J Physiol 580.2 (2007) pp 451–461 451

Voltage-sensitive prestin orthologue expressed inzebrafish hair cells

Jorg T. Albert1, Harald Winter2, Thorsten J. Schaechinger3, Thomas Weber2, Xiang Wang4, David Z. Z. He4,

Oliver Hendrich1, Hyun-Soon Geisler2, Ulrike Zimmermann2, Katrin Oelmann2, Marlies Knipper2,

Martin C. Gopfert1 and Dominik Oliver3

1Volkswagen Foundation Research Group, Institute of Zoology, University of Cologne, 50923 Cologne, Germany2University of Tubingen, Department of Otolaryngology, Tubingen Hearing Research Centre (THRC), Laboratory of Molecular Neurobiology,

Elfriede-Aulhorn-Straße 5, D-72076 Tubingen, Germany3Institute of Physiology II, University of Freiburg, 79104 Freiburg, Germany4Department of Biomedical Sciences, Creighton University School of Medicine, Omaha, NE 68178, USA

Prestin, a member of the solute carrier (SLC) family SLC26A, is the molecular motor that drives

the somatic electromotility of mammalian outer hair cells (OHCs). Its closest reported homo-

logue, zebrafish prestin (zprestin), shares ∼70% strong amino acid sequence similarity with

mammalian prestin, predicting an almost identical protein structure. Immunohistochemical

analysis now shows that zprestin is expressed in hair cells of the zebrafish ear. Similar to

mammalian prestin, heterologously expressed zprestin is found to generate voltage-dependent

charge movements, giving rise to a non-linear capacitance (NLC) of the cell membrane.

Compared with mammalian prestin, charge movements mediated by zprestin display a weaker

voltage dependence and slower kinetics; they occur at more positive membrane voltages, and

are not associated with electromotile responses. Given this functional dissociation of NLC and

electromotility and the structural similarity with mammalian prestin, we anticipate that zprestin

provides a valuable tool for tracing the molecular and evolutionary bases of prestin motor

function.

(Resubmitted 10 January 2007; accepted after revision 25 January 2007; first published online 1 February 2007)

Corresponding author D. Oliver: Email: dominik.oliver@physiologie.uni-freiburg.de, Also: m.gopfert@uni-koeln.de

and marlies.knipper@uni-tuebingen.de

Active mechanical feedback assists hearing in vertebratesand invertebrates (Manley, 2000; Gopfert & Robert, 2003;Gopfert et al. 2005, 2006; for a review, see Manley, 2001;Geleoc & Holt, 2003). A prominent actuator of suchamplificatory feedback is the somatic electromotility ofouter hair cells (OHCs), which, besides active hair bundlemotility (Chan & Hudspeth, 2005; Kennedy et al. 2005;for a review, see Fettiplace, 2006), is widely thought topromote cochlear amplification in mammalian ears (Jia& He, 2005; for a review, see Fettiplace & Hackney,2006). The molecular basis of OHC somatic electro-motility is formed by the unconventional motor proteinnamed prestin (Zheng et al. 2000; Liberman et al. 2002).Prestin molecules are densely packed into the OHC lateralmembrane, where they may form supramolecular motorcomplexes (Navaratnam et al. 2005). These elementarymotors are thought to respond to changes in membranepotential with concerted conformational changes thateventually build up to an overall motion of the OHC soma.The underlying elementary event, i.e. the conformationaltransition of the single prestin molecules between an

expanded and a contracted state, involves the translocationof a charged voltage sensor across the membrane.Prestin motor activity is thus inherently electrogenic, withthe protein converting voltage changes into movements,much like the piezoelectric crystal that was initiallyproposed by Gold (1948) to be at work in the cochlea.The voltage-dependent charge movement conferred byprestin’s voltage sensor can be measured as a non-linearcapacitance (NLC) of the cell membrane. Since thisNLC is linked to motility and can easily be assayedexperimentally, it is often used as a substitute for directmeasurements of the somatic motility of outer hair cellsand prestin-transfected cells (for a review, see Dallos &Fakler, 2002).

Molecularly, prestin is affiliated to the highly versatileSLC26 family of anion transporters (for a review, seeMount & Romero, 2004). Presently, 10 mammalianSLC26A members (A1–A10) are known. The closestreported homologue to mammalian prestin (SLC26A5),as assessed by over-all sequence identity, however, is foundin zebrafish (Weber et al. 2003). The zebrafish prestin

C© 2007 The Authors. Journal compilation C© 2007 The Physiological Society DOI: 10.1113/jphysiol.2007.127993

452 J. T. Albert and others J Physiol 580.2

orthologue, zprestin, shares ∼50% amino acid identitywith mammalian prestin, compared with an amino acididentity of only ∼37% between mammalian prestin andits closest paralogue PAT-1 (SLC26A6). This molecularkinship with the mammalian prestin motor, along with thepresence of zprestin transcripts in the zebrafish auditoryorgan (Weber et al. 2003), have raised the principalquestion whether zprestin constitutes a hair cell motoras well (Weber et al. 2003), thus challenging the generallyheld view that prestin-mediated electromotility is a uniqueproperty of mammalian OHCs (He et al. 2003). To test thishypothesis, we systematically explored the molecular andfunctional properties of zprestin, including its genomicstructure, its expression, and its putative motor function,whereby the latter was assayed by probing for NLC andelectromotility in zprestin-transfected cells. A parallelstudy investigated the role of zprestin in anion transport(Schaechinger & Oliver, in press).

