Identification and cloning of site C splice variants of plasma membrane Ca-ATPase in the gerbil cochlea

Crouch, James J.; Schulte, Bradley A.

doi:10.1016/s0378-5955(96)00132-3

Cited by 21 publications

(17 citation statements)

References 23 publications

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“…5F10 immunoreactivity was absent from hair bundles of mutant mice that lacked PMCA2 expression, suggesting that this isozyme was prominent there (Street et al, 1998;Takahashi and Kitamura, 1999). RT-PCR experiments identified PMCA1-4 gene products, as well as many of their splice variants, in cochlea (Crouch and Schulte, 1996;Furuta et al, 1998); furthermore, in situ hybridization experiments localized PMCA2 transcripts to outer hair cells and PMCA1 to inner hair cells (Furuta et al, 1998). Because PMCA labeling was prominent in outer hair cells and PMCA2 transcripts were localized there and because PMCA2b was the only PMCA2 splice variant identified in cochlea by RT-PCR, PMCA2b was hypothesized to be the stereocilia isoform (Crouch and Schulte, 1995;Furuta et al, 1998).…”

Section: Pmca Isoforms In the Mammalian Vestibular And Auditory Systemsmentioning

confidence: 98%

“…All PMCA isoforms and many of their splice variants are expressed in the mammalian cochlea (Crouch and Schulte, 1996;Furuta et al, 1998). Furthermore, hair cells contain high levels of PMCA (Crouch and Schulte, 1995;Apicella et al, 1997;Street et al, 1998;Yamoah et al, 1998); levels are particularly high in the hair bundle (Crouch and Schulte, 1995;Yamoah et al, 1998).…”

Section: Camentioning

confidence: 99%

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Plasma Membrane Ca²⁺-ATPase Isoform 2a Is the PMCA of Hair Bundles

et al. 2001

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Mechanoelectrical transduction channels of hair cells allow for the entry of appreciable amounts of Ca2+, which regulates adaptation and triggers the mechanical activity of hair bundles. Most Ca2+that enters transduction channels is extruded by the plasma membrane Ca2+-ATPase (PMCA), a Ca2+pump that is highly concentrated in hair bundles and may be essential for normal hair cell function. Because PMCA isozymes and splice forms are regulated differentially and have distinct biochemical properties, we determined the identity of hair bundle PMCA in frog and rat hair cells. By screening a bullfrog saccular cDNA library, we identified abundant PMCA1b and PMCA2a clones as well as rare PMCA2b and PMCA2c clones. Using immunocytochemistry and immunoprecipitation experiments, we showed in bullfrog sacculus that PMCA1b is the major isozyme of hair cell and supporting cell basolateral membranes and that PMCA2a is the only PMCA present in hair bundles. This complete segregation of PMCA1 and PMCA2 isozymes holds for rat auditory and vestibular hair cells; PMCA2a is the only PMCA isoform in hair bundles of outer hair cells and vestibular hair cells and is the predominant PMCA of hair bundles of inner hair cells. Our data suggest that hair cells control plasma membrane Ca2+-pumping activity by targeting specific PMCA isozymes to distinct subcellular locations. Because PMCA2a is the only Ca2+pump present at appreciable levels in hair bundles, the biochemical properties of this pump must account fully for the physiological features of transmembrane Ca2+pumping in bundles.

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Section: Pmca Isoforms In the Mammalian Vestibular And Auditory Systemsmentioning

confidence: 98%

Section: Camentioning

confidence: 99%

Plasma Membrane Ca²⁺-ATPase Isoform 2a Is the PMCA of Hair Bundles

et al. 2001

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“…Products of the PCR were separated on 1% agarose gels and visualized by ethidium bromide staining. PCR products were cloned and sequenced as described previously (Crouch and Schulte 1996). …”

Section: Reverse-transcription Polymerase Chain Reactionmentioning

confidence: 99%

Immunohistochemical Localization of the Na-K-Cl Co-transporter (NKCC1) in the Gerbil Inner Ear

Crouch

Sakaguchi

Lytle

et al. 1997

J Histochem Cytochem.

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165

133

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SUMMARYWe mapped the cellular and subcellular distribution of the Na-K-Cl co-transporter (NKCC) in the adult gerbil inner ear by immunostaining with a monoclonal antibody (MAb T4) generated against human colon NKCC. Heavy immunolabeling was seen in the basolateral plasma membrane of marginal cells in the stria vascularis and dark cells in the vestibular system. Subpopulations of fibrocytes in the cochlear spiral ligament and limbus and underlying the vestibular neurosensory epithelium also stained with moderate to strong intensity, apparently along their entire plasmalemma. Because MAb T4 recognizes both the basolateral secretory (NKCC1) and the apical absorptive (NKCC2) isoforms of the co-transporter, we employed reverse transcription and the polymerase chain reaction (RT-PCR) to explore isoform diversity in inner ear tissues. Using NKCC1 and NKCC2 isoform-specific PCR primers based on mouse and human sequences, only transcripts for NKCC1 were detected in the gerbil inner ear. The presence of abundant NKCC1 in the basolateral plasmalemma of strial marginal and vestibular dark cells confirms conclusions drawn from pharmacological and physiological data. The co-expression of NKCC1 and Na,K-ATPase in highly specialized subpopulations of cochlear and vestibular fibrocytes provides further evidence for their role in recycling K ϩ leaked or effluxed through hair cells into perilymph back to endolymph, as postulated in current models of inner ear ion homeostasis.

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“…Although all four PMCA gene products and many splice variants are expressed in the mammalian cochlea (Crouch and Schulte, 1996;Furuta et al, 1998), only PMCA2 is known to be essential for hearing and balance (Noben-Trauth et al, 1997;Kozel et al, 1998;Street et al, 1998;Takahashi and Kitamura, 1999). PMCA2 with the 2a variant at splice-site C is the only PMCA expressed in frog and rat hair bundles (Dumont et al, 2001).…”

Section: Introductionmentioning

confidence: 99%

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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Identification and cloning of site C splice variants of plasma membrane Ca-ATPase in the gerbil cochlea

Cited by 21 publications

References 23 publications

Plasma Membrane Ca²⁺-ATPase Isoform 2a Is the PMCA of Hair Bundles

Plasma Membrane Ca²⁺-ATPase Isoform 2a Is the PMCA of Hair Bundles

Immunohistochemical Localization of the Na-K-Cl Co-transporter (NKCC1) in the Gerbil Inner Ear

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

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Identification and cloning of site C splice variants of plasma membrane Ca-ATPase in the gerbil cochlea

Cited by 21 publications

References 23 publications

Plasma Membrane Ca2+-ATPase Isoform 2a Is the PMCA of Hair Bundles

Plasma Membrane Ca2+-ATPase Isoform 2a Is the PMCA of Hair Bundles

Immunohistochemical Localization of the Na-K-Cl Co-transporter (NKCC1) in the Gerbil Inner Ear

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

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Plasma Membrane Ca²⁺-ATPase Isoform 2a Is the PMCA of Hair Bundles

Plasma Membrane Ca²⁺-ATPase Isoform 2a Is the PMCA of Hair Bundles