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Hair cells, plasma membrane Ca2+ ATPase and deafness
The International Journal of Biochemistry & Cell Biology 44 (2012) 679–683
Contents lists available at SciVerse ScienceDirect
The International Journal of Biochemistry & Cell Biology journal homepage: www.elsevier.com/locate/biocel
Cells in focus
Hair cells, plasma membrane Ca2+ ATPase and deafness Marta Giacomello a,b,∗ , Agnese De Mario a , Simona Primerano a , Marisa Brini c , Ernesto Carafoli a,c,∗ a
Venetian Institute of Molecular Medicine (VIMM), Via G. Orus, 2, 35129 Padua, Italy Department of Experimental Biomedical Sciences, University of Padua, Padua, Italy c Department of Biological Chemistry, University of Padua, Padua, Italy b
a r t i c l e
i n f o
Article history: Received 23 November 2011 Received in revised form 24 January 2012 Accepted 3 February 2012 Available online 13 February 2012 Keywords: Ca2+ homeostasis Deafness Hair cells Stereocilia
a b s t r a c t Hearing relies on the ability of the inner ear to convert sound waves into electrical signals. The main actors in this process are hair cells. Their stereocilia contain a number of specific proteins and a scaffold of actin molecules. They are organized in bundles by tip-link filaments composed of cadherin 23 and protocadherin 15. The bundle is deflected by sound waves leading to the opening of mechano-transduction channels and to the influx of K+ and Ca2+ into the stereocilia. Cadherin 23 and the plasma membrane calcium ATPase isoform 2 (PMCA2) are defective in human and murine cases of deafness. While the involvement of cadherin 23 in deafness/hearing could be expected due to its structural role in the tiplinks, that of PMCA2 has been discovered only recently. This review will summarize the structural and functional characteristics of hair cells, focusing on the proteins whose mutations may lead to a deafness phenotype. © 2012 Elsevier Ltd. All rights reserved.
1. Introduction Cell facts • Hair cells of the Corti Organ of the inner ear convert sound waves in the endolymph into acoustic signals; inner hair cells transmit the signal to the brain while outer hair cells provide a mechanical feedback, amplifying quiet sounds. • Mechano-electrical transduction channels of hair cells are opened by the movement of the stereocilia bundle and mediate the influx of K+ and Ca2+ into the stereocilia. • Ca2+ is rejected from outer hair cells by a special splice variant of isoform 2 of the plasma membrane Ca2+ pump: in the endolymph, Ca2+ modulates the tip links that are essential for the movement of the stereocilia bundle. • Tip links are formed by the Ca2+ binding proteins cadherin 23 and protocadherin 15. • Mutations of PMCA2 impair the ejection of Ca2+ and cause hereditary deafness in mice and humans: the deafness phenotype could be aggravated by simultaneous mutations in cadherin 23.
Abbreviations: [Ca2+ ], calcium concentration; CaM, calmodulin; EF, hand motif; helix-loop, helix calcium binding motif; ER, endoplasmic reticulum; KO, knock out; MET, mechano-electrical transduction; PMCA, plasma membrane calcium ATPase. ∗ Corresponding authors at: Venetian Institute of Molecular Medicine (VIMM), Via G. Orus, 2, 35129 Padua, Italy. Tel.: +39 049 792 3242; fax: +39 049 827 6125. E-mail address: [email protected] (E. Carafoli). 1357-2725/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2012.02.006
Sound waves are converted into hearing signals through the mechanoelectrical transduction process, that depends on the presence of hair cells in the inner ear. These cells are located in the organ of Corti, within the cochlea, and are arranged in rows: one row of inner hair cells (IHC) and three rows of outer hair cells (OHC) (for a comprehensive recent review, see for instance Schwander et al., 2010 and Fig. 1), all endowed at their apical end with bundles of stereocilia, that protrude into the endolymph connected by filaments known as tip links. The synchronized deflection of the stereocilia by endolymph vibrations produced by sound or head movement induces the opening of the mechano-electrical transduction channels, leading to the entry of K+ and Ca2+ from the endolymph into the stereocilia. The bending of the stereocilia bundle thus generates a mechano-electrical transduction current that depolarizes the cell, resulting in a receptor potential which opens voltage-gated Ca2+ channels in the basal end of the plasma membrane: Ca2+ then enters the IHC and promotes the release of neurotransmitters (glutamate) to trigger action potentials in neighboring afferent nerve terminals. OHC, instead, react to the polarization changes by modify body length to transmit mechanical signals to IHCs. Ca2+ is crucial for the hearing process, even if it composes only the 0.2% of the total mechano-electrical transduction current: it promotes adaptation of the cell to sustained deflections of the stereocilia bundle by a negative feedback on the open probability of the mechano-electrical transduction channels. Ca2+ is exported back to the endolymph by the plasma membrane Ca2+ ATPase (PMCA). The mechano-electrical transduction process is essential for hearing: murine and human cases of deafness are
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Fig. 1. (A) Schematic representation of the Organ of Corti. Inner and outer hair cells are endowed with stereocilia that protrude in the endolymph. They are organized in bundles by the tip links (B and C) that run from a stereocilium to its taller neighbor. The deflection of the stereocilia induces the opening of MET channels, which allow the entry of K+ and Ca2+ in the stereocilia cytoplasm. The tip links are formed by dimers of the Ca2+ binding proteins cadherin 23 (the upper two thirds) and protocadherin 15 (the lower third). The stereocilia are filled with an actin network. A plaque of increased density underlies the point of insertion of the cadherins in the stereocilia. That of the upper stereocilium contains proteins that interact with the cytoplasmic C-terminal portion of cadherin 23 and the actin network: myosin VII, harmonins and SANS. Ca2+ that has entered the stereocilia is extruded to the endolymph by a specific pump (PMCA2, variant w/a). The pump assures the maintenance of the ionic balance that is essential for the operation of tip links and thus for the gating of the MET channels. Ca2+ binding to cadherin 23 regulates the rigidity of the tip links. (D) The three rows of outer hair cells and the single row of inner hair cells (scanning electron micrograph, courtesy of Dr. Robert V. Harrison, Hospital for Sick Children and University of Toronto).
