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J. Biol. Chem., Vol. 276, Issue 33, 31233-31237, August 17, 2001
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From the Departments of Medicine and Physiology,
Cardiovascular Research Institute, University of California, San
Francisco, California 94143-0521
Received for publication, May 14, 2001, and in revised form, June 13, 2001
A role for aquaporins (AQPs) in hearing has been
suggested from the specific expression of aquaporins in inner ear and
the need for precise volume regulation in epithelial cells involved in
acoustic signal transduction. Using mice deficient in selected aquaporins as controls, we localized AQP1 in fibrocytes in the spiral
ligament and AQP4 in supporting epithelial cells (Hensen's, Claudius, and inner sulcus cells) in the organ of Corti. To
determine whether aquaporins play a role in hearing, auditory brain
stem response (ABR) thresholds were compared in wild-type mice and transgenic null mice lacking (individually) AQP1, AQP3, AQP4, and AQP5.
In 4-5-week-old mice in a CD1 genetic background, ABR thresholds in
response to a click stimulus were remarkably increased by >12 db in
AQP4 null mice compared with wild-type mice (p < 0.001), whereas ABR thresholds were not affected by AQP1, AQP3, or AQP5
deletion. In a C57/bl6 background, nearly all AQP4 null mice were deaf,
whereas ABRs could be elicited in wild-type controls. ABRs in AQP4 null
CD1 mice measured in response to tone bursts (4-20 kHz) indicated a
frequency-independent hearing deficit. Light microscopy showed no
differences in cochlear morphology of wild-type versus AQP4
null mice. These results provide the first direct evidence that an
aquaporin water channel plays a role in hearing. AQP4 may facilitate
rapid osmotic equilibration in epithelial cells in the organ of Corti,
which are subject to large K+ fluxes during
mechano-electric signal transduction.
The aquaporins (AQPs)1
are a family of small integral membrane proteins that function as water
transporters. Phenotype analysis of mice lacking aquaporins has
indicated that they play a physiological role in the kidney, central
nervous system, gastrointestinal system, and exocrine glands (reviewed
in Ref. 1). Mice lacking AQP1, AQP2, or AQP3 manifest nephrogenic
diabetes insipidus with defective urinary concentrating ability (2-4),
mice lacking AQP4 have altered brain water balance in response to
injury (5), and mice lacking AQP5 have defective saliva secretion (6).
In humans, mutations in AQP2 cause the autosomal form of hereditary
nephrogenic diabetes insipidus (7). Phenotype studies have also shown
the tissue-specific expression of an aquaporin does not prove its
physiological importance. For example, mice lacking AQP4 in gastric
parietal cells and skeletal muscle have unimpaired stomach acid
secretion (8) and skeletal muscle function (9). The data from aquaporin
null mice suggest that aquaporins are functionally important in tissues
carrying out rapid near isosmolar fluid transport or passive water
transport driven by osmotic gradients (1).
Several aquaporins have been localized in the mammalian inner ear and
have been proposed to play a role in hearing. Reverse transcriptase
polymerase chain reaction analysis of dissected rat inner ear
showed diffuse AQP1 transcript expression, specific expression of AQP2,
AQP3, and AQP4 in the endolymphatic sac, and expression of AQP5 in the
organ of Corti and Reissner's membrane (10). AQP1 was immunolocalized
in guinea pig inner ear in fibrocytes near the bone and lining the
endolymphatic duct and sac (11). Takumi et al. (12) found
only AQP1 and AQP4 in rat inner ear by immunostaining, with AQP4
localized to the basolateral membrane of Hensen's cells and basal
plasma membranes of Claudius cells and inner sulcus cells.
Similar findings were reported by Minami et al. (13).
In one study AQP5 was immunolocalized in cells lining the lateral wall
of rat cochlear duct, including the external sulcus and spiral
prominence (14). Although there is some disagreement among these
expression studies, it appears that several aquaporins are expressed in
the inner ear and thus might participate directly or indirectly in
hearing. Recently, Belyantseva et al. (15) reported
that water permeability in isolated rat cochlear outer hair cells
increased at 8-12 days after birth, corresponding in time to the onset
of hearing. Taken together with evidence that precise cochlear cell
volume regulation is critical to mechano-electric signal transduction
(16), it has been suggested without direct evidence that aquaporins
play an important role in hearing. However, hearing impairment in
humans is not associated with nephrogenic diabetes insipidus caused by
mutations in AQP2 or in reportedly asymptomatic humans lacking AQP1
(17).
