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doi:10.1152/jn.00496.2011 106:2358-2367, 2011. First published 3 August 2011;J NeurophysiolHallworthBenjamin Currall, Danielle Rossino, Heather Jensen-Smith and Richardmammalian prestinresidues in the oligomerization and function of The roles of conserved and nonconserved cysteinyl

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The roles of conserved and nonconserved cysteinyl residues in theoligomerization and function of mammalian prestin

Benjamin Currall, Danielle Rossino, Heather Jensen-Smith, and Richard HallworthDepartment of Biomedical Sciences, Creighton University School of Medicine, Omaha, Nebraska

Submitted 6 June 2011; accepted in final form 27 July 2011

Currall B, Rossino D, Jensen-Smith H, Hallworth R. The roles ofconserved and nonconserved cysteinyl residues in the oligomerization andfunction of mammalian prestin. J Neurophysiol 106: 2358–2367, 2011. Firstpublished August 3, 2011; doi:10.1152/jn.00496.2011.—The creation ofseveral prestin knockout and knockin mouse lines has demonstratedthe importance of the intrinsic outer hair cell membrane proteinprestin to mammalian hearing. However, the structure of prestinremains largely unknown, with even its major features in dispute.Several studies have suggested that prestin forms homo-oligomersthat may be stabilized by disulfide bonds. Our phylogenetic analysisof prestin sequences across chordate classes suggested that the cys-teinyl residues could be divided into three groups, depending on theextent of their conservation between prestin orthologs and paralogs orhomologs. An alanine scan functional analysis was performed of allnine cysteinyl positions in mammalian prestin. Prestin function wasassayed by measurement of prestin-associated nonlinear capacitance.Of the nine cysteine-alanine substitution mutations, all were properlymembrane targeted and all demonstrated nonlinear capacitance. Fourmutations (C124A, C192A, C260A, and C415A), all in nonconservedcysteinyl residues, significantly differed in their nonlinear capacitanceproperties compared with wild-type prestin. In the two most severelydisrupted mutations, substitution of the polar residue seryl for cystei-nyl restored normal function in one (C415S) but not the other(C124S). We assessed the relationship of prestin oligomerization tocysteine position using fluorescence resonance energy transfer. Withone exception, cysteine-alanine substitutions did not significantly alterprestin-prestin interactions. The exception was C415A, one of the twononconserved cysteinyl residues whose mutation to alanine caused themost disruption in function. We suggest that no disulfide bond isessential for prestin function. However, C415 likely participates byhydrogen bonding in both nonlinear capacitance and oligomerization.

hair cell; hearing; molecular motor; disulfide bond; fluorescenceresonance energy transfer

THE COCHLEAR OUTER HAIR CELL (OHC) motor protein prestin isthought to play a major role in mammalian cochlear amplifi-cation (Dallos 2008). Prestin is thought to participate in thegeneration of mechanical energy in the cochlea by means ofreceptor potential-driven OHC length change. The creation ofseveral prestin knockout and knockin mouse lines with hearingloss phenotypes has convincingly demonstrated the importanceof prestin to mammalian hearing (Dallos et al. 2008; Libermanet al. 2002).

Associated with the length change of OHCs is nonlinearcapacitance (NLC), which can also be detected when prestin isexpressed in a cell line such as human embryonic kidney(HEK)-293 cells or opossum kidney cells (Ludwig et al. 2001;Zheng et al. 2000). Briefly, NLC refers to the asymmetric

charging properties of prestin-containing cellular membranes(Iwasa 1993; Santos-Sacchi 1991). In isolated OHCs, NLC andforce generation have proved nearly inseparable. Althoughdeflation of the OHC eliminates length change without alsoeliminating NLC (Kakehata and Santos-Sacchi 1995), no pro-cedure has yet been shown to block NLC without also blockingOHC length change. Thus NLC serves as a proxy measure ofprestin function. NLC is also found in nonplacental mammalprestins in expression systems (Tan et al. 2010), and electro-motility has also been observed in marsupial OHCs (Okoruwaet al. 2008 and our own observations).

Prestin is a transmembrane protein of 744 residues in mam-mals and is a member of an anion transporting protein family,the solute carrier 26, or Slc26a, family (prestin is Slc26a5)(Zheng et al. 2000). The other members of the Slc26 family,where known, are transporters of various species of anion(Dorwart et al. 2008; Ohana et al. 2009). Prestin apparentlydoes not transport anions, although it does require the anionchloride for its conformation change and for NLC (Oliver et al.2001), which may be a relic of a former transporter identity.Consistent with this hypothesis, the nonmammalian prestinhomologs from Gallus gallus and Danio rerio are divalentanion exchangers (Schaechinger and Oliver 2007).

The region, domain, and motif structure of mammalianprestin is shown in Fig. 1A. Of particular importance to ouranalysis are the sulfate transporter (SulP) and the carboxy-terminal STAS (sulfate transporter and anti-sigma factor an-tagonist) domain (Ohana et al. 2009). These features arecommon to nearly all Slc26a5 homologs. Prestin also formshomo-oligomers of unknown stoichiometry, as has been con-sistently shown by Western blot analysis (Zheng et al. 2006)and fluorescence resonance energy transfer (FRET) analysis(Greeson et al. 2006; Navaratnam et al. 2005; Wu et al. 2007).There is evidence that oligomerization is a common feature ofSlc26 family proteins (Detro-Dassen et al. 2008).

