Loss of KCNJ10 protein expression abolishes endocochlear potential and causes deafness in Pendred syndrome mouse model
© Wangemann et al; licensee BioMed Central Ltd. 2004
Received: 16 April 2004
Accepted: 20 August 2004
Published: 20 August 2004
Pendred syndrome, a common autosomal-recessive disorder characterized by congenital deafness and goiter, is caused by mutations of SLC26A4, which codes for pendrin. We investigated the relationship between pendrin and deafness using mice that have (Slc26a4+/+) or lack a complete Slc26a4 gene (Slc26a4-/-).
Expression of pendrin and other proteins was determined by confocal immunocytochemistry. Expression of mRNA was determined by quantitative RT-PCR. The endocochlear potential and the endolymphatic K+ concentration were measured with double-barreled microelectrodes. Currents generated by the stria marginal cells were recorded with a vibrating probe. Tissue masses were evaluated by morphometric distance measurements and pigmentation was quantified by densitometry.
Pendrin was found in the cochlea in apical membranes of spiral prominence cells and spindle-shaped cells of stria vascularis, in outer sulcus and root cells. Endolymph volume in Slc26a4-/- mice was increased and tissue masses in areas normally occupied by type I and II fibrocytes were reduced. Slc26a4-/- mice lacked the endocochlear potential, which is generated across the basal cell barrier by the K+ channel KCNJ10 localized in intermediate cells. Stria vascularis was hyperpigmented, suggesting unalleviated free radical damage. The basal cell barrier appeared intact; intermediate cells and KCNJ10 mRNA were present but KCNJ10 protein was absent. Endolymphatic K+ concentrations were normal and membrane proteins necessary for K+ secretion were present, including the K+ channel KCNQ1 and KCNE1, Na+/2Cl-/K+ cotransporter SLC12A2 and the gap junction GJB2.
These observations demonstrate that pendrin dysfunction leads to a loss of KCNJ10 protein expression and a loss of the endocochlear potential, which may be the direct cause of deafness in Pendred syndrome.
Pendred syndrome is a relatively common autosomal-recessive disorder characterized by deafness and goiter . The syndrome is caused by mutations of the PDS gene SLC26A4, which codes for the protein pendrin . Deafness is congenital and generally profound although sometimes late in onset and provoked by light head injury. Vestibular dysfunction is uncommon. Goiter is variable and generally develops around puberty . The cause of goiter appears to be an impairment of iodide fixation in the follicular lumen due to a reduced rate of iodide transport across the apical membrane of thyroid gland epithelial cells . A positive perchlorate discharge test and an enlarged vestibular aqueduct appear to be the most reliable clinical signs of Pendred syndrome .
Pendrin is an anion exchanger that can transport Cl-, I-, HCO3 - and formate [6, 7]. Expression has been found in the inner ear and thyroid gland consistent with the clinical signs of deafness and goiter [2, 3, 8]. In addition, pendrin expression has been found in the kidney , mammary gland , uterus , testes  and placenta . No expression was found in fetal or adult brain, consistent with a peripheral cause of deafness [2, 11].
Expression of pendrin mRNA in the inner ear has been found in several places including the cochlea, the vestibular labyrinth and the endolymphatic sac . The precise location of pendrin protein expression, however, has not yet been determined. The variability of deafness in Pendred syndrome and the observation that deafness is sometimes late in onset suggest that pendrin dysfunction may not be the direct cause of deafness. It is conceivable that pendrin dysfunction favors changes in the expression levels of proteins that are critical for the maintenance of the hearing function. Detailed studies aimed at identifying the direct cause of deafness in Pendred syndrome have recently become possible due to the generation of a pendrin-specific polyclonal antibody  and the development of Slc26a4-/- mice, which bear a targeted disruption of the mouse Slc26a4 gene . The aim in the present study was to determine the location of pendrin and the cause of deafness in Slc26a4-/- mice.