Methods

All experiments conformed to the European Communityguiding principles on the care and use of animals(86/609/CEE) and were in accordance with theinstitutional guidelines for the use of animals in research.

Animals

Wildtype larvae and adult fish of the AB strain were usedfor immunohistochemistry and in situ hybridizations.Larvae were produced by pair-wise breeding and raisedin embryo medium (5 mm NaCl, 0.17 mm KCl, 0.33 mmCaCl2, 0.33 mm MgSO4) at 30◦C. Adult males were takenfrom laboratory stocks. Zebrafish were maintained andstaged as described elsewhere (Kimmel et al. 1995).

Cloning of zprestin

For measurements of NLC and sequence analysis thezebrafish full-length cDNA was cloned from zebrafishutricle and lagena. Procedures to isolate mRNA aredescribed in Weber et al. (2003). The 3′-end of zebrafishprestin was determined using the GeneRacer Kit(Invitrogen) according to manufacturer’s instructions,using the specific primers Dan3, 5′-GCTGTCAACGATAC-GCTACGTAACG-3′, and Dan3nested, 5′-CGCTACGTA-ACGGCATGACAGTG-3′. The full-length cDNA wasamplified with the forward primer DanF1, 5′-TGCAGC-CATGGAGCACGTAACTGTTAG-3′, and the reverseprimers DanR5, 5′-TTGTGTTCAGTGGATGTTTGGG-TGGAC-3′, respectively, DanR7, 5′-GTTCCAGC-ACAACGCCATCCATATTAG-3′ and cloned intothe pCRII TOPO vector (Invitrogen) followingmanufacturer’s instructions. To generate a GFPfusion-protein the stop codon of the full-length clone wasdeleted by PCR using the primers DanPres USP ApaI,

5′-TCCGGGCCCGCCATGGAGCACGTAACTGTTAG-CGAGG-3′, and DanPres DSP BamHI, 5′-CGCGGAT-CCGTGGATGTTTGGGTGGACGGGAAG-3′. The PCRproduct was cloned into the ApaI and BamHI sitesof the expression vector pEGFP-N3 (BD Biosciences).Correct orientation and reading frame were confirmed bysequence analysis.

Tissue preparation and histology

For immunohistochemistry, adult male fish wereover-anaesthetized with 0.02% 3-aminobenzoic acid ethylester until complete cessation of movements. The anteriorpart of the animal was subsequently dissected andfixed with 4% paraformaldehyde in PBS for 24 h at4◦C. The specimen was then rinsed with PBS anddecalcified with 0.5 m EDTA in PBS (pH 7.8) for 7 daysat 4◦C. Prior to embedding in TissueTek OCT compound(Sakura, Zoeterwounde, the Netherlands) the specimenwas immersed in 25% sucrose in PBS for 12 h at 4◦C.After freezing the specimen at −20◦C, cross sections,25 μm in thickness, were made on a Reichert-Jung 2800Frigocut E (Heidelberg, Germany), collected on Super-frost/Plus slides (Menzel, Braunschweig, Germany), driedfor 1 h at room temperature (RT) and stored at −20◦Cuntil used.

For standard histology, specimens were fixed andembedded as described above. Cryosections were cut to athickness of 10 μm and dried for 2 h. Sections were washedin deionized water, treated with potassium permanganateand oxalic acid, followed by staining with Eosin Y, OrangeG and toluidine blue, dehydration and mounting.

Generation of zprestin-specific antisera

To produce polyclonal primary antibodies againstzprestin (Accession No. NP 958881), rabbits wereimmunized with synthetic peptides correspondingto portions of the N- or C- terminal domainof the zebrafish prestin (N-terminal residues 32–46(LHKRKKTPKPYKLR) for antibody anti-N-zprestin andresidues 617–637 (NGPQKPKHVHTNGQMTEKHIE)for antibody anti-C-zprestin). Antibodies were affinitypurified against corresponding peptides.

Immunohistochemistry

Cross sections of the zebrafish ear were thawed anddried for 1 h at RT. The sections were briefly washedin PBS, permeabilized with 0.5% Triton X-100 in PBSfor 5 min, and blocked for 1 h with blocking solution(5% normal goat serum, 5% bovine serum albumin,in PBS). Goat-polyclonal anti-GFP antibodies werepurchased from Santa Cruz Biotechnology Inc. (sc-5384and sc-5385). Primary antibodies were diluted in thereaction buffer (2% NaCl, 0.5% normal goat serum,

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J Physiol 580.2 Characteristics of zebrafish prestin 453

0.25% bovine serum albumin, 0.1% Triton X-100 in PBS)and incubated overnight at 4◦C. Subsequently, sectionswere washed 3 × 10 min with PBS and incubated withthe secondary antibody AlexaFluor 633 goat anti-rabbit(Molecular Probes, Eugene, USA) at a dilution of 1 : 1000in reaction buffer for 1 h at RT. After a brief wash withPBS, the sections were counterstained with AlexaFluor 488phalloidin (Molecular Probes) at a concentration of2.5 mg ml−1, washed 3 × 10 min with PBS, mountedand imaged using a Zeiss 510 LSM Meta (Oberkochen,Germany) using a 40 × oil lens.