produced by mutations in the structural proteins that compose the tip links and in PMCA. 2. Cell origin and plasticity The cochlea is a spiral shape cavity in which sound waves propagate from the opening of the cochlear duct to the center of the spiral. It is positioned on the basilar membrane, and is surrounded by the Reissner membrane and the stria vascularis, a tissue rich in capillaries. The basilar membrane and the Reissner membrane define
a compartment filled with endolymph, an extracellular medium which has peculiarly high K+ and uniquely low Ca2+ content. The major component of the cochlea is the Organ of Corti, that is positioned between the basilar and tectorial membranes. Sounds cause oscillation of the endolymph and the vibrations are transmitted to the basilar membrane, that transmits them to the sensory receptor cells of the organ of Corti, (IHC and OHC) which run along the whole length of the Cochlea (Schwander et al., 2010). Hair cells are characterized by a bundle of stereocilia that protrude into the endolymph. They are arranged in a staircase of increasing height
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in which each stereocilium is linked to its neighbor through the tip links, formed by cadherin 23 and protocadherin 15, single pass proteins with a long N-terminal domain protruding in the endolymph. IHCs transmit cochlear outputs to the brain, OHCs mainly collect the neural input from encephalic regions. 3. Functions As mentioned in the introduction, the opening of the mechanoelectrical transduction channels in OHC triggers oscillations in cell length (somatic electromotility), produced by the rearrangement of the voltage dependent membrane protein prestin, which provide a mechanical feedback of the hearing range, amplifying quiet sounds more than larger ones. The efferent nerve endings (more than 90% of the OHCs innervations is efferent) release acetylcholine at the basal end of OHCs to promote a hyperpolarizing K+ current that amplifies the electromotility signaling to IHCs. The vibrations are transmitted mechanically to the IHCs, providing the mechanical feedback amplification. The two Ca2+ binding proteins that form the tip links (cadherin 23 and protocadherin 15) contain 27 and 11 cadherin repeats, respectively, with inter-repeat Ca2+ binding sites (Michel et al., 2005; Ahmed et al., 2006; Kazmierczak et al., 2007). The binding of Ca2+ is essential to the integrity of the tip links: its removal leads to their degeneration (Assad et al., 1991; Furness et al., 2008). The mechano-electrical transduction channels are located at the lower end of each tip link and are gated by a spring mechanism that could be the tip-link itself: it conveys tension to the channels and causes the penetration of K+ and Ca2+ (Denk et al., 1995; Goodyear et al., 2005). As mentioned, Ca2+ contributes only about 0.2% of the total current (Ricci and Fettiplace, 1998; Beurg et al., 2010), but the amount is adequate to promote the adaptation process. A PMCA pump exports Ca2+ back to the endolymph is abundantly expressed in the stereocilia of OHC but not in those of IHCs. It controls the homeostasis of Ca2+ in the endolymph and thus the organization and functions of the tip links. 4. Associated pathologies The stereocilia of OHCs contain large amounts of a splice variant of the plasma membrane Ca2+ ATPase isoform 2 (PMCA2), which is one of the two tissue specific PMCA pumps expressed in mammals: together with the PMCA3 pump it is abundantly expressed in the brain and in cells of neuronal derivation (Stauffer et al., 1995; Silverstein and Tempel, 2006; Carafoli, 2011). Alternative splicing of PMCA isoforms occurs at the N (site A) or C terminal (site C) moiety of the pumps. In PMCA2, the site A insertion comprises up to 3 exons. The variants without A site inserts are commonly known as z, while those with 1, 2 or 3 exons have been named x, y, and w, respectively (Brini and Carafoli, 2009; Carafoli, 2011). The site C insertion involves 1 exon in variant a: the variant with no C-site insertion is termed variant b. Since the C-site insert is not in frame, it leads to the truncation of the pump about 50 residues upstream of the original C terminus. The truncation occurs within the binding domain for calmodulin (CaM), which is the main regulator of PMCAs activity. The pump variant resident in the OHC stereocilia is the w/a splice variant, that has three exon inserted at site A and one at site C. Unlike all other PMCA isoforms, the PMCA2 pump has very high activity even in the absence of CaM (Carafoli, 2011). In OHC the w/a pump has been detected only in the plasma membrane of stereocilia, while the resident variant of the pump in the main body of the hair cells is isoform PMCA1 (Grati et al., 2006). No PMCAs have been detected in the stereocilia of IHCs (see above). The ablation of the PMCA2 gene in mice produces equilibrium disorders and defective hearing (Kozel et al., 1998). Mutations of
