1Neuroscience Program, University of Rochester, Rochester, New York 14642; and 2The Bobby R. Alford Department of Otorhinolaryngology and Communicative Sciences, Baylor College of Medicine, Houston, Texas 77030
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ABSTRACT |
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Chen, James W. Y. and
Ruth Anne Eatock.
Major Potassium Conductance in Type I Hair Cells From Rat
Semicircular Canals: Characterization and Modulation by Nitric Oxide.
J. Neurophysiol. 84: 139-151, 2000.
Mammalian vestibular organs have two types of hair cell, type I and
type II, which differ morphologically and electrophysiologically. Type
I hair cells alone express an outwardly rectifying current, IK,L, which activates at relatively negative voltages. We
used whole cell and patch configurations to study
IK,L in hair cells isolated from the sensory
epithelia of rat semicircular canals. IK,L
was potassium selective, blocked by 4-aminopyridine, and permeable to
internal cesium. It activated with sigmoidal kinetics and was
half-maximally activated at 74.5 ± 1.6 mV
(n = 35; range
91 to
50 mV). It was a very
large conductance (91 ± 8 nS at
37 mV; 35 nS/pF for a cell of
average size). Patch recordings from type I cells revealed a candidate
ion channel with a conductance of 20-30 pS. Because
IK,L was activated at the resting potential, the cells had low input resistances (Rm):
median 25 M
at
67 mV versus 1.3 G
for type II cells.
Consequently, injected currents comparable to large transduction
currents (300 pA) evoked small (
10 mV) voltage responses. The cells'
small voltage responses and negative resting potentials
(VR =
81.3 ± 0.2 mV,
n = 144) pose a problem for afferent
neurotransmission: how does the receptor potential depolarize the cell
into the activation range of Ca2+ channels (positive to
60 mV) that mediate transmitter release? One possibility, suggested
by spontaneous positive shifts in the activation range of
IK,L during whole cell recording, is that the activation range might be modulated in vivo. Any factor that reduces the number of IK,L channels open at
VR will increase
Rm and depolarize
VR. Nitric oxide (NO) is an ion channel
modulator that is present in vestibular epithelia. Four different NO
donors, applied externally, inhibited the
IK,L conductance at
67 mV, with mean
effects ranging from 33 to 76%. The NO donor sodium nitroprusside
inhibited channel activity in patches when they were cell-attached but
not excised, suggesting an intracellular cascade. Consistent with an
NO-cGMP cascade, 8-bromo-cGMP also inhibited whole cell
IK,L. Ca2+-dependent NO synthase
is reported to be in hair cells and nerve terminals in the vestibular
epithelium. Excitatory input to vestibular organs may lead, through
Ca2+ influx, to NO production and inhibition of
IK,L. The resulting increase in
Rm would augment the receptor potential, a
form of positive feedback.
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INTRODUCTION |
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The type I hair cell is a relatively recent arrival found only in
the vestibular sensory epithelia of reptiles, birds, and mammals. Its
distinctive properties are just beginning to be understood. Type I hair
cells were first described by Wersäll, who distinguished them
from other hair cells in the vestibular epithelia (type II hair cells)
by cell shape and synaptic contacts (Wersäll 1956) (Fig. 1). Type I cells have a flask shape
and are engulfed by cup-like calyx afferent terminals. Type II cells
come in diverse shapes but tend to be more cylindrical and receive
bouton afferent contacts. Efferent neurons form bouton terminals on
type II hair cells and on the calyx endings around type I cells. More
subtle differences are evident at the ultrastructural level
(Favre and Sans 1983
; Rüsch et al.
1998
).
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Recent work has shown that type I and II hair cells also differ
electrophysiologically. Type I cells alone have a large outwardly rectifying K+ current,
IK,L (also called
IKI), that is unusual in several
respects (Correia and Lang 1990; Rennie and
Correia 1994
; Rüsch and Eatock 1996a
).
Most outward rectifiers in hair cells activate positive to the resting
potential, VR, which is typically
between
70 and
50 mV. The IK,L
conductance (gK,L), however, is
frequently activated at VR; voltages
corresponding to half-maximal activation
(V1/2) can be as negative as
100 mV.
Another unusual property of the activation range is that it varies
widely between cells, by as much as 50 mV (Rüsch and
Eatock 1996a
). Here we present properties of
IK,L in hair cells isolated from the
rat crista, including single-channel properties, that have not
previously been described.
The size of gK,L at
VR largely determines the type I
cell's input resistance, and therefore the gain and time course of its voltage response to the current through the mechanosensitive
transduction channels. As shown here and previously for other type I
hair cells (Rennie et al. 1996; Rüsch and
Eatock 1996b
), when gK,L is
appreciably activated at VR, currents
comparable with the largest transduction currents (hundreds of
picoamperes) depolarize the cell by <10 mV. If similar conditions hold
in vivo, even large transduction currents (<1 nA)
(Géléoc et al. 1997
; Holt et al.
1997
) will not depolarize the cells into the activation range
of the voltage-gated calcium channels that mediate transmitter release
(positive to
60 mV in type I cells as in other hair cells)
(Bao et al. 1999
; Chen and Eatock 1993
).
