Low Endolymph Calcium Concentrations in deafwaddler2J Mice Suggest that PMCA2 Contributes to Endolymph Calcium Maintenance

Wood, Jason; Muchinsky, Sara; Filoteo, Adelaida G.; Penniston, John T.; Tempel, Bruce L.

doi:10.1007/s10162-003-4022-1

Cited by 68 publications

(89 citation statements)

References 43 publications

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“…The mutations impair the longer-term export of Ca 2ϩ , in agreement with the measurements of endolymphatic Ca 2ϩ levels in the dfw mouse model (12), but endolymphatic Ca 2ϩ also influences stereociliary Ca 2ϩ . By controlling the multiple phases of adaptation, Ca 2ϩ influx contributes to setting the resting position of the hair bundle and thus influences the fraction of MET channels open at rest.…”

Section: Discussionsupporting

confidence: 83%

“…The apical surface of hair cells is bathed in endolymph, which is rich in K ϩ but low in Na ϩ and Ca 2ϩ (5). K ϩ carries most the transduction current, but MET channels are Ca 2ϩ selective, i.e., Ca 2ϩ influx is significant even at the low Ca 2ϩ levels of the endolymph, which are much lower than those of other extracellular fluids (6-10): 20-23 M in the rodent cochlea (11,12), 200-250 M in the vestibular system, possibly because of the presence there of calcium carbonate crystals (13,14). Approximately 10% of the MET current may actually be carried by Ca 2ϩ ions (15).…”

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confidence: 99%

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A functional study of plasma-membrane calcium-pump isoform 2 mutants causing digenic deafness

Ficarella

Leva²,

Bortolozzi

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

114

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Ca 2؉ enters the stereocilia of hair cells through mechanoelectrical transduction channels opened by the deflection of the hair bundle and is exported back to endolymph by an unusual splicing isoform (w/a) of plasma-membrane calcium-pump isoform 2 (PMCA2). Ablation or missense mutations of the pump cause deafness, as described for the G283S mutation in the deafwaddler (dfw) mouse. A deafness-inducing missense mutation of PMCA2 (G293S) has been identified in a human family. The family also was screened for mutations in cadherin 23, which accentuated hearing loss in a previously described human family with a PMCA2 mutation. A T1999S substitution was detected in the cadherin 23 gene of the healthy father and affected son but not in that of the unaffected mother, who presented instead the PMCA2 mutation. The w/a isoform was overexpressed in CHO cells. At variance with the other PMCA2 isoforms, it became activated only marginally when exposed to a Ca 2؉ pulse. The G293S and G283S mutations delayed the dissipation of Ca 2؉ transients induced in CHO cells by InsP3. In organotypic cultures, Ca 2؉ imaging of vestibular hair cells showed that the dissipation of stereociliary Ca 2؉ transients induced by Ca 2؉ uncaging was compromised in the dfw and PMCA2 knockout mice, as was the sensitivity of the mechanoelectrical transduction channels to hair bundle displacement in cochlear hair cells.hereditary hearing loss ͉ mutations ͉ calcium homeostasis ͉ calcium transport T he receptive organelle of sensory hair cells in the mammalian cochlea, the stereocilia bundle, protrudes from the cell's apical surface. Inner hair cells transduce mechanical vibrations into electrical signals that are eventually transmitted to the brain to be transformed into hearing signals (1), whereas outer hair cells (OHCs) (2) amplify the vibrations of the basilar membrane (3). Mechanical stimuli that are detected as excitatory deflect a hair bundle, thus increasing tension in the tip link, a filament stretched between the tops of stereocilia. This tension is conveyed to mechanosensitive transduction (MET) channels that open to allow ions into the cell (4). The apical surface of hair cells is bathed in endolymph, which is rich in K ϩ but low in Na ϩ and Ca 2ϩ (5). K ϩ carries most the transduction current, but MET channels are Ca 2ϩ selective, i.e., Ca 2ϩ influx is significant even at the low Ca 2ϩ levels of the endolymph, which are much lower than those of other extracellular fluids (6-10): 20-23 M in the rodent cochlea (11, 12), 200-250 M in the vestibular system, possibly because of the presence there of calcium carbonate crystals (13,14). Approximately 10% of the MET current may actually be carried by Ca 2ϩ ions (15). Ca 2ϩ entering through MET channels is rapidly sequestered by buffers in the stereocilia (16,17) and is shuttled back to endolymph by the plasma membrane Ca 2ϩ pump (PMCA) (18,19), which is very concentrated in the stereocilia membrane (Ϸ2,000 per squared micrometer) (15, 19-21). The PMCA is assumed to increase Ca 2ϩ in the immediate pro...

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Section: Discussionsupporting

confidence: 83%

mentioning

confidence: 99%

A functional study of plasma-membrane calcium-pump isoform 2 mutants causing digenic deafness

Ficarella

Leva²,

Bortolozzi

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

114

View full text Add to dashboard Cite

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“…1, available at www.jneurosci.org as supplemental material). The endolymph concentration of Ca 2ϩ is unusually low (ϳ7 M compared with 23 M in control) in mice with a mutation in PMCA2 (Wood et al, 2004), much closer to the equilibrium concentration of ϳ2 M calculated from the Nernst equation. Because the only cochlear PMCA2 exposed to endolymph is that of the stereocilia (Dumont et al, 2001;Wood et al, 2004), PMCA2-dependent elevated endolymph Ca 2ϩ reports net extrusion by outer hair cells.…”