Riboprobe synthesis and in situ hybridization

zprestin specific riboprobes were in vitro transcribedfollowing standard protocols. Whole-mount in situhybridization procedures were performed accordinga modified method (Harland, 1991; Thisse et al.1993), with the following further modifications.The described hybridization buffer was replacedwith microarray-hyb-buffer (Amersham BioscienceBuckinghamshire, UK), and a different blocking solution(Roche, Grenzach-Wyhlen, Germany) was used.

Electrophysiology

For electrophysiological experiments, the expressionplasmids pEGFP-N1-rprestin or pEGFP-N3-zprestin weretransfected into CHO cells using the Metafectene(Biontex; Munich, Germany) or JetPEI (Polyplus trans-fection; Illkirch, France) transfection reagent according tothe manufacturers’ instructions. Whole-cell patch-clamprecordings were carried out at RT (22–24◦C) withan EPC10 patch clamp amplifier (Heka; Lambrecht,Germany) 24–48 h after transfection. Electrodes werepulled from quartz glass to resistances of 1.5–2.5 M�

and coated with Sylgard. Whole-cell series resistancesranged from 2 to 8 M�. Electrodes were filled with asolution containing (mm): 135 KCl, 3.5 MgCl2, 0.1 CaCl2,5 K2EGTA, 5 Hepes, 2.5 Na2ATP (pH 7.3). In someexperiments a simplified pipette solution (160 CsCl, 1Hepes, 1 K2EGTA, pH 7.3) was used, yielding identicalresults. Extracellular solution was (mm): 144 NaCl, 5.8KCl, 1.3 CaCl2, 0.9 MgCl2, 10 Hepes, 0.7 Na2HPO4, 5.6glucose, adjusted to pH 7.4 with NaOH.

Current and capacitance recordings were made with thePatchmaster acquisition software (Heka). For recordingsof charge transfer, all linear current components weresubtracted using a P/−8 protocol with negative P/overpulses at −100 mV for zprestin and positive P/over pulsesat +50 mV for rat prestin (rprestin) (for a more detaileddescription, please refer to Armstrong & Bezanilla, 1974).Currents were low-pass filtered at cut-off frequencies of3 or 5 kHz and sampled at 50 kHz. Kinetics of chargetransfer were determined by fitting the time courses of

the current relaxations with monoexponential curves. Forthese recordings, serial resistance (Rs) compensation wasapplied to speed up the voltage clamp yielding clamp timeconstants < 20 μs (Cm = 7–15 pF, Rs = 2.2–3.6 M�), andcurrents were low-pass filtered with a cut-off at 10 kHz.Charge was obtained by integration of the averaged trans-ient currents over time and plotted versus membranepotential. Charge–voltage plots were fitted with a two-stateBoltzmann function,

Q(V ) = Qmax

1 + e

(V − V1/2

−α

) (1)

where V is membrane potential, Qmax is maximum voltagesensor charge moved through the membrane electricalfield, V 1/2 is the voltage at half-maximum charge transferandα is the slope factor describing the voltage dependence.

Voltage-dependent capacitance was measured using thelock-in extension of Patchmaster in the sine+DC modewith current filter set to 10 kHz. Voltage ramps from−150 mV (or −130) to +100 mV (duration: 0.5 s) weresummed to the sinusoid command during capacitancemeasurements to obtain voltage dependence. Capacitancetraces were plotted versus membrane voltage and fittedwith the derivative of a Boltzmann function (Deak et al.2005),

C(V ) = Clin + Qmax

αe

(V − V1/2

α

)(1 + e

(V − V1/2

−α

))2(2)

where Clin is the residual linear membrane capacitance.Processing and fitting of data was performed with IgorPro(Wavemetrics, Lake Oswego, OR, USA). All values aregiven as mean ± standard deviation (s.d.).

Motility measurements

HEK293 cells were transiently transfected with theexpression plasmids for zebrafish prestin (pEGFP-N3-zprestin) or gerbil prestin (pEGFP-N2-gprestin; seeZheng et al. 2000 for details). To confirm propermembrane targeting of zprestin, transfected HEK cellswere probed for voltage-dependent charge movementsand a corresponding non-linear capacitance of thecell membrane with an AC technique that has beendescribed elsewhere (Santos-Sacchi et al. 1998). In brief, itutilized a continuous high-resolution (2.56 ms) two-sinevoltage stimulus protocol (10 mV peak at both 390.6and 781.2 Hz), with subsequent FFT-based admittanceanalysis. The high frequency sinusoids were super-imposed on voltage ramp stimuli. Capacitance data werefitted to the first derivative of a two-state Boltzmannfunction eqn (2). The resting membrane potentialsof zprestin-transfected HEK cells were measured bywhole-cell patch-clamp recordings in a separate subset ofcells that was not subjected to motility experiments. Values

