Abstract
Anion exchange proteins (AE) in the inner ear have been the focus of attention for some time. They have been suggested to play a role as anion exchangers for the regulation of endolymphatic pH or as anion exchangers and anchor proteins for the maintenance of the shape and turgor of outer hair cells, and they also have been discussed as a candidate protein for motile hair cell responses that follow high-frequency stimulation. The existence of anion exchangers in hair cells and the specific isoforms which are expressed in hair cells and the organ of Corti is controversial. Using a polyclonal antibody to AE1 (AB1992, Chemicon), we immunoprecipitated a 100 kDa AE polypeptide in isolated outer hair cells which, due to its glycosylation, is comprised of AE2 than AE1 isoforms. We confirmed AE2 expression in outer hair cells with the help of subtype-specific monoclonal and polyclonal antibodies to AE, AE subtype-specific primers and AE subtype-specific cDNA and found glycosylated truncated as well as full-length AE2 isoforms. No AE1 or AE3 subtypes were noted in outer hair cells. In contrast, AE2 and AE3 but not AE1 subtypes were seen in supporting cells of the organ of Corti. Their expression preceded the development of cochlear function, coincident with the establishment of the endocochlear potential and the differentiation of supporting cells. While most developmental processes in the inner ear usually begin in the basal cochlear turn, the AE2 expression in outer hair cells (but not that of AE2 and AE3 in supporting cells) progressed from the apical to the basal cochlear turn, reminiscent of the maturation of frequency-dependency. Irrespective of their presumed individual role as either anion exchanger, anchor protein or motility protein, the differential expression and developmental profile of these proteins suggest a most important role of anion exchange proteins in the development of normal hearing. These findings may also provide novel insights into AE function in general.
Similar content being viewed by others
References
Holley MC, Kalinec F, Kachar B: Structure of the cortical cytoskeleton in mammalian outer hair cells. J Cell Sci 102: 569–580, 1992
Ikeda K, Saito Y, Nishiyama A, Takasaka T: Intracellular pH regulation in isolated cochlear outer hair cells of the guinea-pig. J Physiol (London) 447: 627–648, 1992
Ohnishi S, Hara M, Inoue M, Yamashita T, Kumazawa T, Minato A, Inagaki C: Delayed shortening and shrinkage of cochlear outer hair cells. Am J Physiol 263: C1088–1095, 1992
Ohnishi S, Hara M, Inagaki C, Yamashita T, Kumazawa T: Regulation of Cl- conductance in delayed shortening and shrinkage of outer hair cells. Acta Otolaryngol Suppl 500: 42–45, 1993
Kalinec F, Jaeger RG, Kachar B: Mechanical coupling of the outer hair cell plasma membrane to the cortical cytoskeleton by anion exchanger and 4.1 proteins. In: H. Duifhuis, J. W. Horst, P. van Dijk, S. M. Netten (eds). Proceedings of the Symposium on Biophysics of hair cell sensory systems. World Scientific. Singapore, 1993, pp 175–181
Lim DJ, Kalinec F: Cell and molecular basis of hearing. Kidney Int Suppl 65: S104–113, 1998
Stankovic KM, Brown D, Alper SL, Adams JC: Localization of pH regulating proteins H+-ATPase and Cl-/HCO3 - exchanger in the guinea pig inner ear. Hear Res 114: 21–34, 1997
Alper SL: The band 3-related anion exchanger (AE) gene family. Annu Rev Physiol 53: 549–564, 1991
