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Laboratoire de Biologie Cellulaire et Moléculaire de l'Audition, Equipe Mixte Institut National de la Santé et de la Recherche Médicale 99-27, Université de Bordeaux 2, Centre Hospitalier Universitaire Hôpital Pellegrin, 33076 Bordeaux, France
Submitted 23 December 2002; accepted in final form 9 February 2003
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ABSTRACT |
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ranging between 0.5
and 1 ms). A similar block of a fast outward current was also observed with
the extracellular application of barium ions, which we believe permeate
through Ca2+ channels and block BK channels. In situ
hybridization of Slo antisense riboprobes and immunocytochemistry
demonstrated a strong expression of BK channels in IHCs and spiral ganglion
and to a lesser extent in OHCs. Overall, our results clearly revealed the
importance of BK channels in mammalian cochlear neurotransmission and
demonstrated that at the presynaptic level, fast BK channels are a significant
component of the repolarizing current of IHCs. |
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INTRODUCTION |
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;
Vergara et al. 1998).
Small-conductance Ca2+-activated K+
channels (SK channels) have a small unitary conductance (<20 pS), are
generally voltage independent, are sensitive to the bee venom toxin apamin,
and are activated by an increase in the levels of intracellular
Ca2+ such as occurs during an action potential. SK
channels generally underlie the slow afterhyperpolarization that limits firing
frequency during a train of action potentials (see
Sah 1996). Three mammalian SK
channels (SK1, SK2, SK3) that demonstrate a high degree of homology and high
sensitivity to Ca2+ have been cloned by Köhler et
al. (1996). SK channels, and
in particular SK2 channels, are expressed in cochlear outer hair cells (OHCs)
(Dulon et al. 1998;
Oliver et al. 2000). These SK
channels underlie the fast cholinergic hyperpolarization of cochlear OHCs.
They are functionally coupled to Ca2+-permeable
nicotinic receptors, composed of 
9 and 10 nAChRs subunits
(Elgoyhen et al. 1994,
2001), at the postsynaptic OHC
membrane (Blanchet et al.
1996).
Intermediate Ca2+-activated K+ channels exhibit unit conductance values of 20–80 pS, are voltage independent and insentive to apamin. The molecular basis of this family of potassium channels is poorly defined and they have not yet been described in the mammalian cochlea.
Large Ca2+-activated K+ channels, named BK
or maxi-K channels, display a high unitary conductance (ranging from 75 to 250
pS), are activated by both membrane depolarization and intracellular
Ca2+, and are blocked by scorpion peptidyl toxins such
as charybdotoxin (ChTX) and iberiotoxin (IbTX)
(Galvez et al. 1990;
Miller et al. 1985). BK
channels are generally hypothesized to accelerate action potential
repolarization at synapses and terminate transmitter release (Patillo et al.
2001). These channels are composed of two structurely distinct subunits,
and 
. The subunit is a member of the Slo family
of potassium channels (Knaus et al.
1994) and the gene-encoding Slo was first isolated from
Drosophila (dSlo) using a genetic approach
(Atkinson et al. 1991).
Subsequent cloning experiments have revealed numerous splicing variants of
Slo channels that produce various protein isoforms that differ in
their sensitivity to calcium and voltage
(Adelman et al. 1992).
BK channels are known to play a prominent role in hair cell function of
lower vertebrates such as amphibians, reptiles, and avians (for review see
Fettiplace and Fuchs 1999).
These channels are colocalized with Ca2+ channels at
sites of transmitter release, the hair cell's presynaptic active zones
(Issa and Hudspeth 1994;
Roberts et al. 1990). BK
channels are believed to regulate neurotransmitter release coupled to the
calcium current at the hair cell's active zone as at the nerve-muscle synapse
(Patillo et al. 2001; Robitaille et al.
1993). In electrically tuned hair cells, the number and the
kinetic properties of BK channels (slo splice variants) determine the
resonant frequency of each hair cell along the basilar papilla
(Jones et al. 1999;
Navaratnam et al. 1997;
Ramanathan et al. 1999;
Rosenblatt et al. 1997).
Electrical resonance acts as an electrical filter maximizing the hair cell
response at a specific sound frequency
(Crawford and Fettiplace 1981).
Very little is known, by contrast, about the role of BK channels in mammalian
cochlea neurotransmission. Although Ca2+-activated
K+ conductance has been described in both OHCs
(Housley and Ashmore 1992) and
inner hair cells (IHC) (Dulon et al.
1995; Kros et al.
1998), there is still no direct evidence of Slo protein
expression in mammalian cochlear hair cells. Mature cochlear IHCs, which
constitute the presynaptic terminals connected to the auditory nerve fibers,
do not display electrical resonance. Based on earlier work
(Johnson 1980), Palmer and
Russell (1986) showed
phase-locking (indicative of rapid modulation of neurotransmitter release) up
to several kilohertz with mechanotransduction for these cells. A fast
K+ conductance (IKf), probably involving BK channels and
first appearing at postnatal day 12 in mouse IHCs, is believed to transform
mature mammalian IHCs into high-frequency signal transducers
(Kros et al. 1998). The
purpose of the current study was to characterize the role of BK channels in
sound-evoked auditory nerve action potentials in vivo in the guinea pig
cochlea, using the perfusion of specific toxins into the scala tympani. In
addition, we studied the cellular pattern of expression of Slo
channels in the mammalian inner ear with in situ hybridization of specific
riboprobes and immunocytochemistry. The presence of fast BK channels was also
characterized and confirmed at the presynaptic level on isolated adult guinea
pig IHCs.
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METHODS |
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Experimental animals were female albino guinea pigs (GPs; 250–300 g) with a normal acoustic pinna reflex. The GPs were anesthetized with intra-muscular injections of 1 ml/kg of a mixture of 2 volumes of ketamine chlorhydrate (Ketalar, Parke Davis, 50 mg/ml) and 1 volume of 2% xylazine (Rompun, Bayer). Such anesthesia produced deep sedation of the animal while conserving spontaneous breathing.
Animal handling throughout these experiments was performed with authorization of the French Ministry of Agriculture and in accordance with European Community regulations.
