven cells, these curve-fits yielded values of Qmax=1.76±0.20 pC, V1/2=−57.30±2.98 mV, 1/a˜=kT/ze=28.00±2.13 mV, and Clin=20.50±2.65 pF, respectively (mean ±SEM). The valence can be calculated from a, z=0.91. The average charge density, a possible measure of transiently transfected motor protein density in TSA201 cells, was 5360 square micrometers. The numerical values that characterize the charge movement (V1/2, z) are quite similar to those obtained for OHCs. The maximum charge and charge density are less in the transfected cells, even though the linear capacitance is about the same as that of an average OHC.

[0132] In OHCs, nonlinear capacitive current and electromotility can be reversibly blocked by sodium salicylate. As further evidence that transient currents stem from the transfected motor protein, salicylate was locally applied to cells that had been shown to present charge movement. Sodium salicylate (10 millimolar) reduced the transient currents to 15.5±2.9% of control (FIG. 5D). Of these, 3 cells showed recovery after 2-5 minutes of washing in normal extracellular solution without sodium salicylate (to 88.4±8.8% of control).

[0133] Finally, the existence of nonlinear capacitance was tested in cells that were transfected with both GFP and human pendrin cDNA. None of the GFP-positive cells demonstrated nonlinear capacitance. This result is significant because of the similarity of pendrin and prestin, demonstrating the specificity of the candidate motor protein. It also indicates that the electrophysiological results are not simply the consequence of the introduction of an anion transport protein.

[0134] In TSA201 cells the organized cytoskeletal network is probably missing, thereby making the conversion of mechanical molecular events to gross cellular deformation more difficult. Consequently, even if the motor protein is expressed, gross cellular deformation is expected to be very small. Furthermore, OHCs are efficient producers of axial motility because of their cylindrical shape. Fast mechanical effects occur at constant cell volume and are generally assumed to be the result of a change in cell surface area, due to the aggregate conformational shape changes of large numbers of motor proteins. A spherical cell cannot change its surface area while its volume remains constant, whereas a cylindrical cell can. In order to test that the generally spherical TSA201 cells may produce measurable motility, the cells were distorted to alter their shape by drawing them into a suction pipette (microchamber), thereby producing a dumbbell or hourglass shape (FIG. 6A). Electromotility in about 22% of cells tested was demonstrated. Two of the best responses are shown in FIG. 6B, in contrast to a trace from a non-transfected, non-responsive cell. The usual electrical driving signal was a brief 200 Hertz sinusoid. Sodium salicylate produces a reversible decrease in the electromotile response (FIG. 6C), just as it does in OHCs. In order to examine motility at higher frequencies, comparison between responses at 200 and 1000 Hertz are shown in FIGS. 6D and 6E. When corrected for the characteristics of the recording system, the two responses are essentially identical (FIG. 6E), in accord with OHC data obtained in the past.

[0135] The protein prestin has been shown to convey to a heterologous system novel mechanical responsiveness, almost indistinguishable from that measured in OHCs A portion of the human pres gene has been sequenced and mapped. The location of the motor protein in relation to the other members of its family can be predicted and any relationship to genetic loci known to be related to hearing disorders or deafness can be ascertained. The bacterial artificial chromosome clone (RG107G13) which contains a portion of the pres gene at its 3′ end, has a single known other gene, RELN, which is about 50 kilobases centromeric to the pres gene fragment. This location is approximately 3.7 megabases centromeric to the location of the pendrin (PDS) and DRA genes. Thus, it appears likely that pres is located in an as yet unsequenced portion of 7q31, centromeric to PDS and DRA, and 50 kilobases telomeric to the RELN gene.