As a possible solution to this problem, it has been proposed that
during transduction, K+ accumulates in the long
synaptic cleft between the type I hair cell and the calyx ending,
directly depolarizing both pre- and postsynaptic membranes (Chen
1995
; Goldberg 1996
). This mechanism is proposed
as supplementary to conventional vesicular transmission, which
undoubtedly occurs given the abundance of synaptic ribbons and vesicles
in type I cells (Lysakowski and Goldberg 1997
).
An alternative or additional way around the problem is to reduce the
number of IK,L channels open at the
resting potential by inactivation or by shifting the activation range
positively. A reduction in the number of
K+-selective channels at resting potential would
increase input resistance and depolarize the membrane, both of which
actions would tend to boost receptor potentials into the activation
range of Ca2+ channels. That the voltage
dependence of IK,L is subject to
modulation is indicated by the wide inter-cell variation in activation
range and by spontaneous positive shifts of the activation range
recorded from single cells in whole cell mode (Rüsch and
Eatock 1996a; this study). In vivo,
IK,L might be modulated by second
messengers originating in the hair cell or in neighboring cells: the
anatomy suggests the possibility of retrograde transmission from calyx to hair cell. One retrograde agent that has been proposed by Sans and
colleagues is glutamate. Glutamate puffed onto isolated type I cells
causes intracellular Ca2+ to rise (Devau
et al. 1993
), and synaptic vesicles and presynaptic proteins
have been localized to the calyx ending (Scarfone et al.
1988
).
With this background in mind, we were interested in the possibility of
retrograde modulation of IK,L. Nitric
oxide (NO) has been implicated elsewhere in both retrograde
transmission (Schuman and Madison 1991) and ion channel
modulation (e.g., Bolotina et al. 1994
; Summers
et al. 1999
). Recent histochemical and immunocytochemical localization of Ca2+-dependent forms of nitric
oxide synthase (NOS) shows that NO is made in hair cells and both
afferent and efferent nerve terminals in vestibular sensory epithelia
(see DISCUSSION). A common view holds that NO diffuses
freely and rapidly across membranes, activating soluble guanylate
cyclase to raise cGMP, which can then have diverse effects
(Garthwaite and Boulton 1995
). Here we show that
IK,L is inhibited near resting
potential both by NO-producing agents and by a cGMP analog.
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METHODS |
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Isolation of hair cells
The medium used for preparing isolated hair cells was Leibovitz's L-15 medium (GIBCO BRL, Gaithersburg, MD) with a modified Ca2+ concentration: either 100 µM (low-Ca2+ L-15) or 3.3 mM (high-Ca2+ L-15). Long-Evans rats (age 10-45 days, 16-200 g body wt) were deeply anesthetized with pentobarbital sodium (Nembutal, 100 ml/kg ip) and decapitated. All procedures for handling animals have been approved by institutional animal care review committees. The temporal bones were rapidly dissected out and placed in chilled, oxygenated high-Ca2+ L-15. The ampullae of the semicircular canals were excised and treated for 10-12 min at 37°C with protease XXVII (Sigma, St. Louis, MO; 500 µg/ml in low-Ca2+ L-15) to loosen the bonds between hair bundles and cupulae. They were then immersed in low-Ca2+ L-15 containing papain (Sigma, crude; 500 µg/ml) and L-cysteine (Sigma; 300 µg/ml) for 40 min at 37°C, following which they were left in bovine serum albumin (Sigma; 500 µg/ml in low-Ca2+ L-15 medium) for 40 min at room temperature. One crista (sensory epithelium) was then brushed with a fine probe. The dissociated hair cells were allowed to settle onto the glass floor of the recording chamber. The other cristae were stored in high-Ca2+ L-15 at 6-8°C for later dissociation. All cristae were used within 36 h. No difference was found in the resting potentials and the current amplitudes of cells studied immediately or after storage at 6-8°C. Data from these two groups were pooled. The dissociated cells were viewed at ×600 with Nomarski optics on an inverted microscope (Olympus IMT-2, Olympus Corporation, Lake Success, NY). The recording chamber was continuously perfused with oxygenated high-Ca2+ L-15.
Electrophysiology
WHOLE CELL RECORDINGS.
All recordings were done at room temperature (23-25°C). Whole cell
currents were recorded using the conventional ruptured-patch method.
Borosilicate pipettes were pulled and heat-polished to a final pipette
resistance of between 3 and 5 M in our standard solutions and coated
with silicone elastomer (Sylgard; Dow Corning, Midland, MI). The
pipettes were filled with a solution containing (in mM): 130 KCl, 0.1 CaCl2, 2 MgATP, 11 EGTA, 10 HEPES, and 0.2 Li3GTP. The pH was 7.3 and the osmolality was
~300 mmol/kg as measured with a vapor-pressure osmometer (Wescor,
Logan, UT). The intracellular calcium concentration was estimated with
Eqcal software (Biosoft, Cambridge, England) to be 900 pM. pH was
titrated to 7.3 with 1 N KOH for a final K+
concentration of 159 mM. The standard bath solution was
high-Ca2+ L-15 (330 mmol/kg; pH 7.3). The
junction potential (7 mV) was subtracted from voltages off-line.
SINGLE-CHANNEL RECORDINGS.
All recordings were done at room temperature (23-25°C).