Intracochlear perfusion and sound-evoked potential recordings
After infiltration of local anesthesia of 1% Lidocaïne (Astra), a tracheotomy was performed, and a 2-cm polyethylene tube was inserted and sutured in the trachea. The animal's head was then secured dorsally in a stereotactic apparatus by a mouth-piece and hollow ear-bars. Body temperature was measured with a rectal probe and maintained with a heating blanket at 37 ±1°C (mean ± SD). The bulla of the left ear was approached ventrally and its bony framework opened. Three holes were made into the cochlea: two in the scala vestibuli (SV) and one in the scala tympani (ST). A hole was initially made in the SV at the first turn of the cochlea for the recording electrode. Electrophysiological responses to acoustic stimuli were recorded versus the indifferent and ground needle electrodes in the neighboring tissues. A ST hole was then made in the basal turn of the cochlea for the perfusion catheter. A second SV hole was finally made into the first turn to release perilymph during perfusion to prevent pressure build-up. Only when the recordings at this stage were not significantly different from that performed before the placement of the catheter was the experiment allowed to proceed. The recording electrode was made of Teflon insulated stainless steel wire (125/175 µm bare/coated wire diameters) with a 0.5-mm uncoated end sealed to the Teflon sheath with a drop of cyanoacrylate glue serving both as a stop and seal against the hole in the cochlea wall. The perfusion catheter was made of a 5-mm stainless steel micro-tube of 0.127/0.229 mm inside/outside diameters (Gauge 34, Phymep, France) connected at one of its ends to a polyethylene catheter. The stainless steel microtube was introduced ≤4 mm inside the polyethylene catheter and glued with a drop of cyanoacrylate glue. The steel micro-tube end was introduced inside the cochlea through the hole in the scala tympani of the basal turn. The other end of the polyethylene catheter was connected to a 50 µl Hamilton syringe positioned in an electric syringe pump (Type 101, Phymep). The perfusion rate was set at 1.75 µl/min throughout the experiments.
Electrophysiological measurements were performed on the GP in a double-wall sound-proof room (IAC). The signal-generating and -recording equipment was situated in an adjacent room, except for a battery-operated preamplifier, which was placed inside the sound-proof room. To investigate the effects of the perfused drugs, cochlear microphonic (CM) and whole nerve compound action potentials (CAPs) were measured on responses evoked by tone bursts presented in a Sennheiser speaker (HD 480 II, Germany), mounted in aluminum housing and connected to the left hollow ear bar by silastic tubing. The tone burst stimuli were generated using a PC-based waveform generator (Tucker and Davis Technologies, Gainsville, FL). The tone bursts had a duration of 20 ms, with 2-ms rise/fall times. The frequency used, taking into account the placement of the recording electrode and of the catheter along the cochlear partition (middle of first turn), was 8 kHz. The tone bursts were presented at levels of 30–90 dB SPL at a repetition rate of 12/s. In preliminary experiments, sound levels were calibrated with a Bruel-and-Kjaer 4134 microphone and a calibrated probe-tube placed inside the ear-bar, close to the tympanic membrane. Correct coupling of the ear-bar to the ear was visually controlled in each experiment by observing a slight tympanic membrane displacement when applying pressure at the other end of the ear-bar, before introduction of the silastic tube from the speaker assembly.
In some experiments, measurement of 2f1–f2 distortion products in the cochlear microphonics (DPCM) were made using two simultaneous tonebursts at 8 kHz (f1) and 9.68 kHz (f2) at levels from 30 to 80 dB SPL. These two sounds were presented in two separate Sennheiser speakers connected to the hollow left ear-bar via a Y silastic tubing.
Off-line, using a custom software developed on LabView (National
Instruments), the recordings were automatically analyzed and measured. First,
the electrical compound response recorded from the scala vestibuli
(Fig. 1) was low-pass filtered
(<3 kHz; digital filtering with LabView) and CAP measured from baseline to
first negative trough (N1; Fig.
1B). Then the original response was high-pass filtered
(>3 kHz) and CM amplitude measured (Fig.
1B). In the experiments using two simultaneous tone
bursts, CM amplitudes at f1, f2 and DPCM (2f1–f2) frequencies were
measured on the amplitude frequency spectrum of the raw recorded response. All
analysis and measures were made using digital signal processing through
LabView software. The FFT analysis of the CM signal was so performed and the
amplitude of the peaks at F1, F2, and 2F1-F2 automatically measured in dB re 1
µV RMS (Fig. 1C).
It is worth noting that although the two sounds f1 and f2 were always kept at
the same level SPL, the microphonic spectrogram displayed two primary
components separated by 
20 dB. This was most likely due to changes in
microphonic sensitivity at f1 and f2.
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Inner hair cell preparation and whole cell patch-clamp recordings
Inner hair cells were isolated from adult GP cochleae as previously
described (Dulon et al. 1995;
Sugasawa et al. 1996). The two
lower turns of the organ of Corti were removed from the bony shell of the
cochlea and placed in Hanks' balanced salt solution (HBSS; Sigma), containing
(in mM) 136.9 NaCl, 5.4 KCl, 0.81 MgSO4, 1.26 CaCl2,
0.44 KH2PO4, 0.34 Na2HPO4, 5
N-[2-hydroxyethyl]piperazine-N[prime]-[2-ethanesulfonic
acid] (HEPES), and 5.5 glucose. The Hanks' solution was adjusted to pH 7.35
with 2 mM NaOH and to 300 mosM/kg H20 with 6 mM NaCl. The organ of
Corti, freed from the bony cochlea, was then incubated for 10 min in a
40-µl drop of HBSS containing collagenase (type IV, final concentration:
0.8–1 mg/ml, Sigma). The pieces of organ of Corti were transferred to a
50-µl drop of HBSS in the middle of a glass coverslip sealed on the
perforated bottom of a petri-dish. The cells were then mechanically
dissociated with gentle influx and efflux with a Gilson micropipette and left
to settle for 30 min. The dish was then filled with 3–4 ml of HBSS.