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454 J. T. Albert and others J Physiol 580.2

were taken immediately after rupture of the cell membrane(−8.1 ± 4.1 mV, N = 17) and are thus likely to reflect themembrane potentials of unruptured cells (as used for themotility experiments). Electromotility was probed in themicrochamber configuration. A detailed description ofthe microchamber technique is given elsewhere (He et al.1994). In brief, a suction pipette or microchamber wasused to both mechanically hold the cell and deliver voltagecommands. Microchambers were fabricated from 1.5 mmthin-wall glass tubes (WPI, Inc.) by a Flaming/BrownMicropipette Puller (Sutter Instrument Company, ModelP-97) and heat-polished to an aperture diameter of∼13–15 μm. The microchamber, with a series resistance ofapproximately 0.3–0.4 M�, was mounted in an electrodeholder, which was held by a Leitz 3-D micromanipulator(Leitz, Germany). By moving the microchamber, cells inthe bath could be picked up easily. The inserted cell and themicrochamber formed a resistive seal (3–4 M�) that wasmechanically stable. Motility was measured and calibratedby a photodiode-based measurement system mounted onthe Leica microscope (Jia & He, 2005). The magnifiedimage of the edge of the cell was projected onto a photo-diode through a rectangular slit. Somatic length changes,evoked by voltage stimuli, modulated the light influx tothe photodiode. The photocurrent response was calibratedto displacement units by moving the slit a fixed distance(0.5 μm) with the image of the cell in front of the photo-diode. After amplification, the photocurrent signal was

Figure 1. Exonic and molecular analysis of zprestinA, comparison of the exonic structures of zebrafish prestin (GenBank Acc. No. AAH54604), rat prestin (AJ303372),and mouse pendrin (AF167411) using the exon structure of mouse prestin (AF529192) as a template. Exon sizesidentical to mouse prestin are highlighted in black, differing exons are marked in white. The first coding exonis boxed. Note that exon sizes of zprestin more closely resemble those of mpendrin, whereas the number oftranslated exons is identical to that of mammalian prestins. B, aligned hydrophobicity profiles of zebrafish andmouse prestin (Kyte–Doolittle, window = 9). The strong sequence similarity between mouse and zebrafish prestin(71.7%) extends to the hydrophobicity profile. The mutual agreement is particularly high in the central hydrophobiccore region, presumably corresponding to transmembrane segments.

low-pass filtered by an anti-aliasing filter before beingdigitized by a 16-bit A/D board (Digidata 1322A, AxonInstrument). The photodiode system had a cutoff (3 dB)frequency of 1100 Hz. The sampling frequency was 5 kHz.With an averaging of 400 trials and low-pass filteringset at 400 Hz, movement amplitudes as low as 3 nmcould be detected. The electrical stimulus was a sinusoidalvoltage burst of 100 ms duration. Voltage commands of380 mV (peak to peak) were used. Since the cells wereapproximately 60% inserted into the microchamber, theresultant voltage drops on the extruded segment wereestimated to be 60% of the voltage applied, or 228 mV(cf. Dallos et al. 1991).

Results

Exonic and molecular structure

Functional diversification within a given gene familyis deemed to rely on sequential, genomic duplicationevents (Hughes, 2005). Comparisons of genomic andmolecular structures can therefore be used to gain insightsinto the evolutionary relations within families of genes.We compared the exonic organization of zprestin withsome of its mammalian SLC26A homologues (rprestin,mpendrin; Fig. 1A) using the exon structure of mouseprestin as a template The sizes of exons 4–5, and 7–16are conserved between all four SLC26A members. The

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J Physiol 580.2 Characteristics of zebrafish prestin 455

lengths of exons 6, 17, 18 and 20, as well as thepresence of two untranslated exons are conserved amongthe mammalian prestin genes but not shared by zprestin andmpendrin. The termination of zprestin after exon 20, whichresults in 18 coding exons only, however, is a unique featureof zebrafish and mammalian prestin. Sequence alignmentbetween zprestin and mouse prestin (not shown) revealsan identity of 52.1%, and a strong similarity (i.e. sub-stitution with identical or physico-chemically similaramino acids; alignment calculated with the CustalWprogram (Chenna et al. 2003)) of 71.7%. Hydrophobicityanalysis indicates that this sequence similarity extendsto protein topology (Fig. 1B). The hydrophobic core