Single-channel currents were recorded from membrane patches in either the inside-out or the cell-attached configuration. Inside-out patches
were used to characterize IK,L, and
cell-attached patches were used to study modulation of
IK,L by NO. The external solution was
(in mM): 145 K gluconate, 1.2 MgSO4, 5 HEPES, and
10 EGTA. For cell-attached patches, we assume that the resting membrane potential is 0 mV in this external solution. Recording pipettes (5-8
M) contained a solution similar to the external solution but with 1 mM EGTA and 10 µM Ca2+, for a final estimated
Ca2+ concentration of 1.5 nM (calculated with
Eqcal software).
LOCAL SOLUTION EXCHANGE. Chemicals such as 4-aminopyridine (4-AP, Sigma) or NO donors (see Nitric oxide experiments) were dissolved in high-Ca2+ L-15 and applied to individual cells via wide-bore pipettes (~100 µm tip diameter). Test and control solutions were applied by separate pipettes fed by separate lines.
Data acquisition and analysis
WHOLE CELL DATA.
Whole cell currents were recorded with an amplifier (L/M EPC-7, Adams
and List Associates, Great Neck, NY; or
Axopatch-1D, Axon Instruments, Foster City, CA) and 12-bit A/D and D/A
converters (Scientific Associates, Rochester, NY), controlled by data
acquisition software (DATAQ, JPM Programming, Rochester, NY). The
sampling interval varied from 125 µs to 1.9 ms. Data were low-pass
filtered on-line at a cutoff frequency (3 dB) of 3 kHz (using the
3-pole Bessel filter of the EPC-7) or 5 kHz (using the 4-pole Bessel filter of the Axopatch-1D).
SINGLE-CHANNEL DATA. Single-channel data were recorded with the Axopatch 1D or 200A amplifier (Axon Instruments) and a 12-bit acquisition board (Digidata, Axon Instruments), controlled by pClamp. The sampling interval varied from 50 µs to 1 ms. Data were low-pass filtered on-line at a cutoff frequency of 2 kHz and in some cases were then digitally filtered off-line at 1 kHz. Data were analyzed off-line with pClamp and plotted with Origin.
Nitric oxide experiments
The following NO donors were dissolved in high-Ca2+ L-15 medium: sodium nitroprusside (SNP, Sigma), nitroglycerin (NTG, Tridil, Du Pont Pharmaceutical, Wilmington, DE), 3-morpholinosydnonimine (SIN-1, Molecular Probes, Eugene, OR) and S-nitroso-N-acetylpenicillamine (SNAP, Molecular Probes). These donors are all light sensitive and probably employ photosensitive degradation as one mechanism of NO release. All solutions were prepared under dim light and used within 6 h after they were made. To slow photodegradation, we darkened the room and wrapped aluminum foil around the perfusion apparatus. We used the microscope light to facilitate NO release near the cell of interest. To avoid unwanted effects of leak NO on control recordings, we introduced the local perfusion pipette into the recording chamber after the last control recording was done. Preparations were generally exposed to NO donors or 8-bromo-cGMP just once.
SNP dissolves in solution into Na2+ and
nitroprusside
([Fe(CN)5NO]2)
(Arnold et al. 1984
; Bates et al. 1991
).
Nitroprusside breaks down into NO and other products that may release
minute amounts of cyanide and iron. To control for effects by these
other breakdown products, we added the NO scavenger,
carboxy-2-phenyl-4,4,5,5,-tetramethylimidazoline-3-oxide-1-oxyl (carboxy-PTIO, Calbiochem, La Jolla, CA) (Yoshida et al.
1993
). As shown in RESULTS, carboxy-PTIO largely
eliminated the SNP effect.
NTG
(C3H5N3O9)
releases NO when exposed to thiol-containing compounds (Harrison
and Bates 1993), such as cysteine and dithiothreitol. The L-15
solution in which NTG was dissolved contains cysteine. Because the NTG
stock solution contained ethanol, which might affect channel activity,
we added anhydrous ethanol to control solutions at the same
concentrations as in the NTG test solutions: 1.3% in 1 mM NTG and
0.4% in 0.3 mM NTG.
SNAP
(C7H12N2O4S)
and SIN-1
(C6H10N4O2.HCl)
probably release NO via mechanisms similar to those for NTG
(Lincoln et al. 1997).
NEUROTOXICITY.
One of the oxidation-reduction states of NO (NO·) reacts with
superoxide anion to form peroxynitrite, a neurotoxic agent. Superoxide dismutase (SOD) and catalase (CAT) can prevent the formation of peroxynitrite and H2O2,
respectively. In most but not all of the SNP experiments, SOD (50 IU/ml, Sigma) and CAT (50 IU/ml, Sigma) were added. There was no
obvious difference from SNP data obtained without SOD and CAT. Also,
resting potentials were stable during SNP treatments without SOD and
CAT. These observations are consistent with the report that SNP
releases the neural protective redox state
(NO+) (Lipton et al. 1994). SNP
data obtained with and without SOD and CAT are pooled.
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RESULTS |
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IK,L is strongly correlated with type I morphology
Hair cells were classified before recording as type I if they had flask-shaped bodies and as type II if they had more cylindrical shapes. Rounded cells, which are likely to have lost their shape during the preparation, were not classified.
The morphological and whole cell electrophysiological profiles of hair
cells were correlated. The mean resting potentials, VR, were 81.3 ± 0.2 mV for 144 type I hair cells and
71.3 ± 2.3 mV for 36 type II hair cells.