Inner hair cells were recorded under voltage-clamp configuration with
electrodes pulled from borosilicate glass capillaries (GC150TF-10 Clark
Electromedical), on a Sachs-Flaming horizontal electrode puller (Sutter
Instruments). Recording electrodes were back-filled with the following
internal solution containing (in mM) 158 KCl, 2 MgCl2, 1.1 EGTA, 5
HEPES, and 3.05 KOH, pH 7.20. Patch-clamp recordings were performed as
previously described in detail (Blanchet et
al. 1996) by means of an Axopatch-1D amplifier (Axon Instruments,
Foster City, CA). Axotape and pClamp software (Axon Instruments) were used for
data collection and analysis. Voltage errors attributable to uncompensated
series resistance and liquid junction potential were corrected during data
analysis (Blanchet et al.
1996). All experiments were done at room temperature
(20–22°C).
The test solutions, toxins (from Latoxan) and controls, were applied to
isolated IHCs by a Picospritzer puffer system (Picospritzer II, General Valve,
Fairfield, NJ). Pipettes for the Picospritzer system were pulled in a similar
fashion to the recording patch-clamp pipettes and were placed at
20–40 µm from the cells. The pressure of the perfusion system
was set low enough to avoid any mechanical disturbance of the cell during
current recording.
Cloning rSlo from rat cochlea
To prepare RNA probes for in situ hybridization, we isolated and cloned the
Slo cDNA from rat tissue because the complete sequence was not known in the
GP. Harlan Sprague-Dawley rats (day 7) were deeply anesthetized with a rodent
cocktail (ketamine, 50 mg/kg; rompun, 5 mg/kg; and acepromazine, 1 mg/kg).
After decapitation, the brain was removed and immediately frozen at
–70°C. Total RNA was isolated from frozen samples using TRIZOL
Reagent (Life Technologies), and then reverse transcribed into cDNA using the
Superscript Preamplification System (Life Technologies) from which PCR
amplifications were performed with specific primers. Initially, rSlo
transcripts were amplified from rat brain using a pair of oligonucleotides
designed from mSlo sequence (L16912
[GenBank]
)
(Butler et al. 1993). The sense
and antisense primers were respectively:
5'-CTTGACTCGAAGTGAAGCTGC-3' (nucleotides 2002–2222) and
5'-GGGTGAGGATATTGTCATTG-3' (nucleotides 2877–2896). These
primers bracketed the hydrophobic segments S9 and S10 of mSlo at the
3' region. The PCR consisted of 35 cycles at 94°C for 30 s, 55°C
for 30 s, and 72°C for 1 min 30 s. The PCR products were separated on a
1.5% agarose gel and visualized by ethidium bromide staining. The PCR products
were ligated into the pGEM-T vector (Promega) and sequenced using an ABI 373A
automated DNA sequencing system (ABI). Two splice variants, a short isoform
(homologous to mSlo) and a long isoform (+86 bp compare with
mSlo) were identified and cloned. Only the short isoform was
expressed in the organ of Corti by RT-PCR and was consequently tested for in
situ in the rat inner ear. Our antisense RNA probe spans between the S9 and
S10 region of the KCNMA or mSlo cDNA, a rather well-conserved region between
splice variants. Our probe should theorically recognize all KCNMA splice
variants found in the rat cochlea (Langer
et al. 2003).
In situ hybridization of Slo antisense riboprobes
Sprague-Dawley rats (2 mo old) were used for in situ hybridization.
Complete serial sections from three different animals were analyzed. Tissue
preparation was done as previously described
(Dulon et al. 1998). Briefly,
rats were deeply anesthetized with pentobarbital (70 mg/kg) intraperitoneally
and then perfused transcardially with warm saline followed by acid shift
paraformaldehyde: a two-stage perfusion using equal volumes of 4%
paraformaldehyde (pH 6.5) followed by 4% paraformaldehyde with 0.05%
glutaraldehyde (pH 9.5). Cochleae were dissected from temporal bones and
decalcified in 8% EDTA with 4% paraformaldehyde at 4°C. Frozen sections
(20 µm) were cut and mounted on superfrost plus slides (Fisher Scientific),
air dried, and stored at –20°C for later use.
The protocol for in situ hybridization has been previously described in
detail elsewhere (Simmons et al.
1989). Briefly, tissue samples were permeabilized with 2 mg/ml
proteinase K for 30 min at 37°C. Linearized rat cDNA templates for short
isoforms of rSlo were used to synthesize 35S-labeled
antisense riboprobes using appropriate RNA polymerase and 35S-UTP.
Corresponding sense strand riboprobes were synthesized and used as control to
each probe for nonspecific hybridization. The probes were hybridized to the
tissues and incubated at 56°C overnight. The sections were then treated
with RNase A and high-stringency washes (low salt and high temperatures).
Preliminary evaluation of hybridization was obtained by opposition of slides
to X-ray film for 48–72 h. Afterward, the slides were coated with Kodak
NTP-2 liquid autoradiographic emulsion and exposed at 4°C for 2–4 wk
depending on the strength of the signal obtained on the film. The slides were
developed in Kodak D-19 (2.5 min at 14°C) and fixed in Kodak fixer. The
sections were counterstained through the emulsion with bis-benzamide (0.001%
for 2 min), which preferentially labels cell nuclei but also stains cell
membrane and residual proteins. The sections were examined by fluorescence
microscopy to identify cochlear structures and under dark microscopy to
evaluate the distribution of autoradiographic grains. Only sections that had
been hybridized, dipped, exposed, and developed in the same series were used
for this analysis.
Immunocytochemistry of BKCa channels
Immunolocalization of Slo channels was carried out on surface preparations
of organ of Corti dissected from adult GP using a polyclonal antibody obtained
from Alomone labs. This primary antibody, raised in rabbit against the
C-terminal part (residues 1098–1196) of mouse Slo subunit, was
diluted at 1:100. Fixed cochleae were decalcified and the organ of Corti was
dissected turn by turn. The stria vascularis and the Reisner's membrane were
removed. The different turns of the organ of Corti were then washed, and
incubated in biotinylated anti-rabbit secondary (dilution 1:200; Sigma) for
2.5 h. All incubations were performed by adding triton X-100 at 0.1%. The
signal was amplified with an avidin-biotin-horseradish peroxidase procedure
(Vector's laboratory), and visualized using diaminobenzidine as the
chromogen.