A cysteinyl residue in a polypeptide may contribute totertiary structure by forming disulfide bonds, either within thepolypeptide or between polypeptide strands in oligomers. Cys-teinyl residues may also contribute hydrogen bonding to ter-tiary structure by virtue of their polar character. The mamma-lian prestin sequence includes nine cysteinyl residues. Theirpositions in the two published structures are shown in Fig. 2.Two of the positions are consensus intracellular (C52 andC679) and are therefore readily accessible for inter- or intra-molecular sulfhydryl linkages. A third, C260, may or may notbe included in the membrane. The other six are thought to bein membrane-spanning regions, although this would not com-pletely preclude their participation in sulfhydryl linkages.

Address for reprint requests and other correspondence: R. Hallworth, Dept.of Biomedical Sciences, Creighton Univ., 2500 California Plaza, Omaha, NE68178 (e-mail: [email protected]).

J Neurophysiol 106: 2358–2367, 2011.First published August 3, 2011; doi:10.1152/jn.00496.2011.

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We first examined the evolutionary conservation of cysteinylresidues in prestin and related proteins in mammals and non-mammals. We determined which cysteinyl residues were con-served, either in identity, which may signify the requirementfor a sulfhydryl linkage, or in similarity, which may indicatethat the polar character of the residue is important.

We then performed an alanine scan functional analysis of thenine cysteinyl residues in gerbil prestin. NLC was measured inHEK-293 cells transfected with plasmids expressing mutatedprestins and wild type. Cysteinyl residues were singly mutatedto alanyl residues by site-directed mutagenesis. Each sequencewas conjugated at its carboxy terminus with enhanced greenfluorescent protein (eGFP) to indicate which cells were syn-thesizing prestin. Substitution of alanine at a position occupiedby a cysteine eliminated both sulfhydryl bonding and anyhydrogen bonding essential to function. For those mutations inwhich a large functional effect was observed, we then back-substituted the similarly sized polar residue seryl to distinguish

between polar and sulfhydryl contributions of the originalcysteinyl.

We next determined the importance of each residue toprestin oligomerization by performing FRET analysis. InFRET, the energy conferred to a donor fluorophore by a photonin its excitation wavelength range is partly transferred bynonradiative mechanisms to an acceptor fluorophore, fromwhich a photon is emitted in the acceptor’s emission wave-length range. FRET occurs only if the donor and acceptorfluorophores are within molecular dimensions of each other,and thus the presence of FRET may be taken as an indicationof association. In previous studies, prestin was coupled at itscarboxy terminus to the cyan (CFP) or Venus yellow fluores-cent protein (vYFP) (Greeson et al. 2006; Navaratnam et al.2005; Wu et al. 2007). In this study, we used the monomericteal blue fluorescent protein variant (mTFP), which was chosenbecause it does not bind to itself or other fluorescent proteins(Day et al. 2008). We measured FRET using the acceptor

Fig. 1. Homology analysis of prestin. A: region, domain, and motif designation of the human prestin amino acid sequence: green, transmembrane region; blue,sulfate transporter (SulP) domain; red, sulfate transporter and anti-sigma factor antagonist (STAS) domain; and yellow, SulP motif. B: phylogeny of SulP familymember proteins. The number at each node indicates the bootstrap number out of 100 repetitions. C: multiple sequence alignment of cysteinyl residues. Residueproperties are indicated as follows: gray, small hydrophobic; green, medium-size hydrophobic; cyan, partial positive charge; blue, positive charge; orange, partialnegative charge; red, negative charge; pink, proline; light purple, histidine; and yellow, special property.

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photobleach technique and fluorescence lifetime imaging,which have been demonstrated to yield more reliable resultsthan intensity-based methods (Suhling et al. 2005).

MATERIALS AND METHODS

Sequence comparison. The Homo sapiens prestin sequence(NP_945350.1) was used to retrieve prestin homologs from evolu-tionarily relevant sequences using the Basic Local Alignment SearchTool (BLAST) through the National Center for Biotechnology Infor-mation (www.blast.ncbi.nlm.nih.gov). Retrieved sequences werealigned using the CLC Main Workbench custom alignment algorithm(CLC Bio, Cambridge, MA) with default parameters (Feng andDoolittle 1987). Aligned sequences with large gaps or insertions (�50residues) were rejected. Phylogeny analysis was also performed withCLC Main Workbench using the unweighted pair group method witharithmetic mean with 100 bootstrap replicates.

Plasmid constructs. For the NLC studies, a plasmid containinggerbil prestin cDNA, ligated in-frame to eGFP cDNA (referred to aspgPG) was obtained from Dr. Peter Dallos (Northwestern University,Evanston, IL). Cysteine-substitution mutations were performed usingthe QuickChange II site-directed mutagenesis kit (Agilent Technolo-gies, Santa Clara, CA) according to the manufacturer’s instructions.Correct sequence was confirmed by analysis of the insert performed atthe Creighton University Molecular Biology Core Laboratory.