Mean Cm values, which are proportional to membrane surface area, were 2.6 ± 0.1 pF for 142 type I hair cells and 3.7 ± 0.2 pF for 42 type II hair cells. These numbers are presumably low as many hair cells had lost their hair bundles during preparation.
Morphological cell type correlated strongly with the whole cell
currents evoked by our standard voltage protocol: an iterated series of
50-ms steps from VH = 67 mV. In type
I cells, the voltage steps evoked currents with a large instantaneous
component (see Figs. 6B, 9A, and 10A
for examples). The properties of this instantaneous current, described
in the following text, show that it is
IK,L. In the NO experiments, we
interpret effects of the NO donors on the instantaneous current as
effects on IK,L. Voltage steps
positive to
60 mV evoked sigmoidally activating outward currents
(Fig. 2A). It is not known
whether these currents are through
IK,L channels or other outwardly
rectifying channels, as described in type I hair cells of the mouse
utricle (Rüsch and Eatock 1996a
;
Rüsch et al. 1998
).
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In type II cells, in contrast, voltage steps from 67 mV typically
evoked very small instantaneous currents, reflecting the absence of
gK,L or other large conductances at
67 mV (data not shown). Voltage steps positive to
60 mV evoked
sigmoidally activating outwardly rectifying currents as is typical of
type II hair cells from the vestibular organs of other amniotes
(Eatock et al. 1998
; Lang and Correia
1989
).
We demonstrated the correlation between IK,L and type I morphology in a subset of 88 sequentially recorded cells. Thirty-one of 36 type I hair cells had IK,L (86%), compared with 4 of 29 (11%) of type II cells and 11 of 23 (48%) of the unclassified cells. Whether the few type I cells without IK,L and type II cells with IK,L represent true variation in the sample or misclassification of cell type is not known. The necks of dissociated type I cells sometimes relax; this could lead to their misidentification as type II.
Effects of IK,L on membrane voltage response to injected currents
The presence of a large conductance
(gK,L) at
VR dramatically affects the voltage
responses of type I hair cells to injected currents (Fig.
2C). The median input resistance
(Rm) of 27 type I hair cells at 67
mV was only 25 M
(mean 60 ± 16 M
; Fig. 2D) compared with 1.3 G
(mean 1.5 ± 0.2 G
, data not shown) in
27 type II hair cells. Thus a given input current at
67 mV will evoke
a voltage change in the median type I cell that is just one-fiftieth
that of the median type II cell. Median
Rm and mean Cm values yield membrane time
constants (
m = RmCm) for type I and
type II hair cells of 64 µs and 3.8 ms, respectively, at
67 mV. The
rise time of the receptor potential will therefore be much faster for
type I cells than for type II cells. Differences will be smaller when
values at resting potential are compared because fewer
IK,L channels are open at the average
VR (
81 mV) than at our usual holding
potential of
67 mV. Nevertheless even from
VR, large current injections (360 pA),
comparable with the largest transduction currents measured in mammalian
vestibular hair cells (Géléoc et al. 1997
;
Holt et al. 1997
), may depolarize the type I cells by
<10 mV (Fig. 2C). Similar observations have been made in
type I hair cells from gerbil semicircular canals (Rennie et al.
1996
) and mouse utricles (Rüsch and Eatock
1996b
).
Properties of IK,L
ISOLATION OF IK,L.
Several lines of evidence indicate that negative to 50 mV, most of
the current in type I cells is IK,L.
It is not leak or inwardly rectifying current because it deactivates
with hyperpolarization and is almost completely blocked by 4-AP (see
following text). The criterion used to select cells for analysis
(Gm
1 nS at
97 or
107 mV)
ensures that any contaminating leak or inward rectifying currents are
at most 5% of the total conductance (1 in ~20 nS, the minimum
Gmax estimated for
IK,L; see ACTIVATION). As shown next,
activation curves in this voltage range are consistent with a single
conductance. Although other outwardly rectifying K+ currents have been described in type I hair
cells (Rennie and Correia 1994
; Rüsch and
Eatock 1996a
), they activate positive to
60 mV (typical
half-maximal activation voltages of
30 mV).
REVERSAL POTENTIAL.
The reversal potential of IK,L,
VREV, was estimated from a linear
regression of the instantaneous currents evoked by steps from 67 mV
to voltages on either side of the current reversal. The mean
VREV in 10 cells was
80.2 ± 0.5 mV. The proximity of VREV to the
estimated equilibrium potential for K+,
85 mV,
shows that the instantaneous current was very K+
selective and had little contribution from nonselective leak.
ACTIVATION.
Figure 3A illustrates the slow
sigmoidal activation of IK,L in
response to depolarizing 600-ms steps from
VH = 97 mV.
IK,L took in excess of 600 ms to reach
steady state at the very negative voltages at which it started to
activate, consistent with data from the mouse utricle
(Rüsch and Eatock 1996a
). The activation curve of
IK,L (Fig. 3B) was
generated from the tail currents at
37 mV following the iterated
steps. The curve was fitted with a first-order Boltzmann function
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K CHANNEL BLOCKERS.