The purpose of this study was to investigate the functional role of
aquaporins in hearing. The strategy was to compare auditory brain stem
response (ABR) signals in mice lacking each of the inner ear
aquaporins. We found remarkable hearing impairment in mice lacking AQP4
without anatomical abnormalities. The data provide the first direct
evidence for a role of an aquaporin in hearing, raising the possibility
that AQP4 may be involved in some forms of hearing impairment and that
modulation of AQP4 function in inner ear may be of therapeutic value.
Transgenic Mice--
Transgenic knockout mice deficient in AQP1,
AQP3, AQP4, and AQP5 (individually) in a CD1 genetic background were
generated by targeted gene disruption as described previously (2, 3, 6,
18). Measurements were done in litter-matched wild-type and knockout
mice produced by intercrossing of heterozygous mice. For experiments in
an inbred strain, the AQP4 null genotype was transferred to the C57/bl6
background by >8 back-crosses. For ABR measurements the investigators
were blinded to genotype information until completion of the analysis.
Protocols were approved by the University of California, San Francisco
Committee on Animal Research.
ABR Measurements--
ABR measurements were performed using a
Biopac MP100 work station equipped with differential amplifier
(ERS100B) and stimulator (STM100A) modules. Mice were anesthetized with
ketamine (80 mg/kg) and xylazine (14 mg/kg) by intraperitoneal
injection. Rectal temperature was monitored continuously using a
digital thermistor and maintained at 36-38 °C using a heating
block. Mice were placed in a grounded Faraday cage contained in a
light-proof sound isolation box. The three recording electrodes and
speaker input cables entered a small hole in the box.
Biaural sound stimuli were produced by a broad band speaker (Realistic,
Model 40-1310B) positioned 30 cm from the mice. Click stimuli were
generated by a square pulse of 0.1-ms duration and specified amplitude.
Tone stimuli were generated using a frequency synthesizer and
home-built waveform modulator to give a trapezoidal waveform with 1-ms
rise/fall times and 2-ms flat segment. Sound intensity calibrations
were done using a model C550H measurement microphone (Josephon
Engineering) positioned at the location of the mouse head. For
recording, subdermal stainless steel needle electrodes were placed at
the vertex and ventrolateral to the left and right ears. ABR waveforms
were recorded for 10 ms at a sampling rate of 45,000 Hz using 100-3000
Hz bandpass filter settings. Generally, waveforms from 200 stimuli at a
frequency of 12 Hz were averaged. ABR waveforms were recorded in 10-db
intervals down from a maximum amplitude of 90 db (for click stimuli)
until no waveform could be visualized. Waveforms were stored for
off-line analysis.
Immunocytochemistry and Histological Analysis--
Samples were
fixed by intracardiac perfusion with 4% paraformaldehyde in PBS (pH
7.4). The temporal bone was removed, and the cochlea was post-fixed
overnight in the same fixative solution. After decalcification, the
cochlea was dehydrated and embedded in Tissue-Tek OCT compound for
cryostat sections and in glycol methacrylate for plastic sections. For
histological examination, the cochlea was infiltrated with JB-4 monomer
(Polyscience Inc.), embedded under vacuum at room temperature,
sectioned on a microtome (Sorvall), and stained with toluidine blue.
For immunocytochemistry, 3-4-µm thick cryostat sections were
incubated for 30 min with PBS containing 1% bovine serum albumin and
then with affinity-purified aquaporin antibodies (dilution 1:400-1500)
for 2 h at 23 °C in PBS containing 1% bovine serum albumin.
Slides were rinsed with 2.7% NaC1 and then with PBS and incubated with
a secondary Cy3-conjugated sheep anti-rabbit F(ab)2
fragment (1:200) for visualization by fluorescence microscopy.
Data Analysis--
Data are reported as the mean ± S.E.
with p values determined by analysis of variance.