For the FRET studies, the pmTFP1-C construct containing thecDNA for the donor mTFP was obtained from Allele Biotechnology(San Diego, CA). The Venus construct, which contained the cDNA

for the acceptor vYFP, was obtained from Dr. Atsushi Miyawaki(RIKEN Brain Science Institute, Saitama, Japan) (Nagai et al. 2002).With the use of PCR cloning, the mTFP and vYFP cDNA were clonedinto the pAcGFP-N1 plasmid construct, replacing the cDNA forAequorea coerulescens GFP, to create the pmTFP-N1 and pvYFP-N1constructs. Gerbil prestin cDNA, without the stop codon, was PCR-cloned in-frame to the pmTFP-N1 and pvPYFP-N1 constructs 5= tothe fluorescent protein open reading frame. Cysteine-substitutionmutations for all constructs were performed using the QuickChange IIsite-directed mutagenesis kit (Agilent Technologies) according to themanufacturer’s instructions. Correct sequence was confirmed by se-quence analysis of the insert performed at the Creighton UniversityMolecular Biology Core Facility.

Two other constructs, as FRET positive and negative controls, wereobtained as plasmids from Dr. Jian Zuo. The positive control con-sisted of a plasmid expressing a construct of Cerulean and Venusfluorescent proteins linked by a short amino acid sequence (referred toas pLink). The negative control consisted of a construct of theunrelated SLC family protein SLC38A2, linked by carboxy terminusto Venus fluorescent protein (referred to as p38Y). Both plasmidshave previously been used as controls in this laboratory (Wu et al.2007).

Cell culture. HEK-293 cells were obtained from the AmericanType Culture Collection and were grown in flasks or on 35-mmglass-bottom dishes by using standard methods, without antibiotics.

Transfection. HEK-293 cells were transfected with plasmid(s)when plated cells reached between 80 and 100% confluency using

Fig. 2. Diagram showing the positions of the9 cysteinyl residues in prestin, representedby small gray disks, in the 2 proposed mem-brane topologies (A, based on Deak et al.2005; and B, based on Navaratnam et al.2005).

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Lipofectamine 2000 by following the manufacturer’s protocol (Invit-rogen, Carlsbad, CA). For NLC measurements, cells were examined24–48 h after transfection. In one experimental series (C415S), 10�M salicylate was added to the medium to promote translocation tothe plasma membrane (Kumano et al. 2010). Salicylate blocks prestinNLC, so the salicylate was removed at least 1 h before electrophys-iological measures (Kakehata and Santos-Sacchi 1996; Tunstall et al.1995).

For cotransfection and FRET experiments, if donor and acceptorfluorescence intensity were not approximately equal, plasmid volumeswere adjusted to compensate. Cells were fixed between 24 and 48 hafter transfection using 4% paraformaldehyde in phosphate-bufferedsaline (PBS). After 30 min of fixation, cells were washed with PBSand mounting medium was applied (1:1 vol/vol PBS-glycerol). Cov-erslips (no. 1½ 30-mm round; Warner Instruments, Hamden, CT)were sealed over the cells using rubber cement (Elmer’s Products,Columbus, OH). Plates were stored at 4°C in the dark beforeexperimentation.

Determination of membrane targeting. The incubation mediumsurrounding transfected HEK cells was removed and replaced withPBS containing 2 �g/ml wheat germ agglutinin coupled to the redfluorophore AlexaFluor 568 (WGA-568; Invitrogen). After 10 min ofexposure to WGA-568, the cells were rinsed three times with PBSalone and fixed using 4% paraformaldehyde in PBS (30 min). Thefixed cells were rinsed in PBS, mounted in mounting medium, andcoverslipped and sealed as described above. Cells were examinedusing the LSM 510 META NLO confocal microscope (Carl Zeiss,Thornwood, NY) of the Creighton University Integrated BiomedicalImaging Facility (IBIF). Images were analyzed using ImageJ(http://rsbweb.nih.gov/ij/).

Measurement of nonlinear capacitance. Prestin NLC in HEK cellswas measured as a function of membrane potential 24–48 h aftertransfection. Cells were bathed in a medium designed to blockvoltage-dependent ionic currents. The medium consisted (in mM) of120.0 sodium chloride, 2.0 magnesium chloride, 20.0 tetraethylam-monium chloride, 2.0 cobalt chloride, 10.0 dextrose, and 10.0 HEPES,buffered to pH 7.25 and adjusted to 300 mosmol/l. Cells withclearly membrane-resident fluorescent label were identified on thestage of an Olympus IX-70 inverted microscope (Olympus Amer-ica, Center Valley, PA) using a �100 1.4-numerical aperture (NA)objective. All electrophysiological measurements were performedat room temperature.

The whole cell patch-clamp method was used to measure mem-brane currents evoked by voltage commands. Patch pipettes werepulled from 8250 glass capillaries (A-M Systems, Carlsborg, WA) ona Sutter P-97 electrode puller (Novato, CA) and polished on aNarashige MF-830 polisher (East Meadow, NY). The pipette solutionconsisted (in mM) of 140.0 cesium chloride, 10.0 EGTA, 10.0HEPES, 2.0 magnesium chloride, and 2.0 potassium adenosinetriphosphate. Filled pipette resistances were between 1.5 and 5.0 M�.Membrane currents in response to voltage commands were recordedat the output of a Warner Instruments PC-501A patch-clamp amplifier(Hamden, CT). Voltage commands were generated and currentsdigitized using custom software written in TestPoint (C.E.C., Burl-ington, MA) and a Keithley Instruments (KCPI 3801; Cleveland, OH)analog-to-digital/digital-to-analog board in a personal computer. Cur-rents were low-pass filtered at 5 kHz before digitization.