[0136] There are at present two autosomal nonsyndromic recessive deafness loci mapped to 7q31 in addition to the PDS gene: DFNB14 and DFN3 17. DFNB14 is the most likely candidate to be pres, since it maps centromeric to the PDS gene locus, while DFNB 17 maps to the telomeric side of PDS and DRA. The proof that DFNB 14 is itself distinct from PDS is derived from the fact that, in the kindred analyzed, there were no useful polymorphic markers in a genomic region of 15 centimorgans that contains both the RELN gene and PDS. In this kindred, the PDS exons were sequenced and appeared to be normal, indicating that the causative mutation was distinct from that seen in Pendred's syndrome. The clinical phenotype in this kindred of a congenital, sensorineural, autosomal recessive form of non-syndromic deafness, is consistent with prestin's functional role.

[0137] These results support earlier work on OHCs, demonstrating that neither the subsurface cortical lattice nor the subsurface cisterns are essential for electromotility. It is unlikely that either of these organelles would be present in TSA201 cells, either in their native or transfected form. Demonstration of nonlinear capacitance and motility simplifies the concept of electromechanical changes in OHCs and obviates the need for exotic schemes. Presumed conformational changes of prestin, resulting in a change of cell surface area, produce dimensional changes in the cell. This is the basic electromotile response. It is contemplated that stiffness change may accompany these shape changes in TSA201 cells as they do in OHCs, thus the expression of prestin plays a role in the full electromechanical process.

[0138] While the reliance of the mammalian cochlea on local, OHC-based, amplification is widely accepted, there is no universal agreement about the amplifying mechanism. One view is that, powered by the cell's receptor potential, OHC electromotility provides mechanical feedback and thereby amplification. This view places the motor process in the cell's basolateral membrane and calls for a novel motor protein to drive somatic shape changes. An alternative concept is that amplification arises as a byproduct of the cell's forward transducer process and thus resides in the stereocilia. Whichever mechanism dominates, the existence of electromechanical action in OHCs is indisputable. The novel pres gene herein identified is the gene that codes for a specialized motor protein that produces this electromechanical action when expressed in cells that, in their native form, do not exhibit this phenomenon.

[0139] Isolated OHCs are capable of producing an average maximum axial isometric (stall) force of approximately 6 nanoNewtons (2000, He and Dallos, JARO, 1:64-81). From the number of molecules producing this force it can be estimated that the individual molecular stall force is on the order of 2.4 picoNewtons. In comparison, the stall force of kinesin is 5 to 6 picoNewtons. The potential of prestin to perform as a fast, voltage-driven actuator, individually or in assemblies, forming the basis of futuristic nanomachinery, is substantial.

Example 2

[0140] Isolation of a Novel Gene from Gerbil Outer Hair Cells

[0141] The materials and methods for the experimental procedures employed in this Example are now described. The methods are similar to those employed for Example 1.

[0142] Isolation of Outer Hair Cells (OHCs) and Inner Hair Cells (IHCs)

[0143] Mature gerbils were decapitated immediately following euthanasia. The organ of Corti and the associated basilar membrane were then treated with XX milligrams per milliliter of trypsin for 20 minutes at 37 degrees Celsius. OHCs and IHCs were obtained by gentle trituration of the trypsinized organ of Corti and the basilar membrane. The preparation was mounted on an inverted microscope as described in He, et al (2000, Hearing Res., 145:156-160).

[0144] Solitary OHCs and IHCs were identified by appearance and by the presence of a particular stereocilia configuration, shown in FIG. 12. OHCs and IHCs were then separately isolated by a small glass pipette (30-50 micrometers in tip diameter) mounted on a three dimensional stage micromanipulator. Approximately 1,000 IHCs and 1,000 OHCs were collected from 7 animals.

[0145] PCR Amplification

[0146] Messenger RNAs were isolated from both OHCs and IHCs using 40 microliters of oligo-dT magnetic beads in Lithium Chloride buffer (Dynal). OHC and IHC cDNA pools were created by reverse transcription using 200 units of Superscript II RNAase H reverse transcriptase at 42 degrees Celsius for one hour, followed by amplification with a 5′-Cap and oligo dT-dependent PCR technique according to manufacturer's instructions (Clontech).