The endocochlear potential and the endolymphatic and perilymphatic K+ concentrations were measured in young adult mice (1–4 month of age) that either have (Slc26a4+/+) or lack (Slc26a4-/-) a functional gene for pendrin . The mouse strain 129Sv/Ev (Taconic, Germantown, NY) was chosen as the source of Slc26a4+/+ controls, since Slc26a4-/- mice were propagated in the this strain and generated using a stem cell line derived from 129Sv/Ev. Slc26a4+/+ and Slc26a4-/- were agouti. They did not differ in coat color. Differences in pigmentation were verified using coisogenic age-matched Slc26a4+/+ and Slc26a4-/- that were bred in parallel.
Expression of key proteins involved in the generation of the endocochlear potential and the transport of K+ was studied using confocal immunocytochemistry and quantitative RT-PCR. K+ secretion and the generation of the endocochlear potential were measured using electrophysiological techniques. All experiments were approved by the Institutional Animal Care and Use Committee of Kansas State University.
Animals were deeply anesthetized with sodium pentobarbital (100 mg/kg i.p.) and transcardially perfused with a Cl--free phosphate-buffered Na-gluconate solution containing 4% paraformaldehyde. Temporal bones were removed and cochleae fixed by perilymphatic perfusion, decalcified in EDTA, processed through a sucrose gradient and infiltrated with polyethylene glycol. Mid-modiolar cryosections (12 μm, CM3050S, Leica, Nussloch, Germany) were blocked in PBS with 0.2% Triton-X (PBS-TX) and 10% bovine serum albumin. Slides were incubated overnight at 4°C with primary antibodies in PBS-TX with 1–3% BSA [rabbit anti-pendrin, 1:500 (h766–780); rat anti-ZO-1, 1:100 (Chemicon, Temecula, CA); goat anti-KCNQ1, 1:200 (C20, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-KCNE1, 1:200 (Alomone, Jerusalem, Israel); rabbit anti-KCNJ10, 1:300 (Alomone); rabbit anti-SLC12A2, 1:100 (Chemicon); and rabbit anti-connexin 26, 1:100 (Zymed, San Francisco, CA)]. Slides were washed in PBS-TX and incubated for 1 h at 25°C with appropriate secondary antibodies at a 1:1000 dilution in PBS-TX with 1–3% BSA [donkey anti-rabbit Alexa 488, chicken anti-rat Alexa 594, and chicken anti-goat Alexa 594 (Molecular Probes, Eugene, OR)]. Actin filaments were visualized by staining with Alexa 488 conjugated phalloidin (Molecular Probes). After incubation, slides were washed with PBS-TX, mounted with FluorSave (Calbiochem, La Jolla, CA), and viewed by confocal microscopy (LSM 5 Pascal or LSM 510 Meta, Carl Zeiss, Jena or Göttingen, Germany). Laser scanning brightfield images were collected to document structural preservation, for morphometric analysis and for analysis of pigmentation.
gag gtt cga aga cga tca ga (sense)
83.2 ± 02°C
1.89 ± 0.02
tcg ctc cac caa cta aga ac (antisense)
tgg tgt ggt gtg gta tct gg (sense)
83.2 ± 02°C
1.86 ± 0.02
tga agc agt ttg cct gtc ac (antisense)
ttt gtt cat ccc cat ctc ag (sense)
82.5 ± 02°C
1.85 ± 0.02
gtt gct ggg tag gaa gag (antisense)
ccc gga aac cct tct gtg tc (sense)
83.2 ± 02°C
1.87 ± 0.01
aaa gat gag cac cag gaa cc (antisense)
Template molecules (T) were quantified according to T = P / (F ^Ct) where P is the number of product molecules, F is the fidelity of the reaction and Ct is the cycle at which the number of product molecules reaches a chosen threshold . Fidelity (F) was obtained from the slope (S) of the log-linear phase of the growth curve via a best-fit fifth-order polynomial: F = 7.39 + 3.80 × S + 1.05 × S2 + 0.15 × S3 + 11.38 × 10-3 * S4 + 3.39 × 10-4 × S5. The number of product molecules at threshold (PCt) was determined by amplification of known amounts of 18S rRNA according to PCt = T × F ^Ct. Quantifications of 18S rRNA were used to compare tissue amounts. Genomic contamination of inner ear samples was assessed to be <0.02% by omission of the RT step.