The two-sinusoid method (Kakehata and Santos-Sacchi 1996) wasused to measure membrane capacitance. This method is superior toother methods, including single-sinusoid methods, because it enablesindependent calculation of series resistance, which often varies duringa stimulus protocol. Simultaneous sinusoidal voltage commands offrequencies 195.3 and 390.6 Hz at amplitudes of 10 mV weresuperimposed on membrane potential bias commands that werestepped in 32 intervals of 5 mV, starting at �100 mV. The membraneholding potential in the absence of voltage commands was �70 mV.Membrane potentials were corrected off-line for series resistance error

but were not corrected for the change in liquid junction potential onbreaking into the cell (measured as �4.7 mV). Series resistances weregenerally less than 20 M�. Results were discarded if the cell inputresistance were less than 500 M�.

Corrected capacitance-membrane potential functions were fitted bythe Levenberg-Marquardt algorithm using the program Origin (OriginLab, Northampton, MA) to the following equation (Kakehata andSantos-Sacchi 1996):

C � Cl � �Qz

kT �� b

�1 � b�2� ,

b � exp��ze

kT�V � Vpk�� ,

where Cl is the linear capacitance, V is the membrane potential involts, Vpk is the membrane potential at peak NLC (also in volts), e isthe charge on the electron (coulombs), z is the number of elementarycharges transferred by each molecule between states, Q is the totalnumber of elementary charges transferred between states in a cell, andk and T are Boltzmann’s constant and absolute temperature,respectively.

The results of the curve fits were accepted only if the R2 goodnessof fit value were greater than 0.9 (with one exception, see RESULTS)and the error in any single parameter estimate did not exceed 20% ofthe estimate. The NLC values were normalized for comparison bydividing by the peak NLC value predicted from the curve fit.

Statistical analysis of NLC results. To prevent potential datadiscrepancies due to daily fluctuations in equipment and cell passage,we always compared mutant NLC measurements with a similarnumber of measurements obtained from wild-type transfected cells ofthe same or similar passage number. Differences in cysteine-mutantand wild-type NLC measurements were analyzed using independentsample t-tests (SPSS, Chicago, IL). The t-tests were corrected forunequal variance when a Levene’s test for equality of varianceindicated unequal variances between the test and control (wild type)groups.

Lifetime imaging of donor mTFP fluorescence. Fixed cells wereimaged using a �40 1.4-NA oil-immersion objective on the IBIFconfocal microscope. Cells containing donor alone and/or acceptorfluorophore were imaged using a 512 � 512-pixel field with �4digital magnification. Prebleach lifetime images were captured usingtwo-photon excitation at wavelengths of 820 (for cCFP) or 870 nm(for mTFP) with a titanium-sapphire laser (Chameleon Ultra; Coher-ent, Santa Clara, CA) and time-correlated single photon counting(SPC-830; Becker & Hickl, Nahmitzer Damm, Berlin, Germany).Donor lifetime was calculated using SPCImaging (Becker & Hickl)with either 1 (mTFP) or 2 (cCFP) decay components (lifetime datarange set with minima and maxima of 500 and 3,000 ps and athreshold of 25 photon counts per pixel). The decay matrix distribu-tion over the region of interest was exported. A Gaussian fit of thehistogram was calculated using Origin. Cell lifetimes were rejected ifthe Gaussian fit parameter had a correlation coefficient R2 � 0.9.

FRET analysis of oligomerization. The acceptor photobleach fluo-rescence lifetime imaging version of FRET analysis (apFRET) re-quired five steps: 1) a prebleach spectrum determination to determinethe relative levels of donor and acceptor fluorescence, 2) a prebleachfluorescence lifetime measurement of the acceptor (mTFP or CFP),3) photobleaching of the acceptor (YFP), 4) a postbleach fluorescencelifetime measurement of the acceptor, and 5) a postbleach spectrumdetermination to determine the effectiveness of the acceptor photo-bleach. Prebleach spectral images were determined using excitationwith the 453-nm line of the argon laser of the confocal microscopeand were captured using the META detector in lambda mode. Spectralimages were linearly unmixed into either donor or acceptor based onpreviously acquired donor and acceptor spectra. If the cell image didnot contain approximately equal intensities of donor and acceptor

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fluorescence, then the image was rejected. Acceptor photobleachingwas performed by 30 repetitions of excitation of the region of interestwith the 545-nm laser line of the confocal microscope and band-passfiltered from 565 to 615 nm. Postbleach lifetime and spectral imageswere captured as described above. FRET efficiency was calculatedwith the following equation:

EFRET ��D � �DA

�D,

where EFRET is FRET efficiency, �D is the lifetime of donor alone(after photobleaching), and �DA is the lifetime of the donor in thepresence of an acceptor before photobleaching.