[0147] PCR-Select cDNA Subtraction

[0148] In the subtraction experiment, OHC is the tester cDNA, and IHC is the driver cDNA. The OHC cDNA pool generated from PCR amplification was divided into two groups and then ligated to two different adapters (adapter 1 and 2). The OHC cDNAs were then subjected to two rounds of subtractive hybridization with excess driver cDNA, i.e., the IHC cDNA. After subtraction hybridization, the new hybrids carry different adapters (adapter 1 & 2), allowing them to be preferentially amplified. The hybrids with OHC-specific expressed genes underwent two rounds of selective PCR amplification. The PCR amplification, hybridization and control reaction were according to manufacturer's instructions (Clontech). As shown in FIG. 14, the distribution pattern of OHC cDNA subtracted with IHC is quite different from that of the unsubtracted OHC cDNA pool.

[0149] Construction of OHC Subtracted cDNA Plasmid Library

[0150] The final subtracted OHC cDNA pool was restriction digested with NotI and EagI and cloned into pGEM5Z(−), previously cut with NotI. The plasmid was then transformed into E. coli, produced an OHC subtracted cDNA plasmid library. 1320 Colonies were selected. A sample of these clones was used for T7/Sp6 PCR reaction to confirm the presence and the size of the inserts, as shown in FIG. 15. The PCR amplified cDNA was vacuum-filtered onto four identical nylon membranes (BioRad, Richmond, Va.) for hybridization. Forward-subtracted (OHC-IHC) and reverse-subtracted (IHC-OHC) cDNA, as well as tester (unsubtracted OHC) and driver (unsubtracted IHC) cDNA were radioactively labeled with 32P-dCTP using a random primer labeling method. The cDNA was hybridized with 5×106 counts per minute per milliliter of each individual probe overnight. After a final washing with 0.2×SSC/0.05% SDS, nylon membranes were exposed to autoradiographic film for 8 hours. Results are shown in FIG. 16. Positively hybridizing clones were scored for differential hybridization with forward (OHC-IHC; FIG. 16A) and reverse (IHC-OHC; FIG. 16B) subtracted probes. This differential hybridization was confirmed in most cases by confirming hybridization with tester (OHC; FIG. 16C) and driver (IHC; FIG. 16D) cDNA. In some cases, strongly differential hybridizing clones seen in the forward and reverse pair of blots were also selected for DNA sequencing despite a lack of signal from the tester and driver hybridization blots. 103 Clones were analyzed and selected from the OHC subtracted plasmid library. Of those, 18 clones were known genes, while 32 clones were unknown. Eleven of the unknown clones had evident open reading frames and 21 clones had no apparent coding sequence. Results for known genes are shown in Table 1. 2 TABLE 1 KNOWN GENES NUMBER OF CLONES Collagen alpha-2 (I) 8 Mitochondria cytochrome c oxidase II 3 Acidic Protein Rich in Leucine (APRIL) 2 Mitochondrial tRNA 2 Oncomodulin 2 Adenine nucleotide translocase-2 (Ant2) 1 Acid sphiagomyleinase (ASM)-like 1 phosphodiesterase 3a ATP synthase 1 Collagen alpha-1 (I) 1 Glycerol 3 phosphate acyltransferase 1 Inward rectifier K-channel 1 Mitochondrial DNA control region 1 Mitochondrial 12S RNA 1 Osteonectin (SPARC: secreted protein 1 acidic rich in cysteine) Prolylcarboxypeptidase 1 Receptor tyrosine kinase 1 Ribonucleotide reductase 1 Serine kinase (SRPK2) 1

[0151] Virtual Northern Dot Blot Experiments

[0152] mRNAs were isolated using oligo-dT magnetic beads (Dynal) from thyroid, adult cochlea, newborn cochlea, IHC, OHC, and cultured organ of Corti treated with and without T3. cDNA pools from these tissues were created using the 5′-Cap PCR strategy as described above (Clontech). 0.5 Micrograms of each sample cDNA was mixed with 0.4 molar NaOH and 10 millimolar EDTA and then boiled at 100 degrees Celsius for 10 minutes. The cDNA samples were then vacuum filtered onto nylon membranes (BioRad). The membranes were neutralized with 0.5. molar Tris-HCl before hybridization. Unknown-gene fragments were radioactively labeled with 32P-DATP according to Random Primed stripAble DNA probe Synthesis & Removal Kit™ (Ambion). Results for three genes are shown in FIG. 18.