For cryostat sections, cochleae were put in a gradient of sucrose 10% (2 h), 20% (2 h), and 30% overnight. Cochleae were embedded in OCT medium and were frozen at –20°C. Selected slides were preincubated free floated for immunoreaction. They were rinsed, placed on subbed slides, dehydrated, cleared, and mounted for microscopic observation. Two control experiments to reveal any nonspecific labeling were carried out: omission of the primary antibody from the procedure and preadsorption of the antisera with the antigenic peptide provided by Alomone labs.
In conclusion, we used in the present study three different major procedures to obtain a complete picture of the role of BK channels in the mammalian auditory system. First, we performed in vivo cochlear recordings and intracochlear perfusion of specific toxins to characterize the role of BK channels in sound-evoked cochlear potentials. Second, we worked at the cellular level in vitro to examinate the IHC currents and verify the presence of BK currents at the presynaptic level. Finally, we used in situ hybridization and immunocytochemistry to characterize the expression of mRNA and BK proteins in cochlear tissue.
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RESULTS |
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To test the stability of our preparation, we have first run a large number
of sham experiments where artificial perilymph (HBSS) was perfused (n
= 8). In our experience, CAP and CM were generally stable over time for >2
h. In experiments with the toxins, all GP were preperfused with HBSS for
30 min to verify the stabilty of CAP and CM. We first tested our
perfusion system with the fugu toxin, tetrodotoxin (TTX), a potent blocker of
voltage-dependent Na+ channels. Intracochlear perfusion of TTX is
well known to block auditory nerve activity
(Evans and Klinke 1982;
Konishi and Kelsey 1968;
Zhang et al. 1999). In our
study, TTX was diluted in HBSS and was perfused in the cochlea for 5–15
min at a concentration of 1, 3, or 30 µM. One GPs was tested at each of
these concentrations (the effects of TTX were essentially irreversible). For
the GP tested at 3 µM TTX, a decrease in CAP from 46 to 17 µV was
observed within 3.5 min of perfusion (Fig.
2). The CAP waveform became broader and its latency increased.
These changes were only partially reversible after 3 h of rinsing with HBSS.
On the contrary, CM amplitude, which is believed to essentially reflect the AC
receptor current of hair cells, did not change during TTX perfusion
(Fig. 2). In a similar manner
to Fig. 2, in the other two GPs
tested at 1 and 30 µM of TTX, CAP was also rapidly and completely
suppressed, while CM remained unaffected.
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Intracochlear perfusion of ChTX and IbTX
We first studied the effects of intracochlear perfusion of the scorpion
toxin charybdotoxin (ChTX), a toxin well known to block BK channels
(Garcia et al. 1991). ChTX was
perfused at concentrations of 5 µM (n = 1) and 2 µM (n
= 2). At 2 µM, CAP amplitude decreased by 50–70% within 10–15
min and recovered within 20 min after the end of the ChTX perfusion
(Fig. 3). In this GP, which was
tested with a second ChTX perfusion, there was again a rapid diminution in CAP
but this time we observed an incomplete recovery, presumably due to a slower
removal or washout of the toxin from its targets. At 5 µM, CAP decreased by
68% (from 45 to 14.3 µV) after 3-min perfusion and to 0 µV after 6 min.
After rinsing with HBSS, full recovery of CAP amplitude occurred about 30 min
after the end of the 5 µM ChTX perfusion. This indicated a good washout and
a reversible binding of the toxin on its sites of action. In all animals
tested with ChTX, during the whole experiment CM amplitude fluctuated and
decreased over time by <5 dB. The variations of CM during the toxin
application were not significantly different when compared with the CM changes
observed in untreated animals –1.2 ± 3.4 dB (n = 8).
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We tested the effects of iberiotoxin (IbTX), another scorpion toxin which
is known to specifically block BK channels, unlike ChTX, which can also block
certain other type of potassium channels such as Kv1.3
(Galvez et al. 1990). Results
were obtained from six GPs with different IbTX concentrations: 1 µM
(n = 4) and 2 µM (n = 1), and one GP was perfused
successively with 1, 2, 0.5, and 5 µM. Several perfusions of the same
concentration of toxin were occasionally repeated on the same GP. Between each
IbTX perfusion a continuous rinsing perfusion of HBSS was maintained.
Figure 4 shows recordings and
amplitude changes of CAP in one GP during repeated perfusions with HBSS and 1
µM IbTX. Similar effects to those obtained with ChTX are observed on CM and
CAP during IbTX perfusions. The effects of IbTX were reversible and could be
repeated during a second application (Fig.
4). Maximal reduction in CAP amplitude was observed within 25 min
after starting the perfusion of IbTX. Recovery in CAP amplitude to preIbTX
perfusion levels was complete within 45 min of rinsing with HBSS. In the three
other GPs perfused with 1 µM IbTX, changes in CAP amplitude followed the
same pattern with no change in CM except in one which showed a slight increase
in CM amplitude (by 4.5 dB; Fig.
4). In summary, with 1 µM IbTX, the mean maximum decrease in
CAP amplitude was of 57.9 ± 23%. This maximum CAP decrease occurred
within 26.5 ± 5.7 min (n = 4) while CM was essentially
unaffected (mean variation of –0.5 ± 3.5 dB). The variation of CM
with IbTX was not significantly different when compared with variation of CM
measured over time in IbTX-untreated GP (-1.2 ± 3.4 dB n =
8).
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Successive perfusions with four different concentrations of IbTX (1, 2, 0.5, 5 µM) with HBSS perfusion in between were also carried out in one other GP (Fig. 5). In that case, the tone burst at 8 kHz was presented at four different sound levels (90, 70, 50, and 30 dB SPL). At 0.5 µM IbTX, the reduction in CAP (15–20 µV) was similar at all sound intensities used (Fig. 5B). At 1, 2, and 5 µM IbTX the reduction in CAP increased largely with sound intensity. For 70 and 90 dB SPL, the dose/response curve of block by IbTX, obtained by fitting of the data with a sigmoidal Hill equation, gave an apparent IC50 of 0.4 and 1 µM IbTX, respectively (Fig. 5C).
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We have also tested in two other GPs the effect of an intracochlear
perfusion of apamin (5 µM). Apamin is a specific blocker of another family
of Ca2+-activated K+ channels, the SK
channels (Köhler et al.