Figure 2. Expression analysis of zprestinA, in accordance with zprestin topology predictions GFP fluorescence is distributed around the plasma membraneof zprestin–GFP transfected CHO cells (Left). Without permeabilization, zprestin–GFP is not detected by antibodiesdirected against the C-terminal GFP tag of the zprestin–GFP fusion protein (upper middle). After permeabilization,immunohistochemistry with anti-GFP antibodies produces a clear signal (red) that localizes to the cell membrane(lower middle). Merged images of the GFP (green) and anti-GFP (red) fluorescence channels of non-permeabilized(upper right) and permeabilized (lower right) cells showing co-localization of GFP and anti-GFP fluorescent signalsin permeabilized cells (yellow in lower right). Cells were counterstained with DAPI (blue) (bars = 10 μm). B, zprestinimmunoreactivity co-localizes with the GFP signal in zprestin–GFP transfected HEK cells. C, in situ hybridizationwith a zprestin-specific riboprobe detects transcripts in the ear of wildtype larvae (arrow). No such signals weredetected after hybridization with the sense probe. D, double fluorescence labelling of F-actin (green, phalloidin)and zprestin (red) with anti-zprestin antibodies directed against the N-terminus (upper) and C-terminus (lower) ofthe protein. F-actin localizes to the stereocilia; zprestin appears distributed throughout the hair cells. No signalswere seen in peptide blocked controls (upper inset). Toluidine blue/Eosine Y/Orange G overview staining of a crosssection depicting a row of zebrafish hair cells and overlying remains of the otolith (lower inset). HC, hair cell; SC,stereocilia; Ot, otolith (all bars = 20 μm).

regions of mouse and zebrafish prestin, where sequenceconservation is especially high, closely match (Fig. 1B).Presumably, these central parts of the proteins correspondto regions of transmembrane segments (TMs). In line withthis, computational algorithms yield the same numberof TMs for both prestin orthologues (predictions ofweb-based programs: TMHMM ver.1.0: 10 TMs for bothmouse and zebrafish prestin, LOOPP ver.3.0: 12 TMs forboth mouse and zebrafish prestin; for more informationon prestin membrane topologies see Oliver et al. 2001;Zheng et al. 2001; Deak et al. 2005; Navaratnam et al. 2005).Although the actual membrane topology has not yet beensolved for any of the SLC26A members experimentally,

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456 J. T. Albert and others J Physiol 580.2

Figure 3. Voltage-dependent charge movement by zprestinA, transient currents measured from a CHO cell expressing zprestin–GFP (left) or rprestin–GFP (right) were elicitedby a series of depolarizing voltage steps of 3 ms duration and 10 mV increments starting at −30 mV (zprestin)and −130 mV (rprestin), as are illustrated in the lower panels (dashed lines indicate that larger voltage steps areomitted for clarity). Any residual linear – capacitive or resistive – currents were eliminated using a P/−8 protocol.Each trace is the average of 10 consecutive recordings. Note the inverse transient current at return to the initialvoltage. Non-transfected cells had no detectable transient currents (data not shown). B, charge measured from a

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J Physiol 580.2 Characteristics of zebrafish prestin 457

exonic and molecular analyses further document theclose relationship between zebrafish and mammalianprestin, suggesting that zebrafish and mammalian prestinevolved from a prestin-like ancestor in the course of SLC26evolution.

Heterologously expressed zprestin is targeted to theplasma membrane

The motor function of mammalian prestin cruciallyrelies on its integration into the cell membrane. Suchmembrane localization was observed for zprestin whena zprestin–GFP fusion protein (C-terminal GFP) wasexpressed in HEK cells (Fig. 2B). Cells transfectedwith the empty GFP expression vector displayed onlycytoplasmic fluorescence (not shown). Labelling ofzprestin-transfected cells with a GFP-specific antibody wasonly successful after permeabilization of the cells (Fig. 2A,lower middle and right). Apparently, zprestin, as alsosuggested by topology predictions, shares the intracellularlocalization of the C-terminus previously determined formammalian prestin (Ludwig et al. 2001; Zheng et al. 2001).

zprestin is expressed in hair cells of the zebrafish ear

To test whether zprestin occurs in hair cells of the zebrafishear, its expression was probed by in situ hybridizationand immunohistochemical analysis. Whole-mount in situhybridizations with a zprestin-specific riboprobe detectedtranscripts in the ears of wildtype larvae (Fig. 2C, lower).Hybridization with the sense probe did not reveal anypositive signals (Fig. 2C, upper). Two antibodies, onedirected against the N-terminus and the other directedagainst the C-terminus of the protein, were generatedand used to assay the localization of zprestin in haircells of the zebrafish ear. Hair cells were identified

zprestin-expressing cell at the end of a voltage step (OFF) is plotted against the charge at the onset of the step (ON).A line of unity slope well described the relation, indicating equality of ON- and OFF-charges. C, charge measuredas in A is plotted against step voltage. Data from individual cells were fitted with a 2-state Boltzmann functionand normalized to saturating charge. The plot shows mean normalized data (± S.D.) from 5 cells (zprestin) and 4cells (rprestin). Continuous lines show the best fits to the averaged data with V1/2 = 94.7 mV, α = 53.2 mV forzprestin and V1/2 = −74.6 mV, α = 34.0 mV for rprestin. D, NLC measured from the same CHO cell expressingzprestin–GFP with the different stimulus frequencies indicated. Continuous lines are the best fits to the firstderivative of a 2-state Boltzmann function. For fit results, see Results. Capacitance recordings are presented withlinear membrane capacitance subtracted. E, NLC measured from a rprestin–GFP-expressing cell as described in (C).Fits (continuous lines) yielded peak NLC of 0.75 ± 0.35 pF, Qmax of 111 ± 47 fC, V1/2 of −69 ± 14 mV and slopevalues of 35.5 ± 1.2 mV (at 2 kHz; n = 6). Inset shows the lack of detectable NLC in a representative control cell(recordings for all four frequencies are superimposed). F, frequency dependence of NLC in cells expressing zprestin:peak NLC obtained with the various stimulation frequencies was normalized to peak NLC at 0.5 kHz stimulationfrequency for each cell (n = 7). G, transient currents measured in response to voltage steps (from −140 to −20 mV,rprestin; from −80 to +120 mV, zprestin). Traces are averaged from 4 and 10 repetitive recordings, respectively, andnormalized to peak current. Superimposed curves show monoexponential fits to the current relaxation yieldingtime constants (τON) of 26 μs (rprestin) and 120 μs (zprestin). For recording conditions, see Methods. H, timeconstants of charge transfer for a range of step potentials were obtained as in G. Data are from n = 3 and 6 cellsexpressing rprestin and zprestin, respectively.