[0153] Gamma-gt11 Gerbil Cochlea Library Screening

[0154] The library screening was performed using standard experimental procedure as described in, for example, Sambrook, et al., 1989 (Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, New York).

Example 3

[0155] Prestin is a Cytoplasmic Protein

[0156] N-tagged and C-tagged prestin constructs were created in the same general manner as above and subsequently transiently transfected into TSA201 cells. Immunofluorescence with FITC-labeled secondary antibodies against prestin was used to determine where in the cells the prestin protein is expressed. It has been concluded that prestin is expressed in the cytoplasm of the TSA201 cells.

[0157] Generation of C-tagged and N-tagged Prestin Constructs

[0158] The C-terminal end of prestin was amplified using an oligonucleotide (5′-GCCCTGAATTCCTCGGGTGTGG-3′; SEQ ID NO: 13) which was complementary to the C-terminus of prestin, together with an upstream oligonucleotide primer (5 ′-GGACTACGGACTGATTACTGC-3′; SEQ ID NO: 14). The first primer was modified to create an EcoRI restriction site for ligation into an expression vector. PCR was performed with these primers, resulting in an amplified C-terminal fragment of prestin. The PCR product was cloned into the expression vector pcDNA6/V5-HisA (Invitrogen, Carlsbad, Calif.) to create a full-length C-terminus tagged with a V5 epitope located downstream and in-frame with prestin. C-tagged pendrin was also generated by this method.

[0159] The N-terminal end of prestin was amplified using an oligonucleotide complementary to the N-terminus of prestin (5′-CTGCAGAATTCGGATCATGCCG-3′; SEQ ID NO: 15) and a second downstream oligonucleotide primer (5′-CAACGATGGCTATGGCAATGGC-3′; SEQ ID NO: 16) in a PCR reaction. As above, the first primer was modified to create an EcoRI restriction site for ligation into an expression vector. The amplified PCR product was cloned into the expression vector pcDNA3.1/HisB (Invitrogen) to produce a full-length N-tagged prestin protein with an Xpress epitope located upstream and in-frame with prestin.

[0160] Generation of Prestin-Specific Antibodies

[0161] A peptide consisting of the first twenty N-terminal amino acids (MDHAEENEIPVATQKYHVER; SEQ ID NO: 12) was synthesized commercially and used to immunize two rabbits (SynPep Corporation, Dublin, Calif.). Twenty milliliters of serum from the first bleed was affinity purified by SynPep using standard affinity purification techniques.

[0162] Immunofluorescence

[0163] TSA201 cells transfected with the various prestin constructs were fixed 1 percent paraformaldehyde in PBS for 10 minutes at room temperature, followed by two washes with PBS. Permeabilized cells were incubated for 1 hour at room temperature with 500 nanograms per milliliter anti-prestin in PBS containing 0.1 percent saponin and 2 milligrams per milliliter bovine serum albumin (BSA). The cells were washed and incubated with Cy3-or FITC-conjugated anti-rabbit (or anti-mouse) IgG secondary antibody (Sigma, St. Louis, Mo.) in blocking solution (PBS containing 10 percent goat serum and 2 milligrams per milliliter BSA) containing 0.1 percent saponin. The saponin was used to permeabilize the cell membrane, allowing antibody to bind to intracellular epitopes. The cells were incubated at room temperature for 30 minutes, followed by a 15 minute incubation with 1 microgram per milliliter propridium iodide (PI, Molecular Probes, ), a membrane-impermeant DNA-binding compound used to test plasma membrane integrity. The cells were washed with PBS and mounted using Fluoromount-G™ (Southern Biotechnology Associates, Birmingham, Ala.). Nonpermeabilized cells were similarly treated with the exception of adding saponin to the incubation solution. All cells were then immediately observed using a Nikon Eclipse E400 Microscope.