IK,L channels in type I hair cells
from other organs are blocked by 4-AP (Rennie and Correia
1994; Rüsch and Eatock 1996a
) and are
permeable to Cs+ (Rüsch and Eatock
1996a
). Figure 5 shows that the
instantaneous current evoked by steps from
67 mV to potentials
between
97 and
7 mV was blocked by external application of 5 mM
4-AP. In three cells, the mean percentage inhibition of instantaneous
currents was 92 ± 1%. IK,L
persisted when Cs+ replaced
K+ in the internal solution and was added at 5 mM
to the external solution (n = 5; Fig. 5, D
and E). These results indicate that the conductance at
67
mV is principally gK,L. Later we
exploit this observation to study the effects of NO on
gK,L.
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SINGLE-CHANNEL CURRENTS.
Ion channel activity was recorded in inside-out membrane patches
excised from type I hair cells, in symmetrical K gluconate solutions
(145 mM; n = 10). A good candidate for the
IK,L channel was identified on the
basis of its prevalence and the following properties shared with whole
cell IK,L: voltage range of
activation, K+ selectivity, deactivation
kinetics, sensitivity to 4-AP, and sensitivity to sodium nitroprusside.
These properties are demonstrated in Figs.
68 and 12. Figure 6A shows
channel activity at
60 mV that was inhibited by stepping to
130 mV.
The ensemble average of 17 such traces revealed an inward current that
deactivated with a time constant of 4.4 ms. Whole cell
IK,L recorded from the same cell later
deactivated with a similar time course (Fig. 6B).
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Modulation of IK,L by NO donors
Addition of NO donors to the external solution reduced the
instantaneous currents evoked by voltage steps from 67 mV, which as
shown in the preceding text are principally carried by
IK,L channels. Figure
9, A-D, shows reversible
suppression of the instantaneous current by 1 mM SNP. The residual
outward current in 1 mM SNP (Fig. 9B) is similar in size and
kinetics to the time-varying component of the outward current in
control conditions (Fig. 9A). Whether this residual current
was through IK,L channels or through a
second, more positively activating outward rectifier is not known. We
quantified the inhibition of the instantaneous current by subtracting a
fit of the instantaneous current-voltage (I-V) relation in
SNP from a fit of the instantaneous I-V relation in control
conditions (Fig. 9E), then normalizing the subtracted values
by dividing by the control data. The suppression shown in Fig. 9 was
the most complete in a series of experiments in which we varied the NO
donor or the donor concentration. The concentration of NO is not known;
it is a function of the donor concentration, time since the solution
was made, temperature and light exposure.
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If the NO effect is mediated by a second-messenger cascade, then it may increase gradually. The reversible inhibition of IK,L by another NO donor, NTG, is shown in Fig. 10. After 1 min of NTG treatment, the instantaneous current (IK,L) was substantially reduced, but the time-varying component was still present (Fig. 10B). After wash and recovery of the instantaneous current (Fig. 10C), the cell was treated with NTG for 4.5 min before the recording shown in Fig. 10D, in which the instantaneous current was almost completely suppressed and the time-varying component was substantially reduced. Thus the full effect of NTG took minutes to develop. The mean durations of exposure to the various NO donors ranged from 1 to 4 min. The results of Fig. 10 suggest that mean inhibitory effects might have been greater had exposure durations been longer.
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NO donors may affect channels by mechanisms other than NO release (see
METHODS). As one approach to this problem, we tested four
different NO-producing agents. The fact that all four inhibited the
instantaneous current argues that the effect was mediated by the common
release product, NO. Figure 11
summarizes the inhibition of instantaneous current by four different NO
donors: SNP, NTG, SIN-1, and SNAP, some at more than one concentration.
As another form of control for effects not mediated by NO, we added the
NO scavenger, carboxy-PTIO (200 µM), to solutions containing 1 mM SNP
or 250 µM SNAP (Fig. 11, ). Carboxy-PTIO was very effective at
blocking the inhibitory effects of SNP and SNAP. The mean inhibitory effects of 1 mM SNP without and with carboxy-PTIO were 52 ± 8% (n = 9) and 7 ± 1% (n = 3),
respectively. The mean inhibitory effects of 250 µM SNAP without and
with carboxy-PTIO were 33 ± 6% (n = 8) and
5 ± 3% (n = 4), respectively.
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Mechanism of NO effect
The effects of SNP on channel activity recorded from patches
suggest that the NO effect involves intracellular second messengers. IK,L channels were inhibited by 1 mM
SNP in cell-attached-patch mode (n = 5 patches from 5 cells) but not in excised inside-out patches (n = 10 patches from 10 cells). Figure 12 shows
a patch from which both kinds of recording were obtained. The patch
appeared to have at least two such channels, plus other channels with
much smaller conductances (Fig. 12A). Perfusion of the cell
with 1 mM SNP greatly reduced all channel activity when the patch was
cell-attached (Fig. 12B). Excision of the patch restored
channel activity, despite continued perfusion with SNP (Fig.
12C). This suggests that the SNP effect is mediated by
soluble second messengers that are no longer present in excised
patches. It provides further evidence that the SNP effect is mediated
by an NO cascade and not another byproduct of the breakdown of SNP. The
29-pS channels in the excised patch were reversibly blocked by 5 mM
4-AP (Fig. 12, D and E), consistent with their
being IK,L channels. The
small-conductance channels were more visible after the 29-pS channels
were blocked (Fig. 12D). From their size (~10-pS) and the
fact that they were appreciably activated at 60 mV, these may have
been inwardly rectifying channels. Their absence in Fig. 12B
suggests that they too may be inhibited by NO.