In vitro electrophysiology
Animals were deeply anesthetized with sodium pentobarbital (100 mg/kg i.p.). Stria vascularis without spiral ligament was obtained by microdissection. Currents generated by the stria marginal epithelium were recorded . A Pt-Ir wire microelectrode with a Pt-black tip was positioned 20–30 μm from the apical surface of the epithelium and vibrated at 200–400 Hz by piezo-electric bimorph elements (Applicable Electronics, Forest Dale, MA; ASET version 1.05, Science Wares, East Falmouth, MA). A Pt-black electrode (26-gauge) served as reference in the bath chamber. The signals from the phase-sensitive detectors were digitized (16 bit) at a rate of 0.5 Hz. The output was expressed as current density at the electrode.
In situ electrophysiology
Animals were anesthetized with inactin (thiobutabarbital sodium salt, 140 mg/kg ip). The endocochlear potential and the endolymphatic [K+] were measured with double-barreled microelectrodes . Measurements were made in the basal turn by a round-window approach through the basilar membrane and in the apical turn after thinning the bone over the stria vascularis and picking a small hole (~30 μm). K+-selective electrodes were calibrated in solutions of constant cation (K+ and Na+) concentration of 150 mM. The minor selectivity of the K+ electrodes for Na+ produced a nonlinearity in the calibration curve, which was closely fitted by the Nicolski equation using nonlinear curve-fitting software (OriginLab, Northampton, MA): V = Vi + S × log ([K+] + A × [Na+]), where Vi is an offset term, S is slope, A is selectivity, and [Na+] is Na+ concentration. Calibrations were made immediately after withdrawal of the electrodes from the cochlea. Plasma K+ concentrations were obtained using a blood analyzer (Stat Profile M, Nova Biomedical, Waltham, MA).
Data are presented as mean ± sem; n denotes the number of experiments. Differences were considered significant when p < 0.05.
Results and Discussion
The extent of pendrin expression in spiral prominence cells and stria vascularis was determined by labeling KCNQ1, a K+ channel that is expressed in strial marginal cells, and ZO-1, a tight junction protein that labels basal cells and thereby delineates the boundaries of stria vascularis. Dual labeling experiments demonstrated that pendrin is expressed in spindle-shaped cells, which are surface epithelial cells in stria vascularis adjacent to strial marginal cells near the borders of both the spiral prominence and Reissner's membrane (Fig. 1c).
In situ hybridization in the vestibular labyrinth suggested that pendrin mRNA is expressed in non-sensory cells . Using confocal immunocytochemistry on cryosections, we observed strong expression of the pendrin protein in the apical membrane of vestibular transitional cells in the utricle and ampullae (Fig. 1g,1h,1i). Dual labeling with KCNQ1 demonstrated that pendrin expression was clearly limited to vestibular transitional cells and did not extend to other non-sensory cells such as vestibular dark cells, which were clearly identified by the expression of KCNQ1 and KCNE1 in their apical membranes.
The endocochlear potential is generated by stria vascularis in the lateral wall of the cochlea [17, 23]. The potential is generated across the basal cell barrier of stria vascularis by the K+ channel KCNJ10 located in intermediate cells , which are connected to basal cells by a high density of gap junctions . Marginal cells of stria vascularis, which form the barrier toward endolymph, transport K+ from the intrastrial space into endolymph and keep the K+ concentration low adjacent to the KCNJ10 K+ channel . To determine the cause of the loss of the endocochlear potential in Slc26a4-/- mice, we first determined whether intermediate cells are present in stria vascularis, since a loss of intermediate cells is known to lead to a loss of the endocochlear potential [27, 28]. Intermediate cells of stria vascularis were visualized by their pigmentation. Pigmentation was present in stria vascularis of Slc26a4-/- mice (Fig. 2b), which suggests that intermediate cells are present. Interestingly, pigmentation of stria vascularis was much stronger in Slc26a4-/- than in Slc26a4+/+ mice.