Wang DN: Band 3 protein: structure, flexibility and function. FEBS Lett 346: 26–31, 1994
Elgavish A, Esko JD, Knurr A: Chinese hamster ovary cell mutants deficient in an anion exchanger functionally similar to the erythroid band 3. J Biol Chem 263: 18607–18613, 1988
Brosius FC, Alper SL, Garcia AM, Lodish HF: The major kidney band 3 gene transcript predicts an amino-terminal truncated band 3 polypeptide. J Biol Chem 264: 7784–7787, 1989
Kopito RR: Molecular biology of the anion exchanger gene family. Int Rev Cytol 123: 177–199, 1990
Lindsey AE, Schneider K, Simmons DM, Baron R, Lee BS, Kopito RR: Functional expression and subcellular localization of an anion exchanger cloned from choroid plexus. Proc Natl Acad Sci U S A 87: 5278–5282, 1990
Lee BS, Gunn RB, Kopito RR: Functional differences among nonerythroid anion exchangers expressed in a transfected human cell line. J Biol Chem 266: 11448–11454, 1991
Puceat M, Korichneva I, Cassoly R, Vassort G: Identification of band 3-like proteins and Cl-/HCO3 - exchange in isolated cardiomyocytes. J Biol Chem 270: 1315–1322, 1995
Kudrycki KE, Newman PR, Shull GE: cDNA cloning and tissue distribution of mRNAs for two proteins that are related to the band 3 Cl-/HCO3 - exchanger. J Biol Chem 265: 462–471, 1990
Kopito RR, Lee BS, Simmons DS, Lindsey AE, Morgans CW, Schneider K: Regulation of intracellular pH by a neuronal homolog of the erythrocyte anion exchanger. Cell 59: 927–937
Linn SC, Kudrycki KE, Shull GE: The predicted translation product of a cardiac AE3 mRNA contains an N-terminus distinct from that of the brain AE3 Cl-/HCO3 - exchanger. Cloning of a cardiac AE3 cDNA, organization of the AE3 gene, and identification of an alternative transcription initiation site. J Biol Chem 267: 7927–7935, 1992
Negrini C, Rivolta MN, Kalinec F, Kachar B: Cloning of an organ of Corti anion exchanger 2 isoform with a truncated C-terminal domain. Biochim Biophys Acta 1236: 207–211, 1995
Kalinec F, Kalinec G, Negrini C, Kachar B: Immunolocalization of anion exchanger 2α in auditory sensory hair cells. Hear Res 110: 141–146, 1997
Mhatre AN, Charachon G, Alper SL, Lalwani AK: The guinea pig cochlear AE2 anion exchanger: cDNA cloning and in situ localization within the cochlea. Biochim Biophys Acta 1414: 1–15, 1998
Knipper M, Zimmermann U, Kö pschall I, Rohbock K, Jü ngling S, Zenner HP: Immunological identification of candidate proteins involved in regulating active shape changes of outer hair cells. Hear Res 86: 100–110, 1995
Bennett V: Proteins involved in membrane cytoskeleton association in human erythrocytes: spectrin, ankyrin, and band 3. Meth Enzymol 96: 313–324, 1983
Knipper M, Bandtlow C, Gestwa L, Kö pschall I, Rohbock K, Wiechers B, Zenner HP, Zimmermann U: Thyroid hormone affects Schwann cell and oligodendrocyte gene expression at the glial transition zone of the VIIIth nerve prior to cochlea function. Development 125: 3709–3718, 1998
Knipper M, Gestwa L, Ten-Cate WT, Lautermann J, Brugger H, Maier H, Zimmermann U, Rohbock K, Kö pschall I, Wiechers B, Zenner HP: Distinct thyroid hormone-dependent expression of trkB and p75NGFR in nonneuronal cells during the critical TH-dependent period of the cochlea. J Neurobiol 38: 338–356, 1998
Towbin H, Staehelin T, Gordon J: Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 76: 4350–4354, 1979
Bosman GJ, Renkawek K, Van Workum FP, Bartholomeus IG, De Grip WJ: Involvement of neuronal anion exchange proteins in cell death in Alzheimer's disease. Gerontology 43: 67–78, 1997