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the PMCA2 pump in mice and humans have been linked to deafness. The first deafness inducing mice mutation was a glycine to serine replacement (G283S; the deafwaddler mice, dfw2J; Street et al., 1998). The second mutation linked to equilibrium imbalance and deafness was caused by a lysine to glutamate substitution in the fourth transmembrane domain of PMCA2 (K412E, Wriggle Sagami mouse, wri; Inoue et al., 1993): it impaired the targeting of the pump to the stereocilia (Takahashi and Kitamura, 1999). Other deafness inducing mutations have then been found in PMCA2 in humans, such as the V586M and the G293S (Schultz et al., 2005; Ficarella et al., 2007) and in mice, i.e., the S877F Oblivion and E584K Tommy (Spiden et al., 2008; Bortolozzi et al., 2010) mutations. One last deafness inducing mutation in mice (T692K) has been described without analysis of the pump defect (Tsai et al., 2006). When later performed, the analysis indeed revealed the same pump defect of that of the other mutations (Giacomello et al., 2011). The study of the activity of all pump mutants has been performed in models overexpressing them. The original analysis on the human V586M mutation, however, had been performed on membranes of overexpressing model cells: it had revealed a large decrease of the total PMCA2 pump activity (Schultz et al., 2005). The model cells used to express the pump mutants also expressed the Ca2+ indicator aequorin, in a protocol that permitted a fine dissection of the pump defect. It was then also applied to the human V586M mutant (Giacomello et al., 2011). In the protocol, the pump overexpressing cells were stimulated with the purinergic agonist ATP, which promoted the liberation of Ca2+ from the endoplasmic reticulum (ER) by the newly formed second messenger inositol 1,4,5 trisphosphate (InsP3 ). The arrival of Ca2+ in the cytosol generated a Ca2+ transient peak which was then returned to the baseline by the action of the overexpressed PMCA (the contribution of the SERCA pump to the shape of the transient was negligible, as established in pilot experiments with appropriate inhibitors). The standard defect detected in all pump mutants was an evident delay of the return of the post-peak Ca2+ trace to the baseline (Fig. 2): the mutated PMCA pumps were evidently defective in the ability – which is particularly high in PMCA2, see above – to remove Ca2+ from the cytosol after the phase of pump activation produced by the pulse of Ca2+ liberated from the ER: i.e., the pump had lower ability to function in the non-activated state. The delay in the return of the Ca2+ trace to baseline was predictably more dramatic when the mutation was non-conservative (e.g., in the T692K replacement), or when it would seriously disturb the action of the pump in other ways (e.g., the replacement of a residue within a transmembrane domains in the Oblivion mutation). The mutations also influenced the height of the Ca2+ transient peak, but the effect was more variable. The reduced ability of the mutated pumps to export Ca2+ obviously affected the Ca2+ homeostasis in the endolymph, impairing the ability of the Ca2+ binding proteins of the tip-links to act on the stereocilia bundle: the functional defect of cadherin 23 would disturb the gating of the MET channels (Sotomayor et al., 2010). Cadherin 23 defects are indeed important in the hearing loss phenotypes and in some of the deafness cases listed above mutations in cadherin 23 have been found to accompany the pump mutation. In the V586M human case, for instance (Schultz et al., 2005), deafness was primarily generated by a cadherin F1888S mutation while the PMCA2 mutation, which in this mutant affected the activity of the pump but slightly (Giacomello et al., 2011), only exacerbated the phenotype. Interestingly, in the other human mutation, the G293S pump replacement was present in the healthy mother and the T1999S cadherin 23 mutation in the equally healthy father (Ficarella et al., 2007). Both mutations were instead present in the offspring, which were deaf: a typical example of a digenic disease mechanism. In the Oblivion and Tommy murine mutations, instead, there was no cadherin 23 defect. This shows that both the PMCA
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Fig. 2. Traces showing the effects of mutations of PMCA2 (isoform w/a) on its Ca2+ exporting activity. Each residue substitution in the pump molecule is indicated. The Ca2+ transients were induced in CHO cells overexpressing the mutant pumps and the luminescent Ca2+ indicator aequorin by the InsP3-linked purinergic agonist ATP: all mutant pumps (with the exception of the human V586M,in which the defect was only marginal) were deficient in the ability to export Ca2+ . The long term export of Ca2+ after the peak induced by the promotion of InsP3 was severely affected. The height of the Ca2+ peak was also affected, but only in two PMCA2 mutants.