[0164] In vivo fluorescence experiments were also conducted. Adult Mongolian gerbils were euthanized and decapitated. Cochleae were dissected out and incubated with 4 percent paraformaldehyde in PBS for 1 hour at room temperature (1999, Mammano et al., J. Neurosci., 19(16):6918-6929). The cochleae were then washed 4 times with PBS, followed by a 1-2 hour incubation at room temperature with blocking solution (PBS containing 5 percent goat serum and 2 percent BSA). Blocking solution for permeabilized samples also contained 0.1 percent saponin. Samples were washed again 4 times with PBS and then the tectorial membrane was dissected out. The remaining cochlear sample was divided into three segments by cutting the modiolus. The segments were immunolabeled with antibody and stained with PI as described above. Samples were observed using a laser confocal microscope.

[0165] Immunofluorescence of Native Prestin

[0166] Specificity of anti-prestin antibody was tested using TSA201 cells transiently transfected with the C-tag (V5 epitope) prestin construct. The cells were incubated with both anti-prestin and anti-V5 antibodies as discussed above, followed by extensive washing. The cells were then incubated simultaneously with FITC-labeled anti-rabbit IgG and Cy3-labeled anti-mouse IgG. The anti-prestin antibody attached to the FITC-labeled anti-rabbit IgG, fluoresced as green and the anti-V5 antibody, conjugated with Cy3-anit-mouse IgG, stained red.

[0167] Nonlinear Capacitance Measurements

[0168] The method for measuring nonlinear capacitance is discussed above. Nonlinear capacitance in TSA201 cells was measured in order to determine whether the attached V5 or Xpress epitope tags interfered with the normal function of prestin (i.e., voltage-dependent charge movement).

[0169] The results of the experiments are now described.

[0170] Nonlinear capacitance measurements demonstrated that when TSA201 cells are transfected with N-tagged prestin/GFP or C-tagged prestin/GFP, a typical nonlinear capacitance curve is observed (FIG. 20). This suggests that the V5 and Xpress epitope tags do not interfere with the voltage-dependent charge movement function of prestin.

[0171] Indirect immunofluorescence with FITC-labeled secondary antibody revealed that the anti-Xpress antibody (FIG. 21A) and the anti-V5 antibody (FIG. 21B) expressed green fluorescence in the plasma membrane of about one-third of the cells. This roughly reflects calcium phosphate transfection efficiency (about 30 percent) for TSA201 cells. Propidium Iodide staining was observed in nuclei of all cells. Cells transfected with control vectors did not express green fluorescence.

[0172] Because very few nonpermeabilized cells expressed green fluorescence and propidium iodide staining, it is proposed that these cells lacked plasma membrane integrity (FIGS. 22C and 22D). All of these data suggest that the N and C termini of prestin are located in the cytoplasm of TSA201 cells expressing the synthetic epitope tagged versions of prestin.

[0173] Immunofluorescence studies of native prestin were also performed. FIG. 22 depicts green fluorescence of cells, indicating the presence of the native prestin epitope in the cells. These cells also stained red, confirming the presence of the V5 epitope. The Xpress epitope apparently does not interfere with the immunofluorescent reaction of anti-prestin and its eptiope. These data suggest that the anti-prestin antibody specifically binds with prestin. FIGS. 24A and 24B demonstrate that prestin expression was observed in OHCs, but not in IHCs or other basilar membrane and organ of Corti cells. Prestin is localized in the basolateral wall of OHC as shown in FIGS. 24C and 24D.