Czerwinski M, Usnarska-Zubkiewicz L: Molecular characterization of mouse monoclonal antibody BIII.136 and the epitope recognized by the antibody in human band 3 protein. Hybridoma 14: 217–223, 1995
Demuth DR, Showe LC, Ballantine M, Palumbo A, Fraser PJ, Cioe L, Rovera G, Curtis PJ: Cloning and structural characterization of a human non-erythroid band 3-like protein. EMBO J 5: 1205–1214, 1986
Havenga MJ, Bosman GJ, Appelhans H, De Grip WJ: Expression of the anion exchanger (AE) gene family in human brain. Identification of a new AE protein: AE0. Brain Res Mol Brain Res 25: 97–104, 1994
Renkawek K, Bosman GJ: Anion exchange proteins are a component of corpora amylacea in Alzheimer disease brain. Neuroreport 6: 929–932, 1995
Barker PA, Shooter EM: Disruption of NGF binding to the low affinity neurotrophin receptor p75LNTR reduces NGF binding to TrkA on PC12 cells. Neuron 13: 203–215, 1994
Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254, 1976
Casey JR, Pirraglia CA, Reithmeier RA: Enzymatic deglycosylation of human Band 3, the anion transport protein of the erythrocyte membrane. Effect on protein structure and transport properties. J Biol Chem 267: 11940–11948, 1992
Kudrycki KE, Shull GE: Primary structure of the rat kidney band 3 anion exchange protein deduced from a cDNA. J Biol Chem 264: 8185–8192, 1989
Ashmore JF, Kolston PJ: Hair cell based amplification in the cochlea. Curr Opin Neurobiol 4: 503–508, 1994
Dallos P, Evans BN: High-frequency motility of outer hair cells and the cochlear amplifier. Science 267: 2006–2009, 1995
Cecola RP, Bobbin RP: Lowering extracellular chloride concentration alters outer hair cell shape. Hear Res 61: 65–72, 1992
Sekler I, Kobayashi S, Kopito RR: A cluster of cytoplasmic histidine residues specifies pH dependence of the AE2 plasma membrane anion exchanger Neuron 86: 929–935, 1996
Rubel EW: Ontogeny of structure and function in the vertebrate auditory system. In: M. Jabobson (ed). Development of sensory systems. Springer Verlag. Berlin, Heidelberg, New York, 1978, pp 135–220
Ehret G: Development of hearing and response behavior to sound stimuli: Behavioral studies. In: R. Romand (ed). Development of auditory and vestibular systems. Academic Press. New York, 1983, pp 211–237
Rubel EW, Lippe WR, Ryals BM: Development of the place principle. Ann Otol Rhinol Laryngol 93: 609–615, 1984
Rubel EW, Ryals BM: Development of the place principle: acoustic trauma. Science 219: 512–514, 1983
Eggermont JJ, Ponton CW, Coupland SG, Winkelaar R: Maturation of the traveling-wave delay in the human cochlea. J Acoust Soc Am 90: 288–298, 1991
Sininger YS, Abdala C, Cone-Wesson B: Auditory threshold sensitivity of the human neonate as measured by the auditory brainstem response. Hear Res 104: 27–38, 1997
Jewett DL, Romano MN: Neonatal development of auditory system potentials averaged from the scalp of rat and cat. Brain Res 36: 101–115, 1972
Geal-Dor M, Freeman S, Li G, Sohmer H: Development of hearing in neonatal rats: air and bone conducted ABR thresholds. Hear Res 69: 236–242, 1993
Gimsa J, Ried C: Do band 3 protein conformational changes mediate shape changes of human erythrocytes? Mol Membr Biol 12: 247–254, 1995
Sterkers O, Saumon G, Tran Ba Huy P, Ferrary E, Amiel C: Electrochemical heterogeneity of the cochlear endolymph: effect of acetazolamide. Am J Physiol 246: F47–53, 1984