and cadherin 23 are important actors in the complex process that permits the penetration of Ca2+ within hair cells and its modulation inside them in a way appropriate for the generation of the sound signal. However, depending on the severity of the defect, the development of the deafness phenotype could demand the impairment of both proteins, or of only one. 5. Concluding remarks Thanks to their stereocilia bundles, hair cells are fundamental in the process of auditory sensing. Accordingly, hearing loss may be characterized by the impairment in their activity, due to mutations of the proteins that initiate and regulate the mechano-electrical transduction process: cadherin 23 and the PMCA2 w/a pump, which is responsible for the extrusion of Ca2+ from the OHCs stereocilia. The maintenance of the delicate Ca2+ balance in the hair cells and in the endolymph that bathes their stereocilia is essential for the hearing process. Results on the human PMCA mutations G293S and V586 M which are accompanied by cadherin 23 mutations as well as mice deafness cases in which only the PMCA pump was defective, have revealed that the PMCA2 w/a pump of the stereocilia is key to the temporal and spatial shaping of the Ca2+ movements in the endolymph that are necessary for the production of the sound signals. Acknowledgments This work was supported by grants from the ERA-NET Neuron (nEUROsyn 2008) and from CARIPARO (Project of Excellence, call 2008–2009) to EC, and from the University of Padova (Atheneum project 2008) and from the Italian Ministry of University and Research (PRIN 2008) to MB. ADM and SP were supported by fellowships from the ERA-NET Neuron grant, MG was supported by a Research Fellowship from the CARIPARO grant.
References Ahmed ZM, Goodyear R, Riazuddin S, Lagziel A, Legan PK, Behra M, et al. The tiplink antigen, a protein associated with the transduction complex of sensory hair cells, is protocadherin-15. J Neurosci 2006;26:7022–34. Assad JA, Shepherd GM, Corey DP. Tip-link integrity and mechanical transduction in vertebrate hair cells. Neuron 1991;7:985–94. Beurg M, Nam JH, Chen Q, Fettiplace R. Calcium balance and mechanotransduction in rat cochlear hair cells. J Neurophysiol 2010;104:18–34. Bortolozzi M, Brini M, Parkinson N, Crispino G, Scimemi P, De Siati RD, et al. The novel PMCA2 pump mutation Tommy impairs cytosolic calcium clearance in hair cells and links to deafness in mice. J Biol Chem 2010;285: 37693–703. Brini M, Carafoli E. Calcium pumps in health and disease. Physiol Rev 2009;89:1341–78. Carafoli E. The plasma membrane calcium pump in the hearing process: physiology and pathology. Sci China Life Sci 2011;54:686–90. Denk W, Holt JR, Shepherd GM, Corey DP. Calcium imaging of single stereocilia in hair cells: localization of transduction channels at both ends of tip links. Neuron 1995;15:1311–21. Ficarella R, Di Leva F, Bortolozzi M, Ortolano S, Donaudy F, Petrillo M, et al. A functional study of plasma-membrane calcium-pump isoform 2 mutants causing digenic deafness. Proc Natl Acad Sci USA 2007;104:1516–21. Furness DN, Katori Y, Nirmal Kumar B, Hackney CM. The dimensions and structural attachments of tip links in mammalian cochlear hair cells and the effects of exposure to different levels of extracellular calcium. Neuroscience 2008;154:10–21. Giacomello M, De Mario A, Lopreiato R, Primerano S, Campeol M, Brini M, et al. Mutations in PMCA2 and hereditary deafness: a molecular analysis of the pump defect. Cell Calcium 2011;50:569–76. Goodyear RJ, Marcotti W, Kros CJ, Richardson GP. Development and properties of stereociliary link types in hair cells of the mouse cochlea. J Comp Neurol 2005;485:75–85. Grati M, Aggarwal N, Strehler EE, Wenthold RJ. Molecular determinants for differential membrane trafficking of PMCA1 and PMCA2 in mammalian hair cells. J Cell Sci 2006;119:2995–3007. Inoue Y, Matsumura Y, Inoue K, Ichikawa R, Takayama C. Abnormal synaptic architecture in the cerebellar cortex of a new dystonic mutant mouse, Wriggle Mouse Sagami. Neurosci Res 1993;16:39–48. Kazmierczak P, Sakaguchi H, Tokita J, Wilson-Kubalek EM, Milligan RA, Müller U, et al. Cadherin 23 and protocadherin 15 interact to form tip-link filaments in sensory hair cells. Nature 2007;449:87–91. Kozel PJ, Friedman RA, Erway LC, Yamoah EN, Liu LH, Riddle T, et al. Balance and hearing deficits in mice with a null mutation in the gene encoding plasma membrane Ca2+ ATPase isoform 2. J Biol Chem 1998;273:18693–6.