RESULTS

Determination of conserved cysteinyl residues. More than110 prestin homologs were identified on the basis of a filteredBLAST database search. Sequences that did not containboth a SulP domain and a STAS domain were not includedin the analysis. Homologous sequences were obtained fromspecies in five kingdoms (animals, plants, fungi, amoeba,and bacteria).

We analyzed cysteine conservation by aligning prestin ho-mologs in a subset of sequences (Fig. 1B). Bias toward anyphylogeny class was avoided by selecting single Slc26a5representatives from each chordate class in which a publishedSlc26a5 homolog exists. The analysis data set included Slc26sequences from Caenorhabditis elegans, Strongylocentrotuspurpuratus, Ciona intestinalis, and Dictyostelium discoideum,as well as nonmammalian and mammalian Slc26a5 orthologsequences and mammalian (human) sequences of SLC26 para-logs. The phylogenetic relationships between the selected se-quences are shown in Fig. 1B, as determined using CLC MainWorkbench (see MATERIALS AND METHODS). Particularly note-worthy is the large evolutionary distance between H. sapiensSLC26A5 and its paralog, SLC26A11, greater even than thedistance between mammalian prestin and its C. intestinalis andS. purpuratus homologs.

We then performed a multiple sequence alignment focusedon the cysteinyl residues, also using CLC Main Workbench(Fig. 1C), and organized the results into three groups repre-senting different levels of conservation. The amino-terminalresidue C52, which we allocated to group 1, was identicallyconserved in all prestin orthologs and in nearly all paralogs andhomologs. In the only exceptions, human SLC26A7 and D.discoideum SulP, there were gaps at that position, and we alsonoted poor overall conservation in their amino-terminal se-quences. This high degree of identity conservation suggested arole for C52 in a function common to the entire Slc26 family,such as an ion transport-related function or binding to somemembrane-localization element.

The other cysteinyl residues showed mixed patterns ofconservation that could be organized in two further groups:those residues that were replaced in paralogs and homologs bymainly polar residues (group 2) and those that were replacedby mainly nonpolar residues (group 3). Group 2 consisted ofC196, C381, and C679. For example, the group 2 carboxy-terminal residue C679 was identically conserved in all Slc26a5orthologs and in some closely related paralogs, as determinedby the phylogenetic relationships depicted in Fig. 2B. Someparalogs also had a cysteinyl residue in that position (for

example, H. sapiens SLC26A2), but other polar residues werealso found. In the other members of group 2, the SulP domainresidues C196 and C381, the cysteinyl residue was identicallyconserved in mammalian orthologs (except for the monotremein C196). In the nonmammalian orthologs, paralogs, and ho-mologs, the residue was replaced by one of several small polarresidues. The results suggested that group 2 cysteinyl residuescontribute polar character rather than sulfhydryl linkages tostructure in the Slc26 family proteins.

The group 3 cysteinyl residues C124, C192, C415, and C395(also in the SulP domain) were conserved only in mammalianprestin sequences and were replaced in nonmammalian or-thologs, and in paralogs and homologs, with residues of vari-ous properties, including polar residues, nonpolar aliphaticresidues, charged residues, and even prolyl in C260. The moststriking of these was C415, which was found to be identicallyconserved only in the mammalian prestin orthologs, which arethe ones that have been shown to exhibit large NLC. In thenonmammal orthologs, the paralogs, and the homologs, thiscysteinyl residue was replaced by various medium-size andsmall hydrophobic residues. This position may therefore beimportant in some functional aspect unique to mammalian

Fig. 3. Determination of successful membrane targeting of prestin mutations.A: typical human embryonic kidney (HEK) cell expressing prestin-enhancedgreen fluorescent protein (eGFP) (green) and labeled with WGA-633 (red).Inset: fluorescence intensity profiles of the line in the main image. B: average(�SE) of separation of WGA-633 and prestin in profiles, as in A, for wild-type(WT) and mutated prestins as indicated.

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Slc26a5, such as its membrane potential-dependent conforma-tion change or the associated NLC. The results of replacementof cysteinyl residues in other paralogs and homologs in group3 suggest that neither their polar character nor their ability toform sulfhydryl linkages contribute to conserved, Slc26-relatedaspects of prestin structure or function.

We concluded that the cysteinyl residues in groups 1 and 2are related to function across the entire Slc26 family, whereasthe cysteinyl residues in group 3 more likely contribute tosome mammalian prestin-specific function, such as NLC.

All cysteine-alanine substitution mutations were membranetargeted. For electrophysiological analysis, we transfectedHEK-293 cells with plasmids expressing wild-type prestin orcysteine-substitution prestin mutations as constructs coupled toeGFP. The protein products of wild-type and alanine-substi-tuted prestin-eGFP constructs were synthesized at useful levelswithin 24 h of transfection. All appeared to be membranetargeted. Membrane targeting was confirmed by labeling themembrane of living intact cells with WGA-568, as described inMATERIALS AND METHODS. Confocal microscopic observation oflabeled cells revealed distinct and nearly overlapping WGA-568 label and eGFP fluorescence (Fig. 3A). Measurements oflabel found no significant difference in the relative positions ofeGFP and WGA-568 label in all alanine-substitution con-structs, indicating incorporation of eGFP-coupled prestin intothe plasma membrane (Fig. 3A).