Aquaporin localization in the organ of Corti in the inner ears of
four sets of mice was done by immunostaining using specific antibodies
(Fig. 1). The AQP1 antibody strongly
labeled non-epithelial cells (fibrocytes) in the spiral ligament of
wild-type mice (top left) with no labeling in AQP1 null mice
(top right). The AQP4 antibody labeled supporting Hensen's
cells, inner sulcus cells, and Claudius cells in wild-type mice
(middle left) but not in AQP4 null mice (middle
right). No immunostaining with AQP5 antibody was found
(bottom left and right), despite strong label of
other tissues known to express AQP5 such as salivary gland
(inset). Immunostaining of AQP2 and AQP3 was negative, with
strongly positive controls (mouse kidney, not shown).
Hearing was evaluated functionally in wild-type and aquaporin null mice
of age 4-5 weeks by ABR analysis. Fig. 2
shows representative ABR waveforms in response to click stimuli of
different intensities. As reported in other ABR studies in mice (19),
at least four distinct peaks were identified corresponding to cochlear
nerve activity (wave I) and downstream neural activity (waves II-IV). Decreasing click intensities resulted in a decrease in wave amplitudes. As generally defined, the ABR threshold was identified in each series
of ABR waveforms as the lowest click intensity that produced at least
two clearly visible waves. ABR thresholds for the data in Fig. 2 were
40 db (wild-type mouse) and 50 db (AQP4 null mouse). Control
studies indicated that ABR thresholds were very reproducible in the
same mice measured on different days, with identical ABR thresholds in ~80% of mice and 5 db changes in most remaining mice.
Impaired Hearing in Mice Lacking Aquaporin-4 Water Channels*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (130K):
[in a new window]
Fig. 1.
Immunofluorescence localization of aquaporins
in mouse inner ear. Staining was done using immunopurified
anti-aquaporin antibodies as indicated in wild-type mice
(left) and corresponding aquaporin null mice
(right). Top, AQP1, arrowheads show
staining of fibrocytes. Middle, AQP4, arrowheads
show staining of Hensen's cells, Claudius cells, and inner sulcus
cells; inset shows same section in bright field after
staining. Bottom, AQP5, inset shows positive
control staining of mouse salivary gland. Scale bar, 100 µm.
View larger version (16K):
[in a new window]
Fig. 2.
Representative ABR waveforms measured in
wild-type and AQP4 null mice (age 4-5 weeks) measured in response to
click stimuli of indicated intensities. *, denotes ABR
threshold. See "Results" for further explanations.
Fig. 3 summarizes ABR thresholds measured
in a large series of wild-type and aquaporin null mice. Although
typical variability was found in different mice in the outbred CD1
genetic background, there was a significantly increased ABR threshold
in AQP4 null mice by >12 db (p < 0.001). ABR
thresholds in mice lacking AQP1, AQP3, or AQP5 did not differ
significantly from that in wild-type mice. Further studies were done in
C57/bl6 inbred mice into which the AQP4 null genotype was transferred.
Fig. 3 (right) shows that most AQP4 null mice were deaf,
whereas ABR waveforms could be elicited in matched wild-type mice. The
results indicated remarkably impaired hearing in AQP4 null mice.
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ABR waveforms from wild-type and AQP4 null mice in the CD1 background
were further analyzed. Fig. 4A
shows wave I amplitudes measured at 70, 60, and 50 db click
intensities. At each click intensity, the amplitudes of wave I were
significantly greater for wild-type than for AQP4 null mice. Fig.
4B summarizes amplitude ratios of wave I/wave II and wave
I/wave III for each mouse. There was no significant different in
amplitude ratios in wild-type versus AQP4 null mice.
Together, these results indicate that the hearing impairment arises
from defective cochlear rather than downstream neural function.
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Tone ABR analysis was done to determine whether the hearing impairment
in AQP4 null mice was frequency-dependent. Modulated tone
bursts were created using a custom-built frequency
synthesizer-modulator. Fig. 5A
shows time-domain waveforms along with frequency spectra deduced by
Fourier analysis of single clicks and tone bursts. Fig.
5B shows representative ABR waveforms in response to tone bursts. Qualitatively, ABR thresholds were increased in AQP4 null mice
at all frequencies. Fig. 6 summarizes
click and tone ABR thresholds for a series of wild-type and AQP4 null
mice, indicating that the hearing impairment in AQP4 null mice is not
frequency-specific.