M. Giacomello et al. / The International Journal of Biochemistry & Cell Biology 44 (2012) 679–683 Michel V, Goodyear RJ, Weil D, Marcotti W, Perfettini I, Wolfrum U, et al. Cadherin 23 is a component of the transient lateral links in the developing hair bundles of cochlear sensory cells. Dev Biol 2005;280:281–94. Ricci AJ, Fettiplace R. Calcium permeation of the turtle hair cell mechanotransducer channel and its relation to the composition of endolymph. J Physiol 1998;506:159–73. Schultz JM, Yang Y, Caride AJ, Filoteo AG, Penheiter AR, Lagziel A, et al. Modification of human hearing loss by plasma-membrane calcium pump PMCA2. N Engl J Med 2005;352:1557–64. Schwander M, Kachar B, Müller U. Review series: the cell biology of hearing. J Cell Biol 2010;190:9–20. Silverstein RS, Tempel BL. Atp2b2, encoding plasma membrane Ca2+ -ATPase type 2, (PMCA2) exhibits tissue-specific first exon usage in hair cells, neurons, and mammary glands of mice. Neuroscience 2006;141:245–57. Sotomayor M, Weihofen WA, Gaudet R, Corey DP. Structural determinants of cadherin-23 function in hearing and deafness. Neuron 2010;66:85–100.
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Spiden SL, Bortolozzi M, Di Leva F, De Angelis MH, Fuchs H, Lim D, et al. The novel mouse mutation Oblivion inactivates the PMCA2 pump and causes progressive hearing loss. PLoS Genet 2008;10:e1000238. Stauffer TP, Guerini D, Carafoli E. Tissue distribution of the four gene products of the plasma membrane Ca2+ pump. A study using specific antibodies. J Biol Chem 1995;270:12184–90. Street VA, McKee-Johnson JW, Fonseca RC, Tempel BL, Noben-Trauth K. Mutations in a plasma membrane Ca2+ -ATPase gene cause deafness in deafwaddler mice. Nat Genet 1998;19:390–4. Takahashi K, Kitamura K. A point mutation in a plasma membrane Ca2+ -ATPase gene causes deafness in Wriggle Mouse Sagami. Biochem Biophys Res Commun 1999;261:773–8. Tsai YS, Pendse A, Moy SS, Mohri I, Perez A, Crawley JN, et al. A de novo deafwaddler mutation of PMCA2 arising in ES cells and hitchhiking with a targeted modification of the Ppar ␥ gene. Mamm Genome 2006;17:716–22.
Contents lists available at SciVerse ScienceDirect
The International Journal of Biochemistry & Cell Biology journal homepage: www.elsevier.com/locate/biocel
Cells in focus
Hair cells, plasma membrane Ca2+ ATPase and deafness Marta Giacomello a,b,∗ , Agnese De Mario a , Simona Primerano a , Marisa Brini c , Ernesto Carafoli a,c,∗ a
Venetian Institute of Molecular Medicine (VIMM), Via G. Orus, 2, 35129 Padua, Italy Department of Experimental Biomedical Sciences, University of Padua, Padua, Italy c Department of Biological Chemistry, University of Padua, Padua, Italy b
a r t i c l e
i n f o
Article history: Received 23 November 2011 Received in revised form 24 January 2012 Accepted 3 February 2012 Available online 13 February 2012 Keywords: Ca2+ homeostasis Deafness Hair cells Stereocilia
a b s t r a c t Hearing relies on the ability of the inner ear to convert sound waves into electrical signals. The main actors in this process are hair cells. Their stereocilia contain a number of specific proteins and a scaffold of actin molecules. They are organized in bundles by tip-link filaments composed of cadherin 23 and protocadherin 15. The bundle is deflected by sound waves leading to the opening of mechano-transduction channels and to the influx of K+ and Ca2+ into the stereocilia. Cadherin 23 and the plasma membrane calcium ATPase isoform 2 (PMCA2) are defective in human and murine cases of deafness. While the involvement of cadherin 23 in deafness/hearing could be expected due to its structural role in the tiplinks, that of PMCA2 has been discovered only recently. This review will summarize the structural and functional characteristics of hair cells, focusing on the proteins whose mutations may lead to a deafness phenotype. © 2012 Elsevier Ltd. All rights reserved.
