Excitation of fluorescent dyes inactivates the outer hair cell integral membrane motor protein prestin and betrays its lateral mobility

Santos‐Sacchi, Joseph; Zhao, Hong-Bo

doi:10.1007/s00424-003-1053-8

Cited by 16 publications

(23 citation statements)

References 32 publications

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“…However, there was no prestin labeling in the underlying SSC. This is consistent with previous reports that the OHC motor protein is located within the plasma membrane in the OHC lateral wall Huang and SantosSacchi, 1994;Santos-Sacchi and Zhao, 2003). Figs.…”

Section: Resultssupporting

confidence: 93%

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Prestin is expressed on the whole outer hair cell basolateral surface

Zhu

Zhao

2006

Brain Research

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Prestin has been identified as a motor protein responsible for outer hair cell (OHC) electromotility. Previous experiments revealed that OHC electromotility and its associated nonlinear capacitance resided in the OHC lateral wall and was not detected at the apical cuticular plate and basal region. In this experiment, the distribution of prestin in adult mouse, rat, and guinea pig OHCs was reexamined by use of immunofluorescent staining and confocal microscopy. We found that prestin labeling was located at the whole OHC basolateral wall, including the basal plasma membrane. However, staining at the basal membrane was weak. As compared with the intensity at the lateral wall, the intensities of prestin labeling at the membrane at the nuclear level and basal pole were 80.5% and 61.1%, respectively. Prestin labeling was not found at the cuticular plate and stereocilia. The prestin labeling was also absent in the cytoplasm and nuclei. The OHC lateral wall above the nuclear level is composed of the plasma membrane, cortical lattice, and subsurface cisternae. By costaining with di-8-ANEPPS, prestin labeling was found at the outer layer of the OHC lateral wall, which was further evidenced by use of a hypotonic challenge to separate the plasma membrane from the underlying subsurface cisternae. The data revealed that prestin is expressed at the whole OHC basolateral membrane. Prestin in the basal plasma membrane may provide a reservoir on the OHC surface for prestin-recycling and may also facilitate performing its hypothesized transporter function.

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Section: Resultssupporting

confidence: 93%

“…The lateral diffusion in the OHC lateral wall has been reported (Oghalai et al, 2000;Santos-Sacchi and Zhao, 2003). For some unknown mechanisms, the prestin proteins may be restricted in vivo and could not diffuse down to the nucleus level.…”

Section: Expression Of Prestin In the Ohc Subnuclear Regionmentioning

confidence: 99%

Prestin is expressed on the whole outer hair cell basolateral surface

Zhu

Zhao

2006

Brain Research

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“…The OHCs were freshly isolated from adult guinea pigs (250-400 g) as previously described [10,15,[26][27]. Briefly, the temporal bones were removed after decapitation.…”

Section: Outer Hair Cell Preparationmentioning

confidence: 99%

“…The recording was performed by jClamp (SciSoft, New Haven, CT, USA). The OHC electromotility-associated NLC was measured with a two-sinusoidal wave voltage stimulus in jClamp [27,28]. This voltage stimulus was composed of a ramp command (−150 to +150 mV) summed with twosinusoidal commands (f 1 =390.6 Hz, f 2 = 781.3 Hz, 25 mV peak to peak).…”

Section: Patch Clamp Recording and Nonlinear Capacitance Measurementmentioning

confidence: 99%

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ATP activates P2x receptors and requires extracellular Ca++ participation to modify outer hair cell nonlinear capacitance

Zhao

2008

Pflugers Arch - Eur J Physiol

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Intracochlear ATP is an important mediator in regulating hearing function. ATP can activate ionotropic purinergic (P2x) and metabotropic purinergic (P2y) receptors to influence cell functions. In this paper, we report that ATP can activate P2x receptors directly to modify outer hair cell (OHC) electromotility, which is an active cochlear amplifier determining hearing sensitivity and frequency selectivity in mammals. We found that ATP, but not UTP, a P2y receptor agonist, reduced the OHC electromotility-associated nonlinear capacitance (NLC) and shifted its voltage dependence to the right (depolarizing) direction. Blockage of the activation of P2x receptors by pyridox-alphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS), suramin, and 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) could block the ATP effect. This modification also required extracellular Ca ++ participation. Removal of extracellular Ca ++ abolished the ATP effect. However, chelation of intracellular Ca ++ concentration by a fast calciumchelating reagent 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA, 10 mM) did not affect the effect of ATP on NLC. The effect is also independent of K + ions. Substitution of Cs + for intracellular or extracellular K + did not affect the ATP effect. Our findings indicate that ATP activates P2x receptors instead of P2y receptors to modify OHC electromotility. Extracellular Ca ++ is required for this modification.

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2010

Handbook of Biological Dyes and Stains

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Excitation of fluorescent dyes inactivates the outer hair cell integral membrane motor protein prestin and betrays its lateral mobility

Cited by 16 publications

References 32 publications

Prestin is expressed on the whole outer hair cell basolateral surface

Prestin is expressed on the whole outer hair cell basolateral surface

ATP activates P2x receptors and requires extracellular Ca++ participation to modify outer hair cell nonlinear capacitance

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