1996). These channels, in particular SK2, are known to be highly
expressed in OHCs but not in mature IHCs
(Dulon et al. 1998).
Intracochlear perfusion of apamin did not affect the evoked cochlear
potentials (CAP, CM, or DPCM) while a pre or postperfusion of IbTX (1 µM)
largely reduced CAP in the same animals.
To determine whether the action of IbTX on CAP was arising from a block at the OHCs amplification function, we measured in four additional GPs the effects of the toxin (1 µM) on the inter-modulation distortion products (DPCM) of the cochlear microphonics (CM) in response to two-tone stimuli at five sound intensities (40, 50, 60, 70, 80 dB SPL) (Fig. 6). While CAP rapidly decreased at all sound intensities, the amplitude of CM at f1, f2 and DPCMs (2f1–f2) remained stable during the application of IbTX. Slight changes on f1, f2, and DPCM were only seen 15 min after the drop in CAP. Overall, these results suggested that the diminution of CAP by IbTX was essentially arising from an action at the IHC, IHC/afferent synapse or ganglion cells (see DISCUSSION) rather than at the OHCs function. This assumption was reinforced by an even larger decrease of CAP at high sound intensities such as 80–90 dB SPL (Figs. 5 and 6). At this high sound level, CAP generation is believed to bypass the OHC cochlear amplifier.
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Evidence of fast BK currents in IHCs
We have also studied under whole cell voltage clamp the effects of the same
BK-specific toxins on the time and voltage-dependent conductances of isolated
GP IHCs. Cells were held at –60 mV and current responses to
depolarisation in 10-mV increments were recorded
(Fig. 7A). All
recorded IHCs displayed fast developing noninactivating outward currents which
started to activate above –40 mV (n = 30). The activation time
course of this fast current could be well fitted by a single exponential with
a time constant of 0.6 ± 0.2 ms at 0 mV and 20°C (range:
0.2–0.9 ms). These kinetics are in good agreement with those reported at
20°C in a previous study by Kros and Crawford
(1990). Extracellular
application of IbTX during the voltage-step protocol suppressed, in all IHCs
tested (n = 8), the fast component of outward currents
(Fig. 7B). The
remaining IbTX-insensitive current displayed a kinetic of activation reduced
by about one order of magnitude ( = 5.7 ± 2.2 ms at 0 mV and
20°C; range: 2.1–8.1 ms).
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Several concentrations of IbTX were tested: 0.05, 0.1, and 1 µM. A concentration of IbTX as low as 50 nM showed a maximum block of the fast component, suggesting that the IC50 was in the nanomolar range as expected for a specific action of the toxin on BK channels. The large difference (>1 order of magnitude) in concentration for maximal inhibition between in vivo and in vitro may simply reflect a lower access of the peptide toxin to the BK channels from the perilymph during perfusion in vivo. Indeed, in vivo, the toxin would have to cross the basilar membrane cells and the surrounding supporting cells of the organ of Corti that may act as diffusion barriers to the toxins.
The block by IbTX on the fast IHC currents was completely reversible within
minutes after rinsing with normal HBSS (not shown). The IBTX-sensitive current
was visualized by subtracting currents before and in the presence of IbTX
(Fig. 7C). This
current displayed rapid activation and time inactivation at large depolarized
potentials. The fast IbTX-sensitive currents activated with a fast time
constants () ranging from about 1 ms to <0.5 ms as displayed in
Fig. 7, C and
D, inset. The current-voltage relationship of
the IbTX-sensitive current showed outward currents starting to activate above
–40 mV, with amplitude reaching a plateau at 0 mV and finally outward
rectifying above +10 mV (Fig.
7D). The current showed time inactivation that became
faster with depolarization at potentials >0 mV. In consequence, the
I-V curve displayed a N-shape that was less pronounced at the peak
current (
) than near the steady-state current (
). This faster
inactivation of the BK current at larger depolarization could either be due a
voltage inactivation or due to a faster decrease of calcium near the BK
channels if one considers that these channels are activated consecutively to a
voltage-activated calcium entry or/and release.
A similar fast component of the outward currents was also reversibly
suppressed by 0.1 µM ChTX (n = 5; data not shown) and with the
extracellular application of 4 mM barium ions during the same voltage-step
protocol (n = 3; not shown). On the contrary, extracellular
application of apamin (2 µM), a potent blocker of SK channels
(Köhler et al. 1996), did
not affect the outward currents of IHCs (n = 3; data not shown).
Expression of rSlo
The search for the expression of Slo mRNA was made in the rat cochlea because, unlike GP, the complete cDNA sequence of the gene Slo of mouse and rat is published in GenBank. In situ hybridization of adult rat inner ear sections using 35S-labeled antisense ribiprobes of rSlo showed high level of expression in IHCs and a somewhat lower level of expression in OHCs all along the cochlea (Fig. 8A). A strong expression was also observed in spiral ganglion from the base to the apex of the cochlea. Some expression was also noticed in stria vascularis but at a much lower level. Strong expression of Slo mRNA was also detected in the vestibular sensory epithelium (Fig. 8B).
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The expression of subunits was confirmed at the protein level in
the adult GP organ of Corti by immunocytochemistry
(Fig. 9, A and
B). On surface preparation, strong immunostaining was
observed in IHCs and to a lesser extent in the three rows of outer hair cells.
Similar staining was observed in three GPs. Immunolabelling was stronger at
the base of the cochlea for OHCs and equivalent in all turns for IHCs
(Fig. 9C).
Immunolabelling at the IHC level was confirmed by the identification of
individual labeled whole IHCs at the edge of the surface preparation and in
cryosections (Fig.
9D). No labeling was seen in the afferent fibers below
IHCs, suggesting that BK channels are mainly expressed presynaptically.
Immunoreaction in cryo-sections of whole GP cochlea, confirmed the results
obtained with surface preparations (Fig.
10). In the organ of Corti, IHCs were labeled, particularly at
their basal synaptic pole where strongly labeled dots were observed
(Fig. 10, C and
D). The soma of spiral ganglion neurons were also labeled
while their fibers were unstained, again suggesting that the expression of BK
channels at the IHC-synapse was essentially presynaptic. Deiters cells and
OHCs also were immunoreactive but with somewhat less intensity than IHCs.