Pendrin-expressing surface epithelial cells in the spiral prominence region are located in an area where basal cells, which are interconnected by tight junctions, form additional tight junctions with surface epithelial cells . A discontinuity of this complex junction in Slc26a4-/- mice would explain the absence of the endocochlear potential. To evaluate the presence of this connection, we determined by confocal immunocytochemistry the expression of ZO-1 and of F-actin, which associate with tight junction complexes. ZO-1 and F-actin expression revealed a continuous layer of basal cells, including a junction of basal cells with surface epithelial cells in Slc26a4-/- mice, as observed in normal mice (Fig. 1c,1d,1e,1f,6,7). These observations make it unlikely that the primary cause of the loss of the endocochlear potential is a compromise in the basal cell barrier.
This work was supported by NIH-R01-DC01098 (PW), NIH-R01-DC00212 (DCM), NIH-R01-DK52935 (SMW) and Core facilities funded by NIH-P20-RR017686 (Confocal Microfluorometry and Microscopy Core, Molecular Biology Core) are gratefully acknowledged.
- Pendred V: Deaf-mutism and goitre. Lancet. 1896, 11: 532-10.1016/S0140-6736(01)74403-0.View Article
- Everett LA, Glaser B, Beck JC, Idol JR, Buchs A, Heyman M, Adawi F, Hazani E, Nassir E, Baxevanis AD, Sheffield VC, Green ED: Pendred syndrome is caused by mutations in a putative sulphate transporter gene (PDS). Nat Genet. 1997, 17: 411-422. 10.1038/ng1297-411.PubMedView Article
- Royaux IE, Suzuki K, Mori A, Katoh R, Everett LA, Kohn LD, Green ED: Pendrin, the protein encoded by the Pendred syndrome gene (PDS), is an apical porter of iodide in the thyroid and is regulated by thyroglobulin in FRTL-5 cells. Endocrinology. 2000, 141: 839-845. 10.1210/en.141.2.839.PubMedView Article
- Bidart JM, Mian C, Lazar V, Russo D, Filetti S, Caillou B, Schlumberger M: Expression of pendrin and the Pendred syndrome (PDS) gene in human thyroid tissues. J Clin Endocrinol Metab. 2000, 85: 2028-2033. 10.1210/jc.85.5.2028.PubMed
- Reardon W, OMahoney CF, Trembath R, Jan H, Phelps PD: Enlarged vestibular aqueduct: a radiological marker of Pendred syndrome, and mutation of the PDS gene. QJM. 2000, 93: 99-104. 10.1093/qjmed/93.2.99.PubMedView Article
- Scott DA, Wang R, Kreman TM, Sheffield VC, Karniski LP: The Pendred syndrome gene encodes a chloride-iodide transport protein. Nat Genet. 1999, 21: 440-443. 10.1038/7783.PubMedView Article
- Scott DA, Karniski LP: Human pendrin expressed in Xenopus laevis oocytes mediates chloride/formate exchange. Am J Physiol Cell Physiol. 2000, 278: C207-C211.PubMed
- Everett LA, Morsli H, Wu DK, Green ED: Expression pattern of the mouse ortholog of the Pendred's syndrome gene (Pds) suggests a key role for pendrin in the inner ear. Proc Natl Acad Sci U S A. 1999, 96: 9727-9732. 10.1073/pnas.96.17.9727.PubMedPubMed CentralView Article
- Royaux IE, Wall SM, Karniski LP, Everett LA, Suzuki K, Knepper MA, Green ED: Pendrin, encoded by the Pendred syndrome gene, resides in the apical region of renal intercalated cells and mediates bicarbonate secretion. Proc Natl Acad Sci U S A. 2001, 98: 4221-4226. 10.1073/pnas.071516798.PubMedPubMed CentralView Article
- Rillema JA, Hill MA: Prolactin regulation of the pendrin-iodide transporter in the mammary gland. Am J Physiol Endocrinol Metab. 2003, 284: E25-E28.PubMedView Article