by phalloidin counter staining of their actin-rich hairbundles. The two antibodies produced signals in auditoryhair cells (Fig. 2D). These signals were not seen inpeptide-blocked controls (Fig. 2D, upper inset). Inter-estingly, the antibody stainings indicate that zprestinis distributed throughout the hair cells, hampering theestimation of the degree of membrane localization. Thissubcellular staining pattern contrasts with the exclusivemembrane localization of prestin in mammalian OHCs,but resembles the somatic localization of prestin reportedfor mammalian vestibular hair cells (Adler et al. 2003).Since structural predictions and heterologous expressionanalysis unambiguously identify both mammalian andzebrafish prestin as membrane proteins, the cytoplasmicdistributions are unexpected. Whether they are related toprotein recycling/processing or result from a degradationof membrane proteins in the course of tissue preparation,remains unclear.

zprestin confers NLC to transfected cells

A hallmark of electromotility mediated by mammalianprestin is a voltage-dependent transfer of charges acrossthe cell membrane that is associated with electromotility.Such charge transfer can be measured directly as a trans-ient current at the onset of a voltage pulse (similar to anion channel’s gating current) or as the resultant non-linearcapacitance (NLC) of the cell membrane (Santos-Sacchi,1991). We tested whether a similar charge transferis mediated by zprestin. CHO cells transfected withGFP-tagged zprestin and displaying strong membranefluorescence were selected for the electrophysiologicalmeasurements. When depolarized by a voltage step,zprestin-transfected cells displayed a transient current thatincreased with the amplitude of the voltage step (Fig. 3A,left). Repolarization produced an inverse current of similar

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458 J. T. Albert and others J Physiol 580.2

amplitude. Integration of these transient currents overtime yielded the amount of translocated charge. Thecharges translocated upon the onset and offset of thestimulus were equal, demonstrating a full reversibilityof the charge movement (Fig. 3B). The relation betweencharge and step potential was well described by atwo-state Boltzmann function (Fig. 3C). This behaviourqualitatively agrees with that of mammalian prestin(Fig. 3A, right), yet quantitative differences exist: first,charge transfer by zprestin occurred at much more positivepotentials: voltage at half-maximal charge transfer, V 1/2,was +96 ± 27 mV (n = 5) as opposed to −74 ± 9 mV forrprestin; second, voltage dependence was less pronouncedfor zprestin, where Boltzmann fits yielded about 53 mV foran e-fold change in charge transfer (slope α ≈ 34 mV forrprestin; Fig. 3B). Saturated charge transfer obtained fromthe fits was 65 ± 26 fC for zprestin and 95 ± 27 fC for ratprestin. Membrane fluorescence in zprestin–GFP trans-fected cells was usually somewhat stronger than in cellstransfected with GFP-tagged rprestin as assessed visuallyby wide field fluorescence microscopy. We thus did notfind any evidence for a substantially lower expression levelof zprestin compared with its mammalian counterpart.

Given that zprestin mediates charge transfer, we nexttested for a non-linear capacitance (NLC) of the cellmembrane, which directly results from voltage-dependentcharge transfer and is widely used to characterize thefunction of mammalian prestin (for a review, see Dallos& Fakler, 2002). As shown in Fig. 3D, zprestin-tranfectedcells displayed a NLC similar to the bell-shapedNLC conferred by mammalian prestin (Fig. 3E). NLCapparently resulted from the insertion of zprestin intothe plasma membrane of the cells, as it was neverobserved in non-transfected cells (Fig. 3E, inset). Theamplitude of NLC generated by zprestin strongly decreasedwith increasing stimulus frequency used for detection,vanishing into the noise floor for frequencies greater thanca. 5 kHz (Fig. 3D and F). Such frequency dependenceindicates that the charge transfer underlying the NLC istoo slow to follow high-frequency stimulation. A dropof NLC amplitudes as the stimulus sinusoids approachthe charge transition rates has been observed in OHCs,where the effect was found to occur above 10 kHz (Gale& Ashmore, 1997). In agreement with these observations,NLC generated by rprestin showed up in our experimentsat all stimulus frequencies (Fig. 3E), whereby only a slighthigh-frequency drop of the NLC amplitude was found. Asmammalian prestin operates without attenuation up to atleast 20 kHz (Ludwig et al. 2001), this slight drop is likely toreflect the frequency limits of our instrumentation ratherthan those of prestin-mediated charge tranfer.