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In some systems, NO effects are mediated by cGMP, produced as a consequence of NO activation of guanylate cyclase. We therefore tested the effect of 8-bromo-cGMP, a nonhydrolyzable membrane-permeant cGMP analogue, on the instantaneous currents recorded in whole cell mode with the standard voltage protocol. 8-bromo-cGMP (1 mM) in the external solution inhibited the instantaneous current by 45 ± 7% (n = 10; Fig. 11), consistent with a role for cGMP in the inhibition produced by NO donors.
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DISCUSSION |
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IK,L is a feature of type I hair
cells from all kinds of amniote vestibular organs: the gerbil crista
(Rennie and Correia 1994), mouse utricle
(Rüsch and Eatock 1996a
), all vestibular organs of
the pigeon (Ricci et al. 1996
), and the turtle crista (Brichta et al. 1996
).
IK,L is
K+-selective, activates sigmoidally and
relatively slowly with depolarization, and inactivates slowly. It is
independent of external Ca2+ (Rüsch
and Eatock 1996a
). Here we show that its single-channel conductance is between 20 and 30 pS. In all of these respects, IK,L resembles a delayed rectifier,
but it is unlike most delayed rectifiers in that it has an appreciable
permeability to Cs+ and an activation range that
is unusually negative and variable.
In its activation range and sensitivity to phosphorylation,
IK,L resembles a major conductance,
IK,n, found in some outer hair cells
of the mammalian cochlea (Jagger and Ashmore 1999; Mammano and Ashmore 1996
). It has recently been
suggested that IK,n channels contain
the KCNQ4 subunit (Marcotti and Kros 1999
). C. Chen et al. (1995)
found that 10 mM SNP reduced the
outward current of outer hair cells by 40%; whether the current that
they studied included IK,n is not
known. Another member of the KCNQ family is the neuronal M current,
which is a heteromultimer of KCNQ2 and KCNQ3 (Wang et al.
1998
). Like IK,n and
IK,L, M current dominates the resting
membrane conductance and is sensitive to modulation. Type I hair cells
express KCNQ4 subunits (Hurley et al., 2000), but
whether these contribute to IK,L
channels is not known.
From a functional point of view, the activation range of IK,L is its most intriguing property. The relatively negative range means that many type I hair cells have unusually low input resistances. As a consequence, their voltage responses to injected current have relatively fast rise times and low gains (voltage output per current input). A powerful way to control the gain and time course of the receptor potential, therefore, is to modulate factors that control the number of IK,L channels that are open at the cell's resting potential. The effect of NO donors on IK,L in combination with anatomical data showing NOS at various sites in vestibular epithelia show that NO is a potential endogenous modulator.
Spontaneous shifts of the activation curve
Positive spontaneous shifts of the voltage activation range (Fig.
4) are also seen when IK,L is recorded
in ruptured-patch whole cell mode from hair cells isolated from the rat
utricle (Hurley and Eatock 1999) and from hair cells in
situ in the epithelium of the mouse utricle (Rüsch and
Eatock 1996a
). These shifts suggested to us that the activation
range is under the control of an intracellular factor that diffuses
into the recording pipette rather slowly during whole cell dialysis. In
agreement with this hypothesis, no shift occurs in perforated patch
experiments, which largely preserve the intracellular milieu
(Hurley and Eatock 1999
). Negative shifts in activation
or inactivation curves of other ionic currents, with similar time
courses, have been attributed to changes in the junction potential as
large intracellular anions slowly wash out (Marty and Neher
1983
). A similar trivial mechanism does not explain the
positive shift in V1/2 of
gK,L, however, given that the
V1/2 value of the delayed rectifier in type
II cells does not shift with time (Hurley and Eatock
1999
).
In a recent report on type I cells in the rat utricle, ruptured-patch
recordings of the outwardly rectifying current had more positive
V1/2 values when ATP was omitted from the
pipette solution (Lennan et al. 1999). Preliminary data
from our laboratory suggest that phosphorylation inhibitors positively
shift the activation curve of IK,L
(Hurley and Eatock 1999
). The absence of internal ATP in
the experiments of Lennan et al. may also shift the activation curve
positively by preventing phosphorylation. A comparable effect is seen
in cochlear outer hair cells, where the activation range of
IK,n is negatively shifted by
cAMP-dependent phosphorylation (Jagger and Ashmore
1999
). Phosphorylation induces shifts in the activation curves
of other kinds of K+ channels, although in some
cases the shifts are in the opposite direction (Hoffman and
Johnston 1998
; Thomas et al. 1999
).
Lennan et al. argue that the V1/2 values
reported by us and others for IK,L are
an artifact induced by pipette ATP. ATP is, however, normally present
in cells and is essential to prevent rundown of some ion channels
(Forscher and Oxford 1985). Moreover, the range of
V1/2 values that we obtain in whole cell
recordings from rat utricular type I cells is the same (
90 to
40
mV) whether we record with the perforated-patch method, which preserves
endogenous ATP levels, or the ruptured-patch method (Hurley and
Eatock 1999
).
IK,L channels
MULTIPLE CLOSED STATES.
Several features of the whole cell and single-channel data are
consistent with a multi-state kinetic model, of the form:
C1 ...