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Morphological examination of plastic sections of inner ear was done to
determine whether anatomical differences could account for the impaired
hearing in AQP4 null mice. Fig. 7
(top) shows representative sections from two wild-type and
two AQP4 null mice. The thin (2 µm) plastic sections clearly show an
intact organ of Corti with well demarcated hair cells and supportive
cells. No morphological differences were apparent in sections of inner ear evaluated blindly from four wild-type and four AQP4 null mice. Fig.
7 (bottom) shows a schematic of the mouse cochlea based on the immunocytochemistry and histology studies (see
"Discussion").
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The principal goal of this study was to determine whether aquaporins play a functional role in hearing. Because there was disagreement in the literature about aquaporin expression patterns in mammalian inner ear, and no information was available to our knowledge on mouse inner ear, we determined by immunocytochemistry the expression pattern of aquaporins in mouse inner ear. Knockout mice lacking individual aquaporins served as controls. We found AQP1 protein in non-epithelial cells in spiral ligament and AQP4 in the basolateral plasma membranes of Hensen's cells and inner sulcus cells and the basal plasma membrane of Claudius cells in agreement with previous reports in guinea pig and/or rat. We did not find specific immunostaining of AQP2, AQP3, or AQP5, despite appropriate positive controls using mouse tissues known to express these aquaporins. Nevertheless, recognizing the limitations of antibody detection of aquaporins, ABR analysis was done on wild-type mice and knockout mice lacking AQP1, AQP3, AQP4, and AQP5. Although an AQP2 knock-in mouse model of autosomal recessive nephrogenic diabetes insipidus was recently created (4), these mice were not suitable for ABR analysis because they generally did not survive beyond the first week of life. We found remarkable hearing impairment in AQP4 null mice, with no significant effect of deletion of AQP1, AQP3, or AQP5. The hearing-impaired AQP4 null mice did not show abnormalities in cochlear morphology at the light microscopic level. These results suggest that AQP4-mediated water transport in supportive epithelial cells in the organ of Corti is required for normal hearing.
ABR analysis in mice is an established approach to detect hearing impairment resulting from a variety of genetic and acquired diseases (20-23). The measurements here were done in young adult mice to avoid the age-dependent hearing impairment that occurs in many mouse strains (19, 43), which could confound the interpretation of results. Hearing impairment in AQP4 null mice was found both in an outbred (CD1) and inbred (C57/bl6) genetic background. The ABR includes several waves; the first waves represent cochlear and auditory nerve activity, and the late waves arise from electrical activity in central auditory pathways (24, 25). The AQP4 null mice with elevated ABR thresholds did not have aberrant ABR wave patterns, as quantified by the amplitude ratios of wave I/wave II and wave I/wave III, suggesting that the hearing impairment in AQP4 null mice is caused by defects in the proximal rather than central part of the hearing system. Tone pulse ABR measurements indicated the hearing impairment was not frequency-dependent.
Based on the biology of AQP4, we speculated about its role in hearing. AQP4 is an efficient water-selective transporting protein that does not carry protons, ions, or other small solutes (26). AQP4 was originally cloned from rat lung (27), and subsequently various isoforms in brain (28), stomach (29) and other mammals have been sequenced. Knockout mice lacking AQP4 manifest a mild urinary concentrating defect (18) because of impaired water transport in the inner medullary collecting duct (30). However, AQP4 is most strongly expressed in the nervous system, in astroglia throughout the brain and spinal cord, and in retinal Muller cells (31, 32). AQP4 null mice showed remarkably altered cerebral water balance with protection from brain edema in a hyponatremia model of vasogenic edema and an ischemic stroke model of cytotoxic edema (5). A unique feature of AQP4 is its assembly in membranes in regular square arrays, called orthogonal arrays of particles; these are seen by freeze-fracture electron microscopy in AQP4-transfected cells (33) and are found in the brain, kidney, and muscle of wild-type but not AQP4 null mice (34).