To test more directly whether the charge-transferkinetics of zprestin is slower than that of mammalianprestin, we measured current relaxations in response tovoltage steps in transfected cells. For rprestin-mediated

charge transfer, time constants of charge relaxation wereabout 25 μs, reflecting the temporal limitations set bylow-pass filtering by the amplifier and the voltage-clampspeed. For zprestin-mediated charge transfer, currenttransients were substantially slower. Time constants(around 130 μs, Fig. 3H) were well above the clamp andfilter time constants and, thus, are likely to reflect the actualkinetics of the charge transfer. A time constant of 130 μscorresponds to a corner frequency of 1.2 kHz, which isin reasonable agreement with the observed roll-off of theNLC in the kilohertz range.

Voltage dependence of NLC of mammalian prestinis well characterized by the first derivative of theBoltzmann function that describes charge movement(Santos-Sacchi, 1991). Similarly for zprestin, NLC wasbell-shaped and was described by a Boltzmann derivative(Fig. 3D). However, because only part of the voltagerange (below 100 mV) covered by NLC was accessible tocapacitance measurements at a useful recording quality,the fit parameters were somewhat noisy and someuncertainty remains as to whether a 2-state Boltzmannfunction is the most appropriate description of the voltagedependence of zprestin. Parameter values yielded bythe fits, however, were in reasonable agreement withthose obtained from direct measurements of chargemovement: for stimulus frequencies of 0.5, 1 and2 kHz, Boltzmann fits yielded V 1/2 values of 84 ± 21,85 ± 16 and 93 ± 13 mV, slopes of 79 ± 15, 80 ± 14 and71 ± 20 mV, and peak NLCs of 0.43 ± 0.16, 0.26 ± 0.12and 0.12 ± 0.07 pF, respectively (n = 7). Hence, chargemovement characterizes zprestin as a voltage-sensitiveSLC26A member, the voltage dependence of which israther broad and shifted to potentials largely beyondphysiological membrane voltages. According to our data,the molecular kinetics underlying the charge transferconferred by zprestin is substantially slower than that ofmammalian prestin.

zprestin NLC is not associated with motile responses

zprestin is expressed in hair cells and mediates NLC. Asthe NLC of mammalian prestin is mechanistically linkedto electromotility, zprestin transfected HEK cells wereprobed for motility using a microchamber configuration.For detachment, cells were treated with Ca2+-free medium.Whereas the cells were initially spherical, preventingeffective surface area changes at constant volume, drawingthem into a suction pipette, or microchamber (Dalloset al. 1991) forced them into a dumbbell or hour-glassshape (Fig. 4A). Sinusoidal voltage bursts of ∼168 mVeffective peak-to-peak amplitude and 100 ms durationwere delivered to the cells via the microchamber. Wemeasured 51 randomly selected zprestin-transfected cells,none of which showed any measurable motile response

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J Physiol 580.2 Characteristics of zebrafish prestin 459

(an example of lack of motility is shown in Fig. 4B,upper). When HEK cells were transfected with gerbilprestin (gprestin), in contrast, motile responses wereobtained from 7 of 31 cells tested (Fig. 4, middle).Additional 23 zprestin–GFP transfected cells, all showingclear membrane labelling, were probed for electromotilityusing larger sinusoidal voltage bursts of 228 mV (peakto peak) and stimulation frequencies of 100 Hz and500 Hz (not shown). Given a resting membrane potentialof around −10 mV, a sinusoidal stimulus of 228 mVpeak-to-peak amplitude would result in membrane voltageswings from −124 mV to +104 mV. Such large potentialchanges should be sufficient to evoke motility (if any)even though the V 1/2 of the zprestin-mediated NLC isshifted towards more positive potentials as compared withmammalian prestin. Signs of electromotility, however,were not detected in any of the zprestin-transfected cells.

Discussion

Zebrafish prestin is the closest reported relative ofmammalian prestin. Like its mammalian orthologue,zprestin is expressed in hair cells of the ear and confersNLC to the membranes of transfected cells, similar tothe characteristic electrogenic charge movement thataccompanies the prestin-mediated somatic electromotilityof mammalian OHCs. Comparable voltage-dependentcharge movements are absent from cells transfected withthe closest paralogues of mammalian prestin, SLC26A4(pendrin; Zheng et al. 2000) and SLC26A6 (Oliveret al. 2001), identifying zprestin as the first SLC26member besides mammalian prestin that generates NLC.Notwithstanding these parallels, it seems unlikely thatzprestin gives rise to a somatic motility of hair cells inthe zebrafish ear. First, the distributed expression patternof zprestin in zebrafish hair cells is different from theexclusive membrane localization of prestin in mammalianOHCs, but rather resembles the cytoplasmic distributionreported for prestin in mammalian vestibular hair cells thatneither display somatic electromotility nor NLC. Second,although our expression analysis and electrophysiologicaldata show that zprestin properly localizes to the cellmembrane upon heterologous expression, it nonethelessfails to generate electromotile responses of the trans-fected cells. Hence, though displaying a prestin-like voltagesensitivity, zprestin does not seem to be a prestin-likemotor, supporting the general view that a prestin-mediatedsomatic electromotility is a unique feature of mammalianOHCs.