Cn
O, in which just one
transition is strongly voltage dependent. The activation kinetics are
sigmoidal, consistent with multiple closed states (Rüsch
and Eatock 1996a
). The G-V curve, however,
is well fitted by a first-order Boltzmann function, suggesting that
only one transition is strongly voltage dependent. When recorded at
high temporal resolution, deactivation follows a double-exponential function (Rüsch and Eatock 1996a
). This can
indicate the contribution of more than one channel type or open state
but also can occur for a single channel type with multiple closed
states if the Cn
O
transition has a weak voltage dependence (see discussion in Bezanilla et al. 1994
). Finally, the
single-channel behavior shows long inter-burst intervals and within
bursts both very short closures and intermediate ones (3-40 ms in
trace at
60 mV in Fig. 7A), possibly corresponding to
different closed (and/or blocked or inactivated) states.
IK,L CHANNEL DISTRIBUTION.
Our estimates of single-channel and maximum whole cell conductances
suggest that there are tens of thousands of
IK,L channels per hair cell at a
density of 100-200/µm2. How
IK,L channels are distributed on the
type I hair cell membrane surface is not known. In a freeze-fracture
study of type I hair cells from the guinea pig, Gulley and
Bagger-Sjöbäck (1979) described medium-sized
intramembrane particles that are diffusely distributed over the type I
cell membrane and irregular patches of larger particles (12-14 nm
diam). Either set of particles might include
IK,L channels. The active zones of
frog saccular hair cells display elongate clusters of particles, also
~12 nm diam in freeze fracture replicas, which may be co-localized
Ca2+ and Ca2+-dependent
K+ channels (Roberts et al. 1990
).
In type I cells, the patches of large particles surround
"invaginations" (Gulley and Bagger-Sjöbäck 1979
): zones where the calyx invaginates the hair cell and
where the hair cell and calyx membranes are unusually close together. The function of the invaginations is not known. Although it is attractive to speculate that they are involved in afferent
transmission, only one-quarter of presynaptic ribbons are in close
proximity to invaginations (Lysakowski and Goldberg
1997
). Inspection of micrographs in the Gulley and
Bagger-Sjöbäck paper suggests that some particle clusters
form annuli of ~1,000 particles around an invagination. In the mature
chinchilla crista, the number of invaginations per type I cell varies
from ~10 for peripheral cells to ~50 for central cells
(Lysakowski and Goldberg 1997
). Dividing the estimated
mean number of IK,L channels in our
cells (39,000) by the mean number of invaginations in chinchilla type I
cells (~30) yields ~1,300 channels per invagination, consistent
with the number of large particles per invagination in the guinea pig type I cells. If the particles were the
IK,L channels, the variance in number
of invaginations could account for a good fraction of the variance in
number of channels per cell (Gmax;
10-fold range in our sample).
NO-cGMP cascade in the inner ear?
In one scheme by which NO is proposed to mediate retrograde
neurotransmssion (reviewed by Dawson and Snyder 1994;
Garthwaite and Boulton 1995
), glutamate released from
the presynaptic neuron binds to postsynaptic
N-methyl-D-aspartate (NMDA) receptors, which open and admit Ca2+. The incoming
Ca2+ activates NOS to make NO, which diffuses out
of the postsynaptic terminal to act on neighboring cells. A major
target of NO is believed to be soluble guanylate cyclase (sGC). NO
activates sGC, thereby increasing the production of cGMP, which can
have many actions. In the next paragraphs we review some of the
evidence that the inner ear has the elements of the NO-cGMP cascade.
NMDA RECEPTORS.
The afferent transmitter is either glutamate or another substance that
can activate glutamate receptors (Kataoka and Ohmori 1994). Although fast excitatory transmission is likely to
involve AMPA receptors (see Sewell 1996
for review),
NMDA receptors are found on cultured chick cochlear ganglion neurons
(Yamaguchi and Ohmori 1990
) and appear to participate in
the background discharge of vestibular afferents in amphibians
(Soto et al. 1994
; Zucca et al. 1993
).
According to a preliminary report, both calyx endings and type I hair
cells are immunoreactive for NMDA receptor subunits (Ishiyama et
al. 2000
).
NITRIC OXIDE SYNTHASE.
Various groups have found evidence for NOS in vestibular and cochlear
epithelia. Lysakowski and colleagues have found that efferent terminals
and some hair cells in the rodent vestibular epithelium stain
positively for NADPH diaphorase (Lysakowski and Singer
2000; Singer and Lysakowski 1996
), which in
formaldehyde-fixed neural tissue correlates well with bNOS (reviewed in
Lincoln et al. 1997
). Consistent with the localization
to efferent terminals in the epithelium, a subset of efferent neuron
cell bodies in the brain stem both stains for diaphorase and is
immunoreactive for bNOS.
SOLUBLE GUANYLATE CYCLASE.
For NO's effect on IK,L to be
mediated by cGMP, sGC must be present in type I hair cells; this
remains to be determined, although sGC immunoreactivity has been
localized to vestibular maculae (Hess et al. 1998a).
Whether sGC is present in cochlear hair cells is in dispute
(Fessenden and Schacht 1997
; Hess et al.
1998a
), possibly because Fessenden and Schacht used an enzyme
assay and Hess and colleagues used immunocytochemistry. This recalls
the differences obtained with the diaphorase method, another enzyme assay, and NOS antibodies.