[0174] Immunofluorescence results for permeabilized versus nonpermeabilized organ of Corti samples are shown in FIG. 24. Permeabilized samples are shown in FIGS. 24A and 24B and nonpermeabilized samples are shown in FIGS. 24C and 24D. Some nonpermeabilized cells, as in FIG. 24C, exhibited weak fluorescent staining. These cells may have damaged plasma membranes. The results shown in FIG. 24 further confirm that the N-terminus of native prestin is located in the cytoplasm.

[0175] The results ultimately suggest that both the C- and N-termini are located within the cytoplasm of cells. Both termini are possible sites of interaction with OHC cytoplasmic proteins. The results also suggest that the positive and negative cluster regions near the C-terminus may play a unique role in protein function.

[0176] The disclosure of every patent, patent application, and publication cited herein is hereby incorporated herein by reference in its entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention can be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims include all such embodiments and equivalent variations.

Claims

1. An isolated polynucleotide comprising a portion which anneals with high stringency with at-least twenty consecutive nucleotide residues of a coding region of a mammalian pres gene.

2. The isolated polynucleotide of claim 1, wherein the mammalian pres gene comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 4.

3. An isolated polynucleotide comprising a portion which anneals with high stringency with at least twenty consecutive nucleotide residues of a coding region of a mammalian pres gene, wherein the coding region is one other than a coding region which corresponds to any of exons 1-6 of the human pres gene.

4. An isolated polynucleotide which comprises the coding region of a mammalian prestin gene, wherein the coding region of the gene is at least 75% homologous with the coding region of at least one of the gerbil prestin gene and the murine prestin gene.

5. The isolated polynucleotide of claim 4, further comprising a promoter/regulatory region operably linked with the coding region.

6. An isolated mammalian prestin protein.

7. The isolated protein of claim 6, wherein the mammalian prestin protein is isolated from a mammal selected from the group consisting of a gerbil, a mouse, and a human.

8. The isolated protein of claim 6, wherein the protein has an amino acid sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 3.

9. The isolated protein of claim 6, wherein the protein is substantially purified.

10. An isolated antibody which specifically binds with a mammalian prestin protein.

11. A method of alleviating a hearing disorder in a mammal afflicted with the disorder, the method comprising providing a mammalian prestin protein to cochlear outer hair cells of the mammal, whereby the disorder is alleviated.

12. The method of claim 11, wherein the protein is provided to the cells by providing to the cells a polynucleotide encoding the protein.

13. A method of rendering the surface area of a lipid bilayer susceptible to modulation by membrane potential, the method comprising providing a mammalian prestin protein to the bilayer, whereby the bilayer is rendered susceptible to modulation by membrane potential.

14. The method of claim 13, wherein the lipid bilayer is the plasma membrane of a cell.

15. The method of claim 14, wherein the protein is provided to the bilayer by providing a polynucleotide encoding the protein to the cell and expressing the protein in the cell.

16. A method of modulating the surface area of a lipid bilayer, the method comprising providing a mammalian prestin protein to the bilayer and thereafter modulating the membrane potential, whereby the surface area of the bilayer is modulated.

17. A method of modulating the stiffness of a lipid bilayer which surrounds a relatively fixed volume, the method comprising providing a mammalian prestin protein to the bilayer, and thereafter modulating the membrane potential, whereby the stiffness of the bilayer is modulated.

18. A method of modulating the volume of a porous bilammelar lipid vesicle, the method comprising providing a mammalian prestin protein to the bilayer, and thereafter modulating the membrane potential, whereby the volume of the vesicle is modulated.

19. A method of generating a force between two surfaces, the method comprising

interposing a structure which comprises a lipid membrane comprising prestin and enclosing a relatively fixed volume of fluid between the surfaces;

restraining the ability of the structure to expand in a direction at least partially parallel to at least one of the surfaces; and

altering the membrane potential of the lipid membrane,

whereby the structure impacts upon the two surfaces and a force is generated therebetween.

20. A method for creating an electrical impulse, said method comprising applying an external mechanical force to prestin, thereby creating an electrical impulse.