What might be the reason for the dissociation of NLCand electromotility in zprestin-transfected cells? The areamotor principle (Iwasa, 2001) generally thought to under-lie the prestin-mediated electromotility posits that thisprotein undergoes two sequential molecular events: First a

Figure 4. zprestin-transfected cells do not display somaticelectromotilityA, transfected HEK cells were probed for electromotility in amicrochamber configuration, which allows simultaneous electricalstimulation and displacement recording. The rectangular slit indicatesthe area that was projected onto a photodiode for motilitymeasurements. The stimulus command voltage (Vc) was appliedbetween bath and microchamber compartment. Proportional to theirdegree of insertion into the microchamber, the cell’s resultantmembrane voltage swing was calculated to be ∼60% of Vc. B, lack ofsomatic motility in a zprestin-transfected HEK cell (upper trace). A380 mV (peak-to-peak) voltage burst (lower trace) was applied via themicrochamber. The voltage drop on the extruded segment wasestimated to be 228 mV (peak-to-peak). The cell’s resting membranepotentials normally were around −10 mV. Therefore, the effectivemembrane voltage was estimated to vary from −124 to +104 mV.Note that in contrast, robust electromotiltiy was observed ingprestin-transfected cells (middle trace). C, HEK cells transfected withzprestin display NLC confirming proper membrane targeting ofzprestin in HEK cells. Voltage-dependent capacitance was measuredusing a 2-sine-wave method.

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460 J. T. Albert and others J Physiol 580.2

charged voltage sensor responds to changes in membranepotential by moving across the membrane (Sensing)(Oliver et al. 2001; for a review, see Dallos & Fakler,2002). This, in turn, must trigger a gross conformationalchange of the molecule, resulting in the generationof force (Acting). Finally, these elementary electro-mechanical responses of large arrays of prestin moleculestranslate into whole-cell motility. This latter translationfrom molecular to cellular behaviour may involve inter-actions between prestin monomers (Navaratnam et al.2005) and/or interactions between prestin and the cyto-skeleton (Coupling). Our data show that zprestin respondsto changes in membrane potential with the translocationof charge. This ‘Sensing’ capacity of zprestin, despitequantitative differences, qualitatively resembles that ofmammalian prestin. Whether the charge movementsgenerated by zprestin and mammalian prestin result fromthe same molecular events remains to be examined.The absence of cellular electromotility in the presenceof voltage-dependent charge movements observed forzprestin suggests that either the voltage-induced chargetransfer is not followed by equivalent conformationalreorientations (no ‘Acting’), or, alternatively, that theseconformational changes are not efficiently coupled intothe overall mechanics of the cell (no ‘Coupling’).

As zprestin does not seem to be a motor, what then isits function? With the exception of mammalian prestin,members of the SLC26 protein family have been shown tobe bona fide anion transporters (for a review, see Mount& Romero, 2004). Based on the results presented here, thetransport function of zprestin has been tested in a parallelstudy, revealing that this protein is an electrogenic trans-porter that exchanges sulphate (or oxalate) for chloride(Schaechinger & Oliver, unpublished results). Chlorideions, in turn, have been shown to interfere with boththe voltage dependence and the motor function of themammalian prestin motor (Oliver et al. 2001), suggestingthat the molecular events that lead to electromotilityare mechanistically related to anion transport processes(Oliver et al. 2001, 2006). This idea has recently foundsupport by modelling work suggesting that the motorfunction of mammalian prestin is driven by an electro-genic anion transport cycle (Muallem & Ashmore, 2006).Mediating anion transport and voltage-dependent chargemovements (although quantitatively distinct from thoseof mammalian prestin) but no associated electromotileactivity, zprestin can thus be regarded as the ‘missing link’that may help us understand the sequence of events thathas led to the emergence of a novel type of motor protein inthe course of SLC26 evolution. How the zprestin-mediatedvoltage sensitivity described here relates to anion transportand whether it affects the function of zebrafish hair cellsunder physiological conditions remains to be seen.

The discovery of voltage-dependent charge movementsconferred by zprestin, together with the demonstration

of its electrogenic transport function, make this proteina good candidate for exploring the common grounds ofcharge movement, electromotility and anion transport. Weanticipate that using zprestin as a non-motor backgroundwill help to trace the molecular peculiarities that bringabout the motor function of mammalian prestin.

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Acknowledgements

We thank Sigrun Korsching for providing animals (‘Thanks for

all the fish’) and Karin Rohbock for technical assistance. This

work was supported by grants from the Volkswagen-Foundation

(J.T.A., O.H., M.C.G.), the Deutsche Forschungsgemeinschaft

DFG Kn316/4-1, the Interdisciplinary Center of Clinical

Research Tubingen (M.K.), the National Institute of Health

NIH R01 DC 004696 (D.Z.Z.H.), and the European Union,

LSHG-CT-20054-512063 (‘Eurohear’, D.O.).

C© 2007 The Authors. Journal compilation C© 2007 The Physiological Society