All cysteine-alanine substitution mutants were functional byNLC analysis. We obtained NLC measurements from trans-fected and expressing HEK cells using the methods described.All constructs demonstrated NLC, which confirmed our con-

focal microscopic analysis of prestin incorporation. NLC re-sults were fitted to Boltzmann-derived functions to determinethe NLC parameters, as described in MATERIALS AND METHODS.Plots of averaged normalized NLC values for each of thecysteine-alanine substitution constructs, as a function of mem-brane potential, are shown in Fig. 4. Functions in Fig. 4 werecompared with similarly averaged normalized NLC measure-ments from cells transfected at about the same time withwild-type prestin.

Four cysteine-alanine substitution mutations exhibited sig-nificantly different NLC from wild-type prestin. Our statisticalanalysis of the data is depicted in Fig. 5 and the results arelisted in Table 1. The analysis showed that the NLC parametersVpk and z were insignificantly different from wild-type NLC inthe group 1 and group 2 alanine substitutions. However, all ofthe group 3 alanine substitutions (with 1 exception) exhibitedsignificant positive or negative changes in Vpk, without changesin z.

Group 3 cysteinyl residues were replaced in nonmammalianorthologs, paralogs, and homologs by nonpolar residues, forthe most part. Introducing a nonpolar residue (alanine) in placeof a cysteinyl had therefore disrupted a function specific tomammalian prestin. We then attempted to determine whetherthe polar nature of the cysteinyl residue, rather than its capacityto form covalent bonds, was functionally significant in NLC.The amino acid serine is closest to cysteine in molecularweight and polar nature. We therefore created cysteinyl-serylsubstitutions of prestin-eGFP for the two group 3 positionswith the largest changes in Vpk (C124 and C415, �10 mV).Our reasoning for selecting these two positions was that we

Fig. 4. Normalized nonlinear capacitance (NLC) as a function of membrane potential for cysteine-alanine substitutions (solid squares) compared with wild-typeprestin (open circles). Functions are not corrected for series resistance membrane potential error, which, although small (0.4% or less), means that the plots areshown for illustrative purposes only.

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would have the best chance to detect restoration of normalNLC with recordings from a practical number of cells. We thenmeasured the resulting NLC and compared it with wild-typeprestin (Fig. 5 and Table 1). If the polar character of the residuewere important, we would expect that the NLC parameterswould be restore to wild-type or near wild-type values.

For the C124A substitution, Vpk was hyperpolarized com-pared with wild type (�72.78 mV), and z was not significantlydifferent. For the C124S substitution, Vpk was also significantlyhyperpolarized compared with wild type, although less so thanfor C124A (�67.65 mV), and z was again not significantlydifferent (Fig. 6A and Table 1). In contrast, for the C415Asubstitution, Vpk was depolarized compared with wild type(�32.11 mV), and z was not significantly different. For theC415S substitution, the substitution of serine essentially cor-rected the depolarizing effect of alanine substitution at thatposition (�59.26 mV) (Fig. 6B and Table 1). Thus we con-clude that the polar character of the cysteine at position 415contributes to prestin function.

It should be noted that membrane incorporation levels ofC415S prestin were remarkably low, although synthesis levelsappeared comparable to those of wild-type prestin. Thus, forthis construct, we resorted to promoting membrane incorpora-tion using incubation with salicylate (see MATERIALS AND METH-ODS). Even with this additional step, the fit criterion had to berelaxed to R2 � 0.8 to obtain enough measurements.

FRET efficiency. As explained in the Introduction, oligomer-ization is a feature of mammalian prestin and at least someparalogs and homologs. We reasoned that if particular cystei-nyl residues participate in oligomerization, then wild-typesubunits would interact less efficiently with cotransfected ala-

nyl-substituted subunits than with wild-type subunits. Wetherefore measured FRET efficiency in HEK-293 cells trans-fected with wild-type prestin coupled to the donor (mTFP) andalanine-substituted prestins coupled to the acceptor (vYFP).

First, to establish the limits of our ability to detect FRETusing apFRET, we examined several positive and negativecontrols (Fig. 6A). Two positive controls were used: the pLinkconstruct, in which cCFP is ligated in-frame to vYFP, and thepgPT/pgPY combination, in which gerbil prestin donor andacceptor constructs were cotransfected. FRET was detectablewith both positive controls, although pLink-transfected cellshad significantly greater average FRET efficiencies than cellstransfected with the pgPT/pgPY pair (Fig. 6A; Student’s t-test,P � 0.01).

Several negative controls were also used to establish theminimum detectable FRET efficiency. These included cellstransfected with donor alone (pT and pgPT) and cells trans-fected with a pair not expected to oligomerize (pT/pY andpgPT/p38Y). The average FRET efficiencies obtained for thesetests are also shown in Fig. 6A. The negative control trans-fected cells all had significantly smaller average FRET effi-ciencies than the positive controls (Student’s t-test, P � 0.001),which demonstrated our ability to distinguish between inter-

Fig. 5. Summary of NLC properties of cysteine-alanine substitutions (solidsquares) compared with wild-type prestin (open circles), plotted as mean �SE. A: peak membrane potential (Vpk). B: charge transfer (z). �P � 0.05;�P � 0.001, different from WT.