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DISCUSSION |
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;
Kujawa et al. 1992).
A similar reduction of CAP was observed with ChTX, a toxin also known as a
potent blocker of BK channels but acknowledged to be less specific for BK
channels than IbTX because it can block also other voltage-dependent
K+ channels (Garcia et al.
1991). Apamin, a bee venom toxin specific to SK channels, did not
affect CAP indicating that SK channels are essentially restricted to the OHCs
medial efferent synapse (Dulon et al.
1998). The results obtained with intracochlear perfusion of ChTX
are in good agreement with the recent study of Yoshida et al.
(2001), and we are now
extending these results to IbTX, a BK specific toxin.
One intriguing question is how the toxins gain access to the IHCs via the
perilymph from the scala tympani. There are at least two parameters that give
us some information on the acces of the toxins to IHCs in vivo. First, it is
to be noted that there is a difference of about one order of magnitude between
the toxin concentration showing an effect in vivo (0.5 µM) and in vitro
when applying the toxin directly to isolated IHCs (0.05 µM). This
difference indeed underlined a problem of access when perfusing the toxin via
the perilymph. The concentration to obtain an effect in vivo remained in the
micromolar range, i.e., low enough to consider the reduction of CAP as a
specific block of BK channels. Second, while the toxin has an immediate action
on isolated IHCs in vitro, the beginning of reduction in CAP in vivo showed a
latency of 15 min with a maximum inhibition within 15–30 min,
indicating a slow and progressive access of the toxin to its targets. We don't
know precisely how the toxin reached the IHCs. Because we are perfusing the
scala tympani, the direct route of access could be the basilar membrane, a
structure only composed of one layer of longitudinal cells connected above by
an intercellular substance made up of longitudinal filaments. The basilar
membrane supports directly the organ of Corti. One can speculate that small
peptides such as IbTX bind to the basilar membrane cells and diffuse between
cells to reach the IHC synapse.
In the present study, we further demonstrated that the BK-specific toxin
IbTX blocked a fast outward current in GP IHCs. The IbTX-sensitive current was
activated above –40 mV and was N-shaped, suggesting its dependence on a
Ca2+ influx through voltage-gated
Ca2+ channels. We have demonstrated in a previous study,
using flash UV photorelease of intracellular Ca2+ and
patch-clamp recordings, that GP IHCs express a
Ca2+-actived K+ conductance sensitive to ChTX
and TEA (Dulon et al. 1995).
This is also in good agreement with recent recordings made in developing mice
IHCs by Kros et al. (1998). In
the present study, the block of the fast outward current by barium, a potent
blocker of BK channels (Vergara et al.
1999), also reinforced the idea that the fast current of IHC is
due to the activation of BK channels.
We argue therefore that the reduction in CAP by ChTX and IbTX during
cochlear perfusion in vivo could be explained, at least partly, by an action
at presynaptic BK channels in IHCs. The mechanism by which the block of
presynaptic BK channels may alter neurotransmitter release by IHCs, however,
remains to be elucidated. The pronounced reduction in the waveform of CAP,
produced by IbTX in vivo, seems disproportionate to the partial block of
steady-state potassium current shown in IHCs. This discrepancy suggested that
the underlying mechanisms may have more to do with timing, or kinetics, of the
toxin-sensitive current. We found that the time constant of the membrane
outward currents of IHCs increased in the presence of IbTX from an average of
0.6 to 5.7 ms. These kinetics of the fast and slow currents are in good
agreement with those reported at 20°C by Kros and Crawford
(1990). Assuming that the
membrane behaves as a first-order electrical system, the block of the fast BK
conductance would result in an increase, by about 10-fold, in the corner
frequency of IHCs, i.e., to attenuate the IHCs receptor potential at
frequencies >30 Hz. Any alteration that allows a temporal spread of
transmitter release by individual hair cells would reduce the amplitude of the
CAP (whose amplitude depends on precise coordination of the largest possible
number of individual afferent action potentials).
Because BK channels are known to be co-localized with the calcium channels
that regulate transmitter release in active zones of lower vertebrate hair
cells (see for review Fettiplace and Fuchs
1999), we hypothesize that a similar mechanism occurs in mammalian
IHCs. The fast activation of the outward current and the N-shaped I-V
curve serve as a good argument for a tight coupling with a
Ca2+ influx. In vivo during cochlear perfusion, the
block of the fast repolarizing BK current by IbTX may maintain these IHCs
micro-domains deploarized, breaking the driving force for
Ca2+ entry, and as a result diminish
Ca2+-dependent neurotransmitter release. Such decrease
of transmitter release by presynaptic block of BK channels has been proposed
in nerve-muscle synapses (Patillo et al. 2001).
The assumption of expression of BK channels in IHCs was further reinforced
in our study by the pattern of rSlo mRNA expression that we found in
rat inner ear. Indeed, we show for the first time that rSlo antisense
riboprobes hybridized strongly in IHCs and in spiral ganglion and to a lesser
extent in OHCs. A similar pattern of expression of the Slo protein was
observed by immunocytochemistry in GP organ of Corti. These results are in
agreement with recent studies showing the expression of Slo mRNA by RT-PCR in
the rat cochlea (Brändle et al.
2001) and the immunolocalisation of BK channels in murine spiral
ganglion neurons (Adamson et al.
2002). Furhermore, the pattern of hybridization of antisense
riboprobes derived from the carboxy-terminal domain of rSlo that we
observed in our study resembles the pattern observed in the chicken's cochlea
(Rosenblatt et al. 1997).
Further experiments are now needed to identify whether mammalian cochlea hair
cells express multiple and specific BK splice variants as in the chick
cochlea.
Our results for in situ hybridization and immunocytochemistry suggest that
Slo channels underlie the IbTX-sensitive fast outward current
recorded in isolated IHCs. The expression of Slo in spiral ganglion
neurons also suggest a role for BK channels in the fast repolarization of
action potential in auditory nerve fibers. We believe, however, that the
reduction in CAP that we observed during intracochlear perfusion of IbTX and
ChTX in vivo was not due to an effect at the postsynaptic nerve fibers. First,
the immunolocalisation of BK proteins did not reveal any expression in the
peripheral nerve fibers, and we believe that the soma of the spiral ganlion
neurons would have a poor access to the toxin because they are tightly
surrounded by a myelin shield. Furthermore, we think that the block of BK
channels at the postsynaptic level would on the contrary increase excitability
in the nerve fibers by keeping membrane potential near their action potential
threshold. On the other hand, we have observed that Slo channels are
also expressed in stria vascularis suggesting the presence of BK channels.