The cochlea contains sensory hair cells that are responsible for transducing sound waves into electrical impulses. Hair-like stereocilica on the upper surface of sensory hair cells project into a cavity filled with endolymph fluid (Fig. 7, bottom). Acoustic stimuli reaching the inner ear deflect the stereocilia, resulting in K+ channel activation, K+ flooding from the endolymph into hair cells, hair cell membrane depolarization, and the generation of an electrical signal carried by the auditory nerve to the brain. The K+ concentration in endolymph is high and the Na+ concentration is low, resulting in a high resting endocochlear potential. The maintenance of high K+ concentration in endolymph after acoustic signal transduction requires rapid recycling of K+ into the endolymph (35, 36). Indeed, at least six of the many proteins associated with deafness in humans and mice are probably involved in K+ recycling (37), including the KCNQ4 K+ channel, which is mutated in one type of dominant progressive hearing loss (36). The K+ ions are then taken up by the supporting hair cells, pass through a network of gap junctions that extends from the epithelial supporting cells to the mesenchymal fibrocytes that form the spiral ligament, and are secreted by epithelial marginal cells of the stria vascularis.
AQP4 may be involved in maintaining osmotic balance during
K+ recycling, because AQP4 is expressed selectively in
supporting hair cells in the basolateral plasma membranes of
Hensen's cells and in the basal plasma membrane of Claudius cells. We
propose that the uptake of K+ by Hensen's cells drives
AQP4-mediated water influx and that water exits the supporting cell
syncytium through AQP4-expressing basal membranes of Claudius cells
that face the root of the spiral ligament. The hearing deficit in AQP4
null mice may thus result from altered basal ionic composition of
endolymph and/or outer hair cell volume. The involvement of AQP4 in
facilitating K+ flux in supporting cells may be a general
paradigm in the physiology of neuroexcitable tissues. In the central
nervous system, AQP4 in astroglia is proposed to facilitate
K+ fluxes associated with adjacent neurons (38-40), and in
the eye AQP4 in retinal Muller cells is proposed to facilitate
K+ fluxes associated with adjacent bipolar cells (41). A
close molecular association of AQP4 in orthogonal arrays of particles with K+ channels has been proposed (42). The challenge will
be to establish the cellular and molecular mechanisms by which AQP4
facilitates K+ and water movement in excitable tissues.
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ACKNOWLEDGEMENT |
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We thank Liman Qian for transgenic mouse breeding and genotype analysis.
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FOOTNOTES |
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* This study was supported by National Institutes of Health Grants HL59198, DK35124, HL60288, and DK43840 and Grant R613 from the National Cystic Fibrosis Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Cardiovascular
Research Institute, 1246 Health Sciences East Tower, Box 0521, University of California, San Francisco, San Francisco, CA. 94143-0521. Tel.: 415-476-8530; Fax: 415-665-3847; E-mail:
verkman@itsa.ucsf.edu.
Published, JBC Papers in Press, June 13, 2001, DOI 10.1074/jbc.M104368200
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ABBREVIATIONS |
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The abbreviations used are: AQP, aquaporin; ABR, auditory brain stem response; PBS, phosphate-buffered saline.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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 M.-O. Trowe, H. Maier, M. Schweizer, and A. Kispert Deafness in mice lacking the T-box transcription factor Tbx18 in otic fibrocytes Development, May 1, 2008; 135(9): 1725 - 1734. [Abstract] [Full Text] [PDF]
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 F. Lang, V. Vallon, M. Knipper, and P. Wangemann Functional significance of channels and transporters expressed in the inner ear and kidney Am J Physiol Cell Physiol, October 1, 2007; 293(4): C1187 - C1208. [Abstract] [Full Text] [PDF]
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 J. Ruiz-Ederra, H. Zhang, and A. S. Verkman Evidence against Functional Interaction between Aquaporin-4 Water Channels and Kir4.1 Potassium Channels in Retinal Muller Cells J. Biol. Chem., July 27, 2007; 282(30): 21866 - 21872. [Abstract] [Full Text] [PDF]