1. Introduction Cell facts • Hair cells of the Corti Organ of the inner ear convert sound waves in the endolymph into acoustic signals; inner hair cells transmit the signal to the brain while outer hair cells provide a mechanical feedback, amplifying quiet sounds. • Mechano-electrical transduction channels of hair cells are opened by the movement of the stereocilia bundle and mediate the influx of K+ and Ca2+ into the stereocilia. • Ca2+ is rejected from outer hair cells by a special splice variant of isoform 2 of the plasma membrane Ca2+ pump: in the endolymph, Ca2+ modulates the tip links that are essential for the movement of the stereocilia bundle. • Tip links are formed by the Ca2+ binding proteins cadherin 23 and protocadherin 15. • Mutations of PMCA2 impair the ejection of Ca2+ and cause hereditary deafness in mice and humans: the deafness phenotype could be aggravated by simultaneous mutations in cadherin 23.
Abbreviations: [Ca2+ ], calcium concentration; CaM, calmodulin; EF, hand motif; helix-loop, helix calcium binding motif; ER, endoplasmic reticulum; KO, knock out; MET, mechano-electrical transduction; PMCA, plasma membrane calcium ATPase. ∗ Corresponding authors at: Venetian Institute of Molecular Medicine (VIMM), Via G. Orus, 2, 35129 Padua, Italy. Tel.: +39 049 792 3242; fax: +39 049 827 6125. E-mail address: [email protected] (E. Carafoli). 1357-2725/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2012.02.006
Sound waves are converted into hearing signals through the mechanoelectrical transduction process, that depends on the presence of hair cells in the inner ear. These cells are located in the organ of Corti, within the cochlea, and are arranged in rows: one row of inner hair cells (IHC) and three rows of outer hair cells (OHC) (for a comprehensive recent review, see for instance Schwander et al., 2010 and Fig. 1), all endowed at their apical end with bundles of stereocilia, that protrude into the endolymph connected by filaments known as tip links. The synchronized deflection of the stereocilia by endolymph vibrations produced by sound or head movement induces the opening of the mechano-electrical transduction channels, leading to the entry of K+ and Ca2+ from the endolymph into the stereocilia. The bending of the stereocilia bundle thus generates a mechano-electrical transduction current that depolarizes the cell, resulting in a receptor potential which opens voltage-gated Ca2+ channels in the basal end of the plasma membrane: Ca2+ then enters the IHC and promotes the release of neurotransmitters (glutamate) to trigger action potentials in neighboring afferent nerve terminals. OHC, instead, react to the polarization changes by modify body length to transmit mechanical signals to IHCs. Ca2+ is crucial for the hearing process, even if it composes only the 0.2% of the total mechano-electrical transduction current: it promotes adaptation of the cell to sustained deflections of the stereocilia bundle by a negative feedback on the open probability of the mechano-electrical transduction channels. Ca2+ is exported back to the endolymph by the plasma membrane Ca2+ ATPase (PMCA). The mechano-electrical transduction process is essential for hearing: murine and human cases of deafness are
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Fig. 1. (A) Schematic representation of the Organ of Corti. Inner and outer hair cells are endowed with stereocilia that protrude in the endolymph. They are organized in bundles by the tip links (B and C) that run from a stereocilium to its taller neighbor. The deflection of the stereocilia induces the opening of MET channels, which allow the entry of K+ and Ca2+ in the stereocilia cytoplasm. The tip links are formed by dimers of the Ca2+ binding proteins cadherin 23 (the upper two thirds) and protocadherin 15 (the lower third). The stereocilia are filled with an actin network. A plaque of increased density underlies the point of insertion of the cadherins in the stereocilia. That of the upper stereocilium contains proteins that interact with the cytoplasmic C-terminal portion of cadherin 23 and the actin network: myosin VII, harmonins and SANS. Ca2+ that has entered the stereocilia is extruded to the endolymph by a specific pump (PMCA2, variant w/a). The pump assures the maintenance of the ionic balance that is essential for the operation of tip links and thus for the gating of the MET channels. Ca2+ binding to cadherin 23 regulates the rigidity of the tip links. (D) The three rows of outer hair cells and the single row of inner hair cells (scanning electron micrograph, courtesy of Dr. Robert V. Harrison, Hospital for Sick Children and University of Toronto).