This is in good agreement with previous electrophysiological recordings
(Takeuchi et al. 1992). In our
intracochlear perfusion experiments in vivo, we also need to consider the
possibility that BK toxins alter the standing or evoked current through IHCs
by changing the endocochlear potential (EP). However, such a mechanism appears
unlikely because CM, which is sensitive to change in EP, remained essentially
unaffected during the perfusion of the BK-specific toxins. It has to be noted
that the expression of Slo by OHCs, which was however much less than
that of IHCs, is consistent with electophysiological recordings showing the
expression of Ca2+-activated K+ currents in
these cells (Housley and Ashmore
1992). The absence of effect on DPCM in our study, however,
suggests that IbTX-senstive channels in OHCs have a minimal influence on the
cochlear amplifier.
On the basis of these results, we hypothesize that a fast BK conductance
regulates neurotransmitter release at presynaptic active zones of mammalian
IHCs. These BK channels with fast kinetics, presumably composed of
Slo subunits, allow IHCs to function as high-frequency signal
transducers (Kros et al. 1998;
Palmer and Russel 1986). While we were finishing the writing of our
manuscript, an in situ hybridization study showing BK mRNA expression in the
rat cohlea, and particularly in IHCs, was released by Langer et al.
(2003) in agreement with our
study.
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ACKNOWLEDGMENTS |
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This research was supported by grants from Fondation Pour la Recherche Médicale (FRM Paris France) and Conseil Régional d'Aquitaine.
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FOOTNOTES |
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Address for reprint requests: D. Dulon, EMI 99-27 INSERM, Université de Bordeaux 2, Laboratoire de Biologie Cellulaire et Moléculaire de l'Audition, Hôpital Pellegrin, 33076 Bordeaux, France (E-mail: Didier.dulon{at}bordeaux.inserm.fr).
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REFERENCES |
|---|
|
Adelman JP, Shen KZ, Kavanaugh MP, Warren RA, Wu YN, Lagrutta A, Bond CT, and North RA. Calcium-activated potassium channels expressed from cloned complementary DNAs. Neuron 9: 209–216, 1992.[Web of Science][Medline]
Atkinson NS,
Robertson GA, and Ganetzky B. A component of calcium-activated potassium
channels by the Drosophila Slo locus. Science
253: 551–555,
1991.
Blanchet C,
Erostegui C, Sugasawa M, and Dulon D. Acetylcholine-induced potassium
current of guinea-pig outer hair cells: its dependence on a calcium influx
through nicotinic-like receptors. J Neurosci
16: 2574–2584,
1996.
Brandle U, Frohnmayer S, Krieger T, Zenner HP, Ruppersberg JP, and Maassen MM. Expression of Ca(2+)-activated K(+) channel subunits and splice variants in the rat cochlea. Hear Res 161: 23–28, 2001.[Web of Science][Medline]
Butler A,
Tsunoda S, McCobb DP, Wei A, and Salkoff L. mSlo, a complex mouse gene
encoding "maxi" calcium-activated potassium channels.
Science 261:
221–224, 1993.
Crawford AC and
Fettiplace R. An electrical tuning mechanism in turtle cochlear hair
cells. J Physiol 312:
377–412, 1981.
Dulon D, Luo L, Zhang C, and Ryan AF. Expression of small-conductance calcium-activated potassium channels (SK) in outer hair cells of the rat cochlea. Eur J Neurosci 10: 907–915, 1998.[Web of Science][Medline]
Dulon D, Sugasawa M, Blanchet C, and Erostegui C. Direct measurement of Ca(2+)-activated K+ currents in inner hair cells of the guinea-pig cochlea using photolabile Ca2+ chelators. Pfluegers 430: 365–373, 1995.
Elgoyhen AB,
Johnson DS, Boulter J, Vetter DE, and Heinemann S. 9: an
acetylcholine receptor with novel pharmacological properties expressed in rat
cochlear hair cells. Cell 79:
705–715, 1994.[Web of Science][Medline]
Elgoyhen AB,
Vetter DE, Katz E, Rothlin CV, Heinemann SF, and Boulter J. alpha
10: a determinant of nicotinic cholinergic receptor function in mammalian
vestibular and cochlear mechanosensory hair cells. Proc Natl Acad
Sci USA 98:
3501–3506, 2001.
Evans EF and
Klinke R. The effects of intracochlear cyanide and tetrodotoxin on the
properties of single cochlear nerve fibres in the cat. J
Physiol 331:
385–408, 1982.
Fettiplace R and Fuchs PA. Mechanisms of hair cell tuning. Annu Rev Physiol 61: 809–834, 1999.[Web of Science][Medline]
Galvez A,
Gimenez-Gallego G, Reuben JP, Roy-Contancin L, Feigenbaum P,
Kaczorowski GJ, and Garcia ML. Purification and characterization of a
unique, potent, peptidyl probe for the high conductance calcium-activated
potassium channel from venom of the scorpion Buthus tamulus. J Biol
Chem 265:
11083–11090, 1990.
Garcia ML, Galvez A, Garcia-Calvo M, King VF, Vazquez J, and Kaczorowski GJ. Use of toxins to study potassium channels. J Bioenerg Biomembr 23: 615–646, 1991.[Web of Science][Medline]
Hinrichsen RD. Calcium-dependent potassium channels: distribution and function. In: Calcium-Dependent Potassium Channels. Austin, TX: Landes, 1993, p. 16–31.
Housley GD and
Ashmore JF. Ionic currents of outer hair cells isolated from the
guinea-pig cochlea. J Physiol
448: 73–98,
1992.
Issa NP and
Hudspeth AJ. Clustering of Ca2+ channels and
Ca(2+)-activated K+ channels at fluorescently labeled pre-synaptic
active zones of hair cells. Proc Natl Acad Sci USA
91: 7578–7582,
1994.