Section: Segregation Of Pmca Isoforms In the Cochleasupporting

confidence: 67%

Splice-Site A Choice Targets Plasma-Membrane Ca2+-ATPase Isoform 2 to Hair Bundles

Hill

Williams

LeMasurier

et al. 2006

Journal of Neuroscience

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Localization of mechanotransduction in sensory hair cells to hair bundles requires selective targeting of essential proteins to specific locations. Isoform 2 of the plasma-membrane Ca 2ϩ -ATPase (PMCA2), required for hearing and balance, is found exclusively in hair bundles. We determined the contribution of splicing at the two major splicing sites (A and C) to hair-cell targeting of PMCA2. When PMCA2 isoforms were immunoprecipitated from purified hair bundles of rat utricle, 2w was the only site A variant detected; moreover, immunocytochemistry for 2w in rat vestibular and cochlear tissues indicated that this splice form was located solely in bundles. To demonstrate the necessity of the 2w sequence, we transfected hair cells with PMCA2 containing different variants at splice sites A and C. Although native hair bundles exclusively use the 2a form at splice-site C, epitope-tagged PMCA2w/a and PMCA2w/b were both concentrated in bundles, indicating that site C is not involved in bundle targeting. In contrast, PMCA2z/a was excluded from bundles and was instead targeted to the basolateral plasma membrane. Bundle-specific targeting of PMCA2w/a tagged with green fluorescent protein (GFP) was diminished, suggesting that GFP interfered with splice-site A. Together, these data demonstrate that PMCA2w/a is the hairbundle isoform of PMCA in rat hair cells and that 2w targets PMCA2 to bundles. The 2w sequence is thus the first targeting signal identified for a hair-bundle membrane protein; moreover, the striking distribution of inner-ear PMCA isoforms dictated by selective targeting suggests a critical functional role for segregated pathways of Ca 2ϩ transport.

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“…Operating in many places (e.g. gut, kidney, placenta, teeth, bones, oviduct, lung, inner ear), active transport is used to control the amount of calcium in body fluids and so impacts on nutrition, biomineralization, fertility, respiration, and hearing (1,2). Superior control is achieved by passaging calcium actively through cells rather than passively between them, but this comes at the risk of cytotoxicity should the ability to regulate intracellular calcium be overburdened.…”

mentioning

confidence: 99%

Calbindin Independence of Calcium Transport in Developing Teeth Contradicts the Calcium Ferry Dogma

Turnbull

Looi

Mangum

et al. 2004

Journal of Biological Chemistry

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Cytosolic calcium-binding proteins termed calbindins are widely regarded as a key component of the machinery used to transport calcium safely across cells. Acting as mobile buffers, calbindins are thought to ferry calcium in bulk and simultaneously protect against its potentially cytotoxic effects. Here, we contradict this dogma by showing that teeth and bones were produced normally in null mutant mice lacking calbindin 28kDa . Structural analysis of dental enamel, the development of which depends critically on active calcium transport, showed that mineralization was unaffected in calbindin 28kDa -null mutants. An unchanged rate of calcium transport was verified by measurements of 45 Ca incorporation into developing teeth in vivo. In enamelforming cells, the absence of calbindin 28kDa was not compensated by other cytosolic calcium-binding proteins as detectable by 45 Ca overlay, two-dimensional gel, and equilibrium binding analyses. Despite a 33% decrease in cytosolic buffer capacity, cytotoxicity was not evident in either the null mutant enamel or its formative cells. This is the first definitive evidence that calbindins are not required for active calcium transport, either as ferries or as facilitative buffers. Moreover, in challenging the broader notion of a cytosolic route for calcium, the findings support an alternative paradigm involving passage via calcium-tolerant organelles.The active transport of calcium across cells holds widespread importance in medicine and biology, yet the underlying mechanisms remain unclear. Operating in many places (e.g. gut, kidney, placenta, teeth, bones, oviduct, lung, inner ear), active transport is used to control the amount of calcium in body fluids and so impacts on nutrition, biomineralization, fertility, respiration, and hearing (1, 2). Superior control is achieved by passaging calcium actively through cells rather than passively between them, but this comes at the risk of cytotoxicity should the ability to regulate intracellular calcium be overburdened.Mechanistically, active transport is considered in three steps: the entry of calcium to the cell, transit across it, and extrusion at the other side. The transit step has received the most attention over several decades, being considered rate-limiting and having key molecular players defined. However, recent molecular characterization of calcium entry channels has transformed the field by providing a new mechanistic focus for vitamin D-restricted transport (3, 4). With these advances reigniting interest in therapeutic applications, it is important to revisit what happens following calcium entry.The 30-year-old paradigm that calcium is ferried through cytosol by mobile calcium-binding proteins (calbindins) remains widely accepted (3-9). Calbindins are thought to facilitate the naturally poor diffusion of calcium in cytosol and simultaneously buffer calcium at safe concentrations. Comprehensively supporting this view, tight correlations between calbindin expression and vitamin D-dependent transport were found in intestine...

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Low Endolymph Calcium Concentrations in deafwaddler2J Mice Suggest that PMCA2 Contributes to Endolymph Calcium Maintenance

Cited by 68 publications

References 43 publications

A functional study of plasma-membrane calcium-pump isoform 2 mutants causing digenic deafness

A functional study of plasma-membrane calcium-pump isoform 2 mutants causing digenic deafness

Splice-Site A Choice Targets Plasma-Membrane Ca2+-ATPase Isoform 2 to Hair Bundles

Calbindin Independence of Calcium Transport in Developing Teeth Contradicts the Calcium Ferry Dogma

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