Table 1. Averaged Vpk and z values of prestin NLC of wild-typeprestin and prestin mutations, their statistically significantdifferences from wild type in the same batch, and theirconservation group status

Conserved Mutation Vpk, mV P Value z P Value

Group 1 C52AControl �48.33 0.901Test �58.45 0.051 0.902 0.986

Group 2 C196AControl �52.38 0.809Test �54.71 0.615 0.918 0.082

C381AControl �50.25 0.913Test �59.34 0.211 0.931 0.907

C679AControl �53.48 0.918Test �45.25 0.055 0.936 0.740

Group 3 C124AControl �49.94 0.923Test �72.78 �0.001 0.944 0.849

C192AControl �52.23 0.817Test �63.05 0.006 0.954 0.077

C260AControl �50.98 0.861Test �60.22 0.025 0.882 0.766

C395AControl �59.34 0.850Test �51.41 0.053 0.797 0.438

C415AControl �53.74 0.944Test �32.11 �0.001 0.937 0.928

Group 3 C124SControl �52.03 0.866Test �67.65 0.007 0.997 0.124

C415SControl �47.62 0.949Test �59.26 0.132 0.821 0.139

Values are averaged peak membrane potential (Vpk) and charge transfer (z)for prestin nonlinear capacitance (NLC) of wild-type prestin (control) andprestin mutations. See text for descriptions of conservation groups 1–3.

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acting and noninteracting fluorophores. Cells transfected withpgPT had significantly greater FRET efficiencies than the othernegative controls (Student’s t-test, P � 0.05, P � 0.001). Theother three negative controls were indistinguishable. For theremainder of the FRET experiments, the FRET efficiency ofpgPT/pA38Y cotransfected cells was considered the lowerlevel detection of FRET (EFRET � 1.70%) and the FRETefficiency of pgPT/pgPY transfected cells was considered thenormal FRET efficiency of gerbil prestin subunits (EFRET �7.64%).

To test interaction between wild-type and cysteine-substitutionmutations, FRET efficiency was measured between wild-typedonor subunits and cysteine-mutated acceptor subunit. The aver-age FRET efficiencies are shown in Fig. 7B. All cysteine mutantsshowed no difference between pgPT/pgPY-transfected cells andgPT/cysteine-alanine mutant acceptor construct, except for thecells transfected with pgPT/pgPY-C415A (Dunnett’s 2-way t-test,P � 0.001). The average FRET efficiency of pgPT/pgPY-C415A-transfected cells was indistinguishable from the lower limit ofFRET detection (Dunnett’s 1-way t-test, P � 0.05). Thus weconclude that only the group 3 residue C415 contributes to prestinoligomerization.

DISCUSSION

Our results point to a significant functional role specifi-cally in mammalian prestin for the cysteine at position 415.Mutation of this position to the nonpolar amino acid alaninedepolarized the peak membrane potential of NLC (Vpk)without changing the charge transfer (z). Mutation of thesame position to the polar amino acid serine had no effect.Both mutations were functional in that they underwent theconformation change associated with NLC. This finding,

and the preservation of normal function with the serinesubstitution, eliminates the possibility that the cysteine isrequired for a sulfhydryl linkage. However, it does noteliminate the possibility that one exists. Indeed, the fact thatthe alanine substitution reduced or eliminated FRET be-tween it and wild-type prestin suggests a dramatic change inthe properties of the assembled molecules. FRET could be

Fig. 6. A: normalized NLC of C124A (solid squares), C124S (shaded squares),and WT prestin (open circles) as a function of membrane potential.B: normalized NLC of C415A (solid squares), C415S (shaded squares), andWT prestin (open circles) as a function of membrane potential. Functions arenot corrected for series resistance membrane potential error.

Fig. 7. Averaged fluorescence resonance energy transfer (FRET) efficiencies ofcontrol and prestin construct-transfected cells. A: FRET efficiencies of cellstransfected with negative controls (left) or positive controls (right) [ANOVAagainst negative control (3, 108, P � 0.01) and Student’s t-tests]. *P � 0.05;***P � 0.001. B: comparison of FRET efficiencies of positive (left) ornegative control (dashed line) to WT/mutant donor/acceptor-cotransfectedcells (right) [ANOVA against positive control (9, 320, P � 0.001), ANOVAagainst negative control (9, 295, P � 0.001), and Dunnett’s 2-sided t-tests].***P � 0.001. Error bars indicate SE. See text for description of groups 1–3.

Fig. 8. Total nonlinear charge transfer as a function of linear capacitance for117 cells synthesizing WT prestin (slope � 0.0058 pC/pF, R � 0.118).

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reduced by uncoupling of the subunits or by a conformationchange that sufficiently separates the carboxy-terminal fluo-rophores from each other. Our results cannot distinguishbetween these possibilities. We can, however, assert that thecysteine at position 415 is involved in both NLC andoligomerization in mammalian prestin.