Busa WB, Nuccitelli R: Metabolic regulation via intracellular pH. Am J Physiol 246: R409–438, 1984
Glowatzki E, Fakler G, Brä ndle U, Rexhausen U, Zenner HP, Ruppersberg JP, Fakler B: Subunit-dependent assembly of inwardrectifier K+ channels. Proc R Soc Lond B Biol Sci 261: 251–261, 1995
Fakler B, Schultz JH, Yang J, Schulte U, Brä ndle U, Zenner HP, Jan LY, Ruppersberg JP: Identification of a titratable lysine residue that determines sensitivity of kidney potassium channels (ROMK) to intracellular pH. EMBO J 15: 4093–4099, 1996
Gunn RB, Dalmark M, Tosteson DC, Wieth JO: Characteristics of chloride transport in human red blood cells. J Gen Physiol 61: 185–206, 1973
Funder J, Wieth JO: Chloride transport in human erythrocytes and ghosts: a quantitative comparison. J Physiol (Lond) 262: 679–698, 1976
Hinojosa R: A note on development of Corti´ s organ. Acta Otolaryngol (Stockh) 84: 238–251, 1977
McPhee JR, Van De Water TR: Structural and functional development of the ear. In: A.F. Jahn, J. Santos-Sacchi (eds). Physiology of the Ear. Raven Press. New York, 1988, pp 221–242
Roth B, Bruns V: Postnatal development of the rat organ of Corti. General morphology, basilar membrane, tectorial membrane and border cell. Anat Embryol 185: 559–569, 1992
Spicer SS, Smolka AJ, Schulte BA: Distribution of H+-K+-ATPase in the adult gerbil inner ear and changes in ist expression during development. Assoc Res Otolaryngol. Abstr 18: 559, 1995
Woolf NK, Ryan AF, Harris JP: Development of mammalian endocochlear potential: normal ontogeny and effects of anoxia. Am J Physiol 250: R493–498, 1986
Rybak LP, Whitworth C, Scott V: Development of endocochlear potential and compound action potential in the rat. Hear Res 59: 189–194, 1992
Sadanaga M, Morimitsu T: Development of endocochlear potential and its negative component in mouse cochlea. Hear Res 89: 155–161, 1995
Sato Y, Santos-Sacchi J: Cell coupling in the supporting cells of Corti's organ: sensitivity to intracellular H+ and Ca2+. Hear Res 80: 21–24, 1994
Kay MM, Goodman J, Lawrence C, Bosman G: Membrane channel protein abnormalities and autoantibodies in neurological disease. Brain Res Bull 24: 105–111, 1990
Bosman GJ, Van Workum FP, Renkawek K, Van Kalmthout PJ, Bartholomeus IG, De Grip WJ: Proteins immunologically related to erythrocyte anion transporter band 3 are altered in brain areas affected by Alzheimer's disease. Acta Neuropathol 86: 353–359, 1993
Mohandas N, Winardi R, Knowles D, Leung A, Parra M, George E, Conboy J, Chasis J: Molecular basis for membrane rigidity of hereditary ovalocytosis. A novel mechanism involving the cytoplasmic domain of band 3. J Clin Invest 89: 686–692, 1992
Schofield AE, Reardon DM, Tanner MJ: Defective anion transport activity of the abnormal band 3 in hereditary ovalocytic red blood cells. Nature 355: 836–838, 1992
McPherson RA, Donald DR, Sawyer WH, Tilley L: Proteolytic digestion of band 3 at an external site alters the erythrocyte membrane organisation and may facilitate malarial invasion. Mol Biochem Parasitol 62: 233–242, 1993
Tanner MJ: Molecular and cellular biology of the erythrocyte anion exchanger (AE1). Semin Hematol 30: 34–57, 1993
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Zimmermann, U., Köpschall, I., Rohbock, K. et al. Molecular characterization of anion exchangers in the cochlea. Mol Cell Biochem 205, 25–37 (2000). https://doi.org/10.1023/A:1007002916772
Issue Date:
DOI: https://doi.org/10.1023/A:1007002916772