- Suzuki K, Royaux IE, Everett LA, Mori-Aoki A, Suzuki S, Nakamura K, Sakai T, Katoh R, Toda S, Green ED, Kohn LD: Expression of PDS/Pds, the Pendred syndrome gene, in endometrium. J Clin Endocrinol Metab. 2002, 87: 938-941. 10.1210/jc.87.2.938.PubMedView Article
- Lacroix L, Mian C, Caillou B, Talbot M, Filetti S, Schlumberger M, Bidart JM: Na+/I- symporter and Pendred syndrome gene and protein expressions in human extra-thyroidal tissues. Eur J Endocrinol. 2001, 144: 297-302.PubMedView Article
- Bidart JM, Lacroix L, Evain-Brion D, Caillou B, Lazar V, Frydman R, Bellet D, Filetti S, Schlumberger M: Expression of Na+/I- symporter and Pendred syndrome genes in trophoblast cells. J Clin Endocrinol Metab. 2000, 85: 4367-4372. 10.1210/jc.85.11.4367.PubMed
- Everett LA, Belyantseva IA, Noben-Trauth K, Cantos R, Chen A, Thakkar SI, Hoogstraten-Miller SL, Kachar B, Wu DK, Green ED: Targeted disruption of mouse Pds provides insight about the inner-ear defects encountered in Pendred syndrome. Hum Mol Genet. 2001, 10: 153-161. 10.1093/hmg/10.2.153.PubMedView Article
- Pfaffl M, Meyer HH, Sauerwein H: Quantification of insulin-like growth factor-1 (IGF-1) mRNA: development and validation of an internally standardised competitive reverse transcription-polymerase chain reaction. Exp Clin Endocrinol Diabetes. 1998, 106: 506-513.PubMedView Article
- Lee JH, Chiba T, Marcus DC: P2X2 receptor mediates stimulation of parasensory cation absorption by cochlear outer sulcus cells and vestibular transitional cells. J Neurosci. 2001, 21: 9168-9174.PubMed
- Marcus DC, Wu T, Wangemann P, Kofuji P: KCNJ10 (Kir4.1) potassium channel knockout abolishes endocochlear potential. Am J Physiol Cell Physiol. 2002, 282: C403-C407.PubMedView Article
- Okamura HO, Sugai N, Suzuki K, Ohtani I: Enzyme-histochemical localization of carbonic anhydrase in the inner ear of the guinea pig and several improvements of the technique. Histochem Cell Biol. 1996, 106: 425-430. 10.1007/s004180050060.PubMedView Article
- Lim DJ, Karabinas C, Trune DR: Histochemical localization of carbonic anhydrase in the inner ear. Am J Otolaryngol. 1983, 4: 33-42.PubMedView Article
- Vetter DE, Mann JR, Wangemann P, Liu Z, McLaughlin KJ, Lesage F, Marcus DC, Lazdunski M, Heinemann SF, Barhanin J: Inner ear defects induced by null mutation of isk gene. Neuron. 1996, 17: 1251-1264. 10.1016/S0896-6273(00)80255-X.PubMedView Article
- Anniko M, Nordemar H: Embryogenesis of the inner ear. IV. Post-natal maturation of the secretory epithelia of the inner ear in correlation with the elemental composition in the endolymphatic space. Arch Otorhinolaryngol. 1980, 229: 281-288.PubMedView Article
- Sadanaga M, Morimitsu T: Development of endocochlear potential and its negative component in mouse cochlea. Hear Res. 1995, 89: 155-161. 10.1016/0378-5955(95)00133-X.PubMedView Article
- Wangemann P: K+ cycling and the endocochlear potential. Hear Res. 2002, 165: 1-9. 10.1016/S0378-5955(02)00279-4.PubMedView Article
- Takeuchi S, Ando M: Voltage-dependent outward K+ current in intermediate cell of stria vascularis of gerbil cochlea. Am J Physiol. 1999, 277: C91-C99.PubMed
- Takeuchi S, Ando M: Dye-coupling of melanocytes with endothelial cells and pericytes in the cochlea of gerbils. Cell Tissue Res. 1998, 293: 271-275. 10.1007/s004410051118.PubMedView Article