The cysteine at position 415 is one of our group 3 cysteinylresidues, which are replaced in paralogs and orthologs by avariety of nonpolar residues. The group 3 cysteine residueswere predicted, from their lack of conservation, to be involvedin mammalian prestin-specific functions, which proved to bethe case. For all but one of the group 3 cysteinyl residues,mutation to alanyl resulted in a change in Vpk of NLC (depo-larizing for C415, hyperpolarizing for the others) withoutchanging z. We suggest that these residues contribute to mask-ing (or unmasking) of the voltage sensor governing the prestinconformation change but do not move during that conforma-tion change, at least not orthogonally to the plane of themembrane.

Mutation of the highly conserved group 1 cysteine atposition 52 had no effect on NLC. The same applied to thegroup 2 cysteinyl residues, which are replaced by polarresidues in nonmammalian prestin and in paralogs andhomologs. The group 2 cysteinyl residues are not apparentlyrequired to form sulfhydryl linkages for functionally normalmammalian prestin, although, as before, we cannot dismissthe possibility that they are present. They also are notrequired for oligomerization. They may participate in hy-drogen bonding, as may their polar substitutes in other Slc26family members, but if so, it may contribute to somefamily-wide function such as ion transport or binding tomembrane-localization elements.

All of the group 3 cysteinyl residues, with the exception ofC395, appear to participate in NLC to some degree, since thesubstitution of alanyl modified Vpk, without, however, modi-fying z. Modifying Vpk alone suggests an effect on the voltagesensor, without modification of the conformation change, or atleast that part of the conformation change that contributes toNLC. The cysteinyl may be masking, or unmasking, thevoltage sensor to some extent. Current hypotheses attribute theNLC to either movement of a bound chloride ion (like atransporter) or movement of charged or polar residues, modu-lated by chloride ions in an allosteric manner (Oliver et al.2001; Rybalchenko and Santos-Sacchi 2003). If the boundchloride hypothesis applied, we might infer that the affinity ofprestin for the chloride ion had been modified by alanylsubstitution, but the translocation of chloride was unaltered. Ifthe allosteric modulation hypothesis applied, we might inferthat either the chloride binding site, or the voltage sensor, hadbeen modified by alanyl substitution. Without further informa-tion, it is not yet possible to distinguish between these hypoth-eses.

We do not find the apparent hyperpolarized shift in Vpk inwild-type prestin relative to isolated hair cells (Bai et al. 2010;McGuire et al. 2010). Our average Vpk value for wild-typeprestin, �51 mV, is much closer to reported Vpk values forOHCs and prestin-transfected HEK cells.

The percentage FRET obtained with pgPT/pgPY, at 7%, issubstantial and comparable to recent observations by us andother laboratories (McGuire et al. 2010; Navaratnam et al.2005; Wu et al. 2007). The higher FRET percentage of the

pLink construct (20%) is likely a consequence of the forced 1:1donor-acceptor ratio and the short distance between the donorand acceptor fluorophores in the construct. The higher FRETefficiency of gPT alone may be due to donor-to-donor FRET(Koushik and Vogel 2008) in the dense puncta of fluorescentlabel that are normally seen in HEK cells (Rajagopalan et al.2007 and our own observations).

We have chosen not to report Q/Cl results as a measure ofmembrane incorporation, as others have done (Bai et al.2010; McGuire et al. 2010), because they do not have thesignificance ascribed to them. In transfected cell experi-ments, the cells selected for examination are a vanishinglysmall subset of those available to the experimenter and areundoubtedly selected to be those with the clearest membranelabel. Even then, the experimenter does not sample a fixednumber of cells but continues until a satisfactory number ofrecordings has been achieved. Thus the sample population isfar from unbiased. Furthermore, Cl is a poor predictor of Qeven for a single construct. In our analysis of 107 cells thatwere visibly synthesizing wild-type prestin (coupled toeGFP) and incorporating it into their membranes, Q wasonly weakly correlated with Cl (Fig. 8). We conclude thatcomparisons of membrane incorporation between mutationsusing Q/Cl are not reliable. This is not to say that there areno differences among constructs in membrane incorpora-tion. For example, as we described, the C415S construct wasrobustly synthesized but poorly incorporated into the mem-brane, so much so that a frustratingly large number of cellshad to be examined to provide even the limited data shown.

ACKNOWLEDGMENTS

We thank Dr. Atsushi Miyawaki (RIKEN Brain Science Institute, Japan),Dr. Jian Zuo (St. Jude Children’s Research Hospital, Memphis, TN), and Dr.Peter Dallos (Northwestern University, Evanston, IL) for gifts of cDNA. Wethank Dr. Jing Zheng and Dr. Peter Dallos of Northwestern University, and Dr.Kirk Beisel and Dr. Venkatesh Govindarajan of Creighton University, forvalued help in getting these experiments started. We acknowledge the use ofthe confocal microscope facility of the Integrated Biomedical Imaging Facilityof Creighton University.

Present address of B. Currall: Department of Pathology, Brigham andWomen’s Hospital, Harvard Medical School, Boston, MA.

GRANTS

This work was supported by National Science Foundation-Nebraska EPSCoR(Experimental Program to Stimulate Corporate Research) Grant EPS-0701892(to R. Hallworth). Research was conducted in a facility constructed withsupport from National Center for Research Resources (NCRR) ResearchFacilities Improvement Program C06 RR17417-01. The Integrated BiomedicalImaging Facility is supported in part by NCRR Grant P20 RR16469.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

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