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 K. J. Watson, I. Kim, A. F. Baquero, C. A. Burks, L. Liu, and T. A. Gilbertson Expression of Aquaporin Water Channels in Rat Taste Buds Chem Senses, June 1, 2007; 32(5): 411 - 421. [Abstract] [Full Text] [PDF]
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 Y. Wang and E. Tajkhorshid Molecular Mechanisms of Conduction and Selectivity in Aquaporin Water Channels J. Nutr., June 1, 2007; 137(6): 1509S - 1515S. [Abstract] [Full Text] [PDF]
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 H.-F. Huang, R.-H. He, C.-C. Sun, Y. Zhang, Q.-X. Meng, and Y.-Y. Ma Function of aquaporins in female and male reproductive systems Hum. Reprod. Update, November 1, 2006; 12(6): 785 - 795. [Abstract] [Full Text] [PDF]
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 A. S. Verkman More than just water channels: unexpected cellular roles of aquaporins J. Cell Sci., August 1, 2005; 118(15): 3225 - 3232. [Abstract] [Full Text] [PDF]
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M. C. Papadopoulos and A. S. Verkman Aquaporin-4 Gene Disruption in Mice Reduces Brain Swelling and Mortality in Pneumococcal Meningitis J. Biol. Chem., April 8, 2005; 280(14): 13906 - 13912. [Abstract] [Full Text] [PDF]
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 M. H. Levin and A. S. Verkman Aquaporin-Dependent Water Permeation at the Mouse Ocular Surface: In Vivo Microfluorimetric Measurements in Cornea and Conjunctiva Invest. Ophthalmol. Vis. Sci., December 1, 2004; 45(12): 4423 - 4432. [Abstract] [Full Text] [PDF]
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T. Da and A. S. Verkman Aquaporin-4 Gene Disruption in Mice Protects against Impaired Retinal Function and Cell Death after Ischemia Invest. Ophthalmol. Vis. Sci., December 1, 2004; 45(12): 4477 - 4483. [Abstract] [Full Text] [PDF]
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 D. K. Binder, M. C. Papadopoulos, P. M. Haggie, and A. S. Verkman In Vivo Measurement of Brain Extracellular Space Diffusion by Cortical Surface Photobleaching J. Neurosci., September 15, 2004; 24(37): 8049 - 8056. [Abstract] [Full Text] [PDF]
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E. Solenov, H. Watanabe, G. T. Manley, and A. S. Verkman Sevenfold-reduced osmotic water permeability in primary astrocyte cultures from AQP-4-deficient mice, measured by a fluorescence quenching method Am J Physiol Cell Physiol, February 1, 2004; 286(2): C426 - C432. [Abstract] [Full Text]
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L. E. Ball, M. Little, M. W. Nowak, D. L. Garland, R. K. Crouch, and K. L. Schey Water Permeability of C-Terminally Truncated Aquaporin 0 (AQP0 1-243) Observed in the Aging Human Lens Invest. Ophthalmol. Vis. Sci., November 1, 2003; 44(11): 4820 - 4828. [Abstract] [Full Text] [PDF]
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P. L. Splinter, A. I. Masyuk, and N. F. LaRusso Specific Inhibition of AQP1 Water Channels in Isolated Rat Intrahepatic Bile Duct Units by Small Interfering RNAs J. Biol. Chem., February 14, 2003; 278(8): 6268 - 6274. [Abstract] [Full Text] [PDF]
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 Z. Borok and A. S. Verkman Lung Edema Clearance: 20 Years of Progress: Invited Review: Role of aquaporin water channels in fluid transport in lung and airways J Appl Physiol, December 1, 2002; 93(6): 2199 - 2206. [Abstract] [Full Text] [PDF]
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 D. Zhang, L. Vetrivel, and A.S. Verkman Aquaporin Deletion in Mice Reduces Intraocular Pressure and Aqueous Fluid Production J. Gen. Physiol., May 28, 2002; 119(6): 561 - 569. [Abstract] [Full Text] [PDF]
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T. Ma, M. Hara, R. Sougrat, J.-M. Verbavatz, and A. S. Verkman Impaired Stratum Corneum Hydration in Mice Lacking Epidermal Water Channel Aquaporin-3 J. Biol. Chem., May 3, 2002; 277(19): 17147 - 17153. [Abstract] [Full Text] [PDF]
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J. Li, R. V. Patil, and A. S. Verkman Mildly Abnormal Retinal Function in Transgenic Mice without Muller Cell Aquaporin-4 Water Channels Invest. Ophthalmol. Vis. Sci., February 1, 2002; 43(2): 573 - 579. [Abstract] [Full Text] [PDF]
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 B. L. de Groot and H. Grubmuller Water Permeation Across Biological Membranes: Mechanism and Dynamics of Aquaporin-1 and GlpF Science, December 14, 2001; 294(5550): 2353 - 2357. [Abstract] [Full Text] [PDF]
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