MECHANISM OF NO ACTION.
The inhibition by NO donors and 8-bromo-cGMP of the
IK,L conductance at 67 mV may
reflect a reduction in the total number of channels
(Gmax) or a positive shift of the
activation curve so that fewer channels are open at
67 mV (reduction
in Po), or both. Our preliminary
efforts to resolve this question produced inconsistent results. One
problem is the lability of the position of the activation curve in
ruptured-patch recording. Behrend et al. (1997)
found
that cGMP reduces both Po and
Gmax in multi-channel cell-attached
patches from type I cells that were also dissociated from the rat
crista. No effect was seen in excised patches, indicating that cGMP was
not directly affecting the IK,L
channels. cGMP may affect IK,L via
cGMP-dependent protein kinase or phosphodiesterases of which there are
multiple types (reviewed in Garthwaite and Boulton
1995
). Depending on the relative amounts of each cGMP-dependent enzyme in the type I hair cell, NO can induce complex intracellular reactions merely by increasing the intracellular level of cGMP.
NO modulation of afferent transmission
In type I hair cells from a variety of organs, either
enzymatically dissociated or in situ in semi-intact epithelia,
IK,L produces low input resistances
and unusually negative resting potentials. Preliminary data indicate
that the activation range of voltage-gated Ca2+
channels in type I hair cells is positive to 60 mV (Bao et al. 1999
) as it is in other hair cells. Therefore it is not clear how type I cells generate receptor potentials large enough to activate
conventional chemical transmitter release in vivo. Our data suggest
that NO released within the vestibular epithelium would reduce
IK,L and consequently would both move
the resting potential positive and enhance the receptor potential.
NO may be active as far as ~300 µm from the source
(Garthwaite and Boulton 1995), a distance that would
include much of the crista. Thus NO produced by hair cells or afferent
or efferent terminals might affect
IK,L in any given type I hair cell. It has been argued that NO produced in one cell acts principally on NO
targets in other cells (Lincoln et al. 1997
) because
Ca2+ influx that stimulates NOS in a cell
simultaneously inhibits sGC and stimulates cGMP phosphodiesterase in
that same cell. In that case, IK,L
channels would be most affected by NO produced outside the hair cell,
in neighboring hair cells or in nerve terminals. Excitatory head
movements would produce Ca2+ flow into hair cells
and afferent terminals through multiple routes, which might then
activate NOS in either or both sites, producing NO and inhibiting
IK,L in type I hair cells in the vicinity.
The evidence that NOS is present in vestibular efferent neurons is
particularly intriguing, as it suggests a way for efferents to modulate
type I cells without physically contacting them (Fig. 1). Activation of
efferent neurons would cause Ca2+ influx through
voltage-gated Ca2+ channels in the efferent
terminals. The influx would promote the release of conventional
neurotransmitter, chiefly acetylcholine, from the efferent terminal
onto the calyx ending but would also stimulate NO production by NOS in
the efferent terminals. The NO would readily cross the calyx to the
type I cell and inhibit IK,L, making
the resting potential more positive (enhancing background transmitter
release) and increasing the receptor potential (enhancing response
gain). These effects are consistent with efferent actions in the
mammalian vestibular system. Shocking the vestibular efferents has
predominantly excitatory effects on vestibular afferents, increasing
background discharge rates and in some cases increasing the gain of the
response (spike rate per unit of stimulus) (Goldberg and
Fernandez 1980). In the axolotl inner ear, there is evidence for an excitatory effect on afferent discharge by endogenous NO. Inhibition of NO production reduces both the background and evoked discharge of vestibular afferents (Flores et al. 1996
).
In summary, our results show that NO could substantially affect the
excitability of type I hair cells by inhibiting the dominant conductance near resting potential. But in the intact epithelium, NO
actions may be more complex. As a simple example, NO activates L-type
Ca2+ channels in some cell types (Kurenny
et al. 1994) but inhibits them in other cell types
(Summers et al. 1999
). A preliminary report suggests
that NO reduces the open probability of Ca2+
channels in hair cells of the frog saccule (Rodriguez-Contreras et al. 2000
). In type I hair cells, NO activation of
Ca2+ channels would act synergistically with NO
suppression of IK,L to enhance
afferent transmission, whereas NO inhibition of
Ca2+ channels would antagonize the
IK,L effect.
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ACKNOWLEDGMENTS |
---|
We thank Dr. Michael Strupp for help and participation in early experiments on IK,L channels, in particular the experiment featured in Fig. 6. We thank M. Vollrath and Drs. Karen Hurley and Anna Lysakowski for helpful comments on the manuscript.
This research was supported by National Institute on Deafness and Other Communication Disorders Grant DC-02290 and by funds from the Karim Al-Fayed Neurobiology of Hearing Laboratory.
Present address of J.W.Y. Chen: Dept. of Neurology, UCLA School of Medicine, Los Angeles, CA 90095.
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FOOTNOTES |
---|
Address for reprint requests: R. A. Eatock, Dept. of Otolaryngology, Baylor College of Medicine, One Baylor Plaza, Houston TX 77030 (E-mail: eatock{at}bcm.tmc.edu).
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.
Received 15 September 1999; accepted in final form 17 March 2000.
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REFERENCES |
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