produced by mutations in the structural proteins that compose the tip links and in PMCA. 2. Cell origin and plasticity The cochlea is a spiral shape cavity in which sound waves propagate from the opening of the cochlear duct to the center of the spiral. It is positioned on the basilar membrane, and is surrounded by the Reissner membrane and the stria vascularis, a tissue rich in capillaries. The basilar membrane and the Reissner membrane define
a compartment filled with endolymph, an extracellular medium which has peculiarly high K+ and uniquely low Ca2+ content. The major component of the cochlea is the Organ of Corti, that is positioned between the basilar and tectorial membranes. Sounds cause oscillation of the endolymph and the vibrations are transmitted to the basilar membrane, that transmits them to the sensory receptor cells of the organ of Corti, (IHC and OHC) which run along the whole length of the Cochlea (Schwander et al., 2010). Hair cells are characterized by a bundle of stereocilia that protrude into the endolymph. They are arranged in a staircase of increasing height
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in which each stereocilium is linked to its neighbor through the tip links, formed by cadherin 23 and protocadherin 15, single pass proteins with a long N-terminal domain protruding in the endolymph. IHCs transmit cochlear outputs to the brain, OHCs mainly collect the neural input from encephalic regions. 3. Functions As mentioned in the introduction, the opening of the mechanoelectrical transduction channels in OHC triggers oscillations in cell length (somatic electromotility), produced by the rearrangement of the voltage dependent membrane protein prestin, which provide a mechanical feedback of the hearing range, amplifying quiet sounds more than larger ones. The efferent nerve endings (more than 90% of the OHCs innervations is efferent) release acetylcholine at the basal end of OHCs to promote a hyperpolarizing K+ current that amplifies the electromotility signaling to IHCs. The vibrations are transmitted mechanically to the IHCs, providing the mechanical feedback amplification. The two Ca2+ binding proteins that form the tip links (cadherin 23 and protocadherin 15) contain 27 and 11 cadherin repeats, respectively, with inter-repeat Ca2+ binding sites (Michel et al., 2005; Ahmed et al., 2006; Kazmierczak et al., 2007). The binding of Ca2+ is essential to the integrity of the tip links: its removal leads to their degeneration (Assad et al., 1991; Furness et al., 2008). The mechano-electrical transduction channels are located at the lower end of each tip link and are gated by a spring mechanism that could be the tip-link itself: it conveys tension to the channels and causes the penetration of K+ and Ca2+ (Denk et al., 1995; Goodyear et al., 2005). As mentioned, Ca2+ contributes only about 0.2% of the total current (Ricci and Fettiplace, 1998; Beurg et al., 2010), but the amount is adequate to promote the adaptation process. A PMCA pump exports Ca2+ back to the endolymph is abundantly expressed in the stereocilia of OHC but not in those of IHCs. It controls the homeostasis of Ca2+ in the endolymph and thus the organization and functions of the tip links. 4. Associated pathologies The stereocilia of OHCs contain large amounts of a splice variant of the plasma membrane Ca2+ ATPase isoform 2 (PMCA2), which is one of the two tissue specific PMCA pumps expressed in mammals: together with the PMCA3 pump it is abundantly expressed in the brain and in cells of neuronal derivation (Stauffer et al., 1995; Silverstein and Tempel, 2006; Carafoli, 2011). Alternative splicing of PMCA isoforms occurs at the N (site A) or C terminal (site C) moiety of the pumps. In PMCA2, the site A insertion comprises up to 3 exons. The variants without A site inserts are commonly known as z, while those with 1, 2 or 3 exons have been named x, y, and w, respectively (Brini and Carafoli, 2009; Carafoli, 2011). The site C insertion involves 1 exon in variant a: the variant with no C-site insertion is termed variant b. Since the C-site insert is not in frame, it leads to the truncation of the pump about 50 residues upstream of the original C terminus. The truncation occurs within the binding domain for calmodulin (CaM), which is the main regulator of PMCAs activity. The pump variant resident in the OHC stereocilia is the w/a splice variant, that has three exon inserted at site A and one at site C. Unlike all other PMCA isoforms, the PMCA2 pump has very high activity even in the absence of CaM (Carafoli, 2011). In OHC the w/a pump has been detected only in the plasma membrane of stereocilia, while the resident variant of the pump in the main body of the hair cells is isoform PMCA1 (Grati et al., 2006). No PMCAs have been detected in the stereocilia of IHCs (see above). The ablation of the PMCA2 gene in mice produces equilibrium disorders and defective hearing (Kozel et al., 1998). Mutations of
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the PMCA2 pump in mice and humans have been linked to deafness. The first deafness inducing mice mutation was a glycine to serine replacement (G283S; the deafwaddler mice, dfw2J; Street et al., 1998). The second mutation linked to equilibrium imbalance and deafness was caused by a lysine to glutamate substitution in the fourth transmembrane domain of PMCA2 (K412E, Wriggle Sagami mouse, wri; Inoue et al., 1993): it impaired the targeting of the pump to the stereocilia (Takahashi and Kitamura, 1999). Other deafness inducing mutations have then been found in PMCA2 in humans, such as the V586M and the G293S (Schultz et al., 2005; Ficarella et al., 2007) and in mice, i.e., the S877F Oblivion and E584K Tommy (Spiden et al., 2008; Bortolozzi et al., 2010) mutations. One last deafness inducing mutation in mice (T692K) has been described