- Wangemann P, Liu J, Marcus DC: Ion transport mechanisms responsible for K+ secretion and the transepithelial voltage across marginal cells of stria vascularis in vitro. Hear Res. 1995, 84: 19-29. 10.1016/0378-5955(95)00009-S.PubMedView Article
- Schrott A, Melichar I, Popelár J, Syka J: Deterioration of hearing function in mice with neural crest defect. Hear Res. 1990, 46: 1-8. 10.1016/0378-5955(90)90134-B.PubMedView Article
- Carlisle L, Steel K, Forge A: Endocochlear potential generation is associated with intercellular communication in the stria vascularis: structural analysis in the viable dominant spotting mouse mutant. Cell Tissue Res. 1990, 262: 329-337.PubMedView Article
- Luciano L, Reiss G, Reale E: The junctions of the spindle-shaped cells of the stria vascularis: A link that completes the barrier between perilymph and endolymph. Hear Res. 1995, 85: 199-209. 10.1016/0378-5955(95)00047-8.PubMedView Article
- Johnstone BM, Patuzzi R, Syka J, Sykova E: Stimulus-related potassium changes in the organ of Corti of guinea-pig. J Physiol (Lond). 1989, 408: 77-92.View Article
- Zidanic M, Brownell WE: Fine structure of the intracochlear potential field. I. The silent current. Biophys J. 1990, 57: 1253-1268.PubMedPubMed CentralView Article
- Casimiro MC, Knollmann BC, Ebert SN, Vary JC, Greene AE, Franz MR, Grinberg A, Huang SP, Pfeifer K: Targeted disruption of the Kcnq1 gene produces a mouse model of Jervell and Lange-Nielsen Syndrome. Proc Natl Acad Sci U S A. 2001, 98: 2526-2531. 10.1073/pnas.041398998.PubMedPubMed CentralView Article
- Dixon MJ, Gazzard J, Chaudhry SS, Sampson N, Schulte BA, Steel KP: Mutation of the Na-K-Cl co-transporter gene Slc12a2 results in deafness in mice. Hum Mol Genet. 1999, 8: 1579-1584. 10.1093/hmg/8.8.1579.PubMedView Article
- Spector GJ, Carr C: The ultrastructural cytochemistry of peroxisomes in the guinea pig cochlea: a metabolic hypothesis for the stria vascularis. Laryngoscope. 1979, 89: 1-38.PubMedView Article
- Takumi Y, Matsubara A, Tsuchida S, Ottersen OP, Shinkawa H, Usami S: Various glutathione S-transferase isoforms in the rat cochlea. Neuroreport. 2001, 12: 1513-1516. 10.1097/00001756-200105250-00042.PubMedView Article
- Usami S, Hjelle OP, Ottersen OP: Differential cellular distribution of glutathione–an endogenous antioxidant–in the guinea pig inner ear. Brain Res. 1996, 743: 337-340. 10.1016/S0006-8993(96)01090-6.PubMedView Article
- Gratton MA, Wright CG: Hyperpigmentation of chinchilla stria vascularis following acoustic trauma. Pigment Cell Res. 1992, 5: 30-37.PubMedView Article
- Koyama Y, Kimura Y, Hashimoto H, Matsuda T, Baba A: L-lactate inhibits L-cystine/L-glutamate exchange transport and decreases glutathione content in rat cultured astrocytes. J Neurosci Res. 2000, 59: 685-691. 10.1002/(SICI)1097-4547(20000301)59:5<685::AID-JNR12>3.3.CO;2-Q.PubMedView Article
- Tang XD, Santarelli LC, Heinemann SH, Hoshi T: Metabolic regulation of potassium channels. Annu Rev Physiol. 2004, 66: 131-159. 10.1146/annurev.physiol.66.041002.142720.PubMedView Article
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1741-7015/2/30/prepub
This article is published under license to BioMed Central Ltd. This is an open-access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.