Johnson DH. The relationship between spike rate and synchrony in responses of auditory-nerve fibers to single tones. J Acoust Soc Am 68: 1115–1122, 1980.[Web of Science][Medline]
Jones EM,
Gray-Keller M, and Fettiplace R. The role of
Ca2+-activated K+ channel spliced variants in
the tonotopic organization of the turtle cochlea. J
Physiol 518:
653–665, 1999.
Kemp DT and Brown AM. Ear canal acoustic and round window electrical correlates of 2f1–f2 distortion generated in the cochlea. Hear Res 13: 39–46, 1984.[Web of Science][Medline]
Knaus HG,
Garcia-Calvo M, Kaczorowski GJ, and Garcia ML. Subunit composition of the
high conductance calcium-activated potassium channel from smooth muscle, a
representative of the mSlo and slowpoke family of potassium channels.
J Biol Chem 269:
3921–3924, 1994.
Köhler M,
Hirschberg B, Bond CT, Kinzie JM, Marrion NV, Maylie J, and Adelman JP.
Small-conductance, calcium-activated potassium channels from mammalian brain.
Science 273:
1709–1714, 1996.
Konishi T and Kelsey E. Effect of tetrodotoxin and procaine on cochlear potentials. J Acoust Soc Am 43: 471–480, 1968.[Web of Science][Medline]
Kros CJ and
Crawford AC. Potassium currents in inner hair cells isolated from the
guinea-pig cochlea. J Physiol
421: 263–291,
1990.
Kros CJ, Ruppersberg JP, and Rusch A. Expression of a potassium current in inner hair cells during development of hearing in mice. Nature 394: 281–284, 1998.[Medline]
Kujawa SG, Fallon M, and Bobbin RP. Intracochlear salicylate reduces low-intensity acoustic and cochlear microphonic distortion products. Hear Res 64: 73–80, 1992.[Web of Science][Medline]
Langer P, Grunder S, and Rusch A. Expression of Ca2+-activated BK channel mRNA and its splice variants in the rat cochlea. J Comp Neurol 455: 198–209, 2003.[Web of Science][Medline]
Miller C, Moczydlowski E, Latorre R, and Phillips M. Charybdotoxin, a protein inhibitor of single Ca2+-activated K+ channels from mammalian skeletal muscle. Nature 313: 316–318, 1985.[Medline]
Navaratnam DS, Bell TJ, Tu TD, Cohen EL, and Oberholtzer JC. Differential distribution of Ca2+-activated K+ channel splice variants among hair cells along the tonotopic axis of the chick cochlea. Neuron 19: 1077–1085, 1997.[Web of Science][Medline]
Palmer AR and Russell IJ. Phase-locking in the cochlear nerve of the guinea-pig and its relation to the receptor potential of inner hair-cells. Hear Res 24: 1–15, 1986.[Web of Science][Medline]
Oliver D, Klocker N, Schuck J, Baukrowitz T, Ruppersberg JP, and Fakler B. Gating of Ca2+-activated K+ channels controls fast inhibitory synaptic transmission at auditory outer hair cells. Neuron 26: 595–601, 2000.[Web of Science][Medline]
Pattillo JM, Yazejian B, DiGregorio DA, Vergara JL, Grinnell AD, and Meriney SD. Contribution of pre-synaptic calcium-activated potassium currents to transmitter release regulation in cultured Xenopus nerve-muscle synapses. Neuroscience 102: 229–240, 2001.[Web of Science][Medline]
Ramanathan K,
Michael TH, Jiang GJ, Hiel H, and Fuchs PA. A molecular mechanism for
electrical tuning of cochlear hair cells. Science
283: 215–217,
1999.
Robitaille R, Garcia ML, Kaczorowski GJ, and Charlton MP. Functional colocalization of calcium and calcium-gated potassium channels in control of transmitter release. Neuron 11: 645–655, 1993.[Web of Science][Medline]
Roberts WM, Jacobs RA, and Hudspeth AJ. Colocalization of ion channels involved in frequency selectivity and synaptic transmission at presynaptic active zones of hair cells. J Neurosci 10: 3664–3684, 1990.[Abstract]
Rosenblatt KP, Sun ZP, Heller S, and Hudspeth AJ. Distribution of Ca2+-activated K+ channel isoforms along the tonotopic gradient of the chicken's cochlea. Neuron 19: 1061–1075, 1997.[Web of Science][Medline]
Sah P. Ca(2+)-activated K+ currents in neurons: types, physiological roles and modulation. Trends Neurosci 19: 150–154, 1996.[Web of Science][Medline]
Simmons DM, Arriza JL, and Swanson LW. A complete protocol for in situ hybridization of messenger RNAs in brain and other tissues with radio-labeled single-stranded RNA probes. J Histotechnol 12: 169–181, 1989.
Sugasawa M,
Erostegui C, Blanchet C, and Dulon D. ATP activates nonselective cation
channels and calcium release in inner hair cells of the guinea-pig cochlea.
J Physiol 491:
707–718, 1996.
Takeuchi S, Marcus DC, and Wangemann P. Ca(2+)-activated nonselective cation, maxi K+ and Cl– channels in apical membrane of marginal cells of stria vascularis. Hear Res 61: 86–96, 1992.[Web of Science][Medline]
Vergara C,
Alvarez O, and Latorre R. Localization of the K+ lock-In and
the Ba2+ binding sites in a voltage-gated
calcium-modulated channel. Implications for survival of K+
permeability. J Gen Physiol
114: 365–376,
1999.
Vergara C, Latorre R, Marion NV, and Adelman JP. Calcium-activated potassium channels. Curr Opin Neurobiol 8: 321–329, 1998.[Web of Science][Medline]
Yoshida N,
Liberman MC, Brown MC, and Sewell WF. Fast, but not slow, effects of
olivocochlear activation are resistant to apamin. J
Neurophysiol 85:
84–88, 2001.
Zhang SY,
Robertson D, Yates G, and Everett A. Role of L-type Ca(2+) channels in
transmitter release from mammalian inner hair cells. I. Gross sound-evoked
potentials. J Neurophysiol 82:
3307–3315, 1999.
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