without analysis of the pump defect (Tsai et al., 2006). When later performed, the analysis indeed revealed the same pump defect of that of the other mutations (Giacomello et al., 2011). The study of the activity of all pump mutants has been performed in models overexpressing them. The original analysis on the human V586M mutation, however, had been performed on membranes of overexpressing model cells: it had revealed a large decrease of the total PMCA2 pump activity (Schultz et al., 2005). The model cells used to express the pump mutants also expressed the Ca2+ indicator aequorin, in a protocol that permitted a fine dissection of the pump defect. It was then also applied to the human V586M mutant (Giacomello et al., 2011). In the protocol, the pump overexpressing cells were stimulated with the purinergic agonist ATP, which promoted the liberation of Ca2+ from the endoplasmic reticulum (ER) by the newly formed second messenger inositol 1,4,5 trisphosphate (InsP3 ). The arrival of Ca2+ in the cytosol generated a Ca2+ transient peak which was then returned to the baseline by the action of the overexpressed PMCA (the contribution of the SERCA pump to the shape of the transient was negligible, as established in pilot experiments with appropriate inhibitors). The standard defect detected in all pump mutants was an evident delay of the return of the post-peak Ca2+ trace to the baseline (Fig. 2): the mutated PMCA pumps were evidently defective in the ability – which is particularly high in PMCA2, see above – to remove Ca2+ from the cytosol after the phase of pump activation produced by the pulse of Ca2+ liberated from the ER: i.e., the pump had lower ability to function in the non-activated state. The delay in the return of the Ca2+ trace to baseline was predictably more dramatic when the mutation was non-conservative (e.g., in the T692K replacement), or when it would seriously disturb the action of the pump in other ways (e.g., the replacement of a residue within a transmembrane domains in the Oblivion mutation). The mutations also influenced the height of the Ca2+ transient peak, but the effect was more variable. The reduced ability of the mutated pumps to export Ca2+ obviously affected the Ca2+ homeostasis in the endolymph, impairing the ability of the Ca2+ binding proteins of the tip-links to act on the stereocilia bundle: the functional defect of cadherin 23 would disturb the gating of the MET channels (Sotomayor et al., 2010). Cadherin 23 defects are indeed important in the hearing loss phenotypes and in some of the deafness cases listed above mutations in cadherin 23 have been found to accompany the pump mutation. In the V586M human case, for instance (Schultz et al., 2005), deafness was primarily generated by a cadherin F1888S mutation while the PMCA2 mutation, which in this mutant affected the activity of the pump but slightly (Giacomello et al., 2011), only exacerbated the phenotype. Interestingly, in the other human mutation, the G293S pump replacement was present in the healthy mother and the T1999S cadherin 23 mutation in the equally healthy father (Ficarella et al., 2007). Both mutations were instead present in the offspring, which were deaf: a typical example of a digenic disease mechanism. In the Oblivion and Tommy murine mutations, instead, there was no cadherin 23 defect. This shows that both the PMCA
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Fig. 2. Traces showing the effects of mutations of PMCA2 (isoform w/a) on its Ca2+ exporting activity. Each residue substitution in the pump molecule is indicated. The Ca2+ transients were induced in CHO cells overexpressing the mutant pumps and the luminescent Ca2+ indicator aequorin by the InsP3-linked purinergic agonist ATP: all mutant pumps (with the exception of the human V586M,in which the defect was only marginal) were deficient in the ability to export Ca2+ . The long term export of Ca2+ after the peak induced by the promotion of InsP3 was severely affected. The height of the Ca2+ peak was also affected, but only in two PMCA2 mutants.
and cadherin 23 are important actors in the complex process that permits the penetration of Ca2+ within hair cells and its modulation inside them in a way appropriate for the generation of the sound signal. However, depending on the severity of the defect, the development of the deafness phenotype could demand the impairment of both proteins, or of only one. 5. Concluding remarks Thanks to their stereocilia bundles, hair cells are fundamental in the process of auditory sensing. Accordingly, hearing loss may be characterized by the impairment in their activity, due to mutations of the proteins that initiate and regulate the mechano-electrical transduction process: cadherin 23 and the PMCA2 w/a pump, which is responsible for the extrusion of Ca2+ from the OHCs stereocilia. The maintenance of the delicate Ca2+ balance in the hair cells and in the endolymph that bathes their stereocilia is essential for the hearing process. Results on the human PMCA mutations G293S and V586 M which are accompanied by cadherin 23 mutations as well as mice deafness cases in which only the PMCA pump was defective, have revealed that the PMCA2 w/a pump of the stereocilia is key to the temporal and spatial shaping of the Ca2+ movements in the endolymph that are necessary for the production of the sound signals. Acknowledgments This work was supported by grants from the ERA-NET Neuron (nEUROsyn 2008) and from CARIPARO (Project of Excellence, call 2008–2009) to EC, and from the University of Padova (Atheneum project 2008) and from the Italian Ministry of University and Research (PRIN 2008) to MB. ADM and SP were supported by fellowships from the ERA-NET Neuron grant, MG was supported by a Research Fellowship from the CARIPARO grant.
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