Department of Physiology, University of Wisconsin, Madison, Wisconsin 53706
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ABSTRACT |
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Bal, Ramazan and
Donata Oertel.
Potassium Currents in Octopus Cells of the Mammalian Cochlear
Nucleus.
J. Neurophysiol. 86: 2299-2311, 2001.
Octopus cells in the posteroventral cochlear nucleus (PVCN) of
mammals are biophysically specialized to detect coincident firing in
the population of auditory nerve fibers that provide their synaptic
input and to convey its occurrence with temporal precision. The
precision in the timing of action potentials depends on the low input
resistance (~6 M) of octopus cells at the resting potential that
makes voltage changes rapid (
~ 200 µs). It is the
activation of voltage-dependent conductances that endows octopus cells
with low input resistances and prevents repetitive firing in response
to depolarization. These conductances have been examined under whole
cell voltage clamp. The present study reveals the properties of two
conductances that mediate currents whose reversal at or near the
equilibrium potential for K+ over a wide range of
extracellular K+ concentrations identifies them
as K+ currents. One rapidly inactivating
conductance, gKL, had a threshold of
activation at
70 mV, rose steeply as a function of depolarization with half-maximal activation at
45 ± 6 mV (mean ± SD), and was fully activated at 0 mV. The low-threshold
K+ current (IKL)
was largely blocked by
-dendrotoxin (
-DTX) and partially blocked
by DTX-K and tityustoxin, indicating that this current was mediated
through potassium channels of the Kv1 (also known as shaker
or KCNA) family. The maximum low-threshold K+
conductance (gKL) was large, 514 ± 135 nS. Blocking IKL with
-DTX
revealed a second K+ current with a higher
threshold (IKH) that was largely
blocked by 20 mM tetraethylammonium (TEA). The more slowly inactivating conductance, gKH, had a threshold for
activation at
40 mV, reached half-maximal activation at
16 ± 5 mV, and was fully activated at +30 mV. The maximum high-threshold
conductance, gKH, was on average
116 ± 27 nS. The present experiments show that it is not the
biophysical and pharmacological properties but the magnitude of the
K+ conductances that make octopus cells unusual.
At the resting potential,
62 mV, gKL
contributes ~42 nS to the resting conductance and mediates a resting
K+ current of 1 nA. The resting outward
K+ current is balanced by an inward current
through the hyperpolarization-activated conductance,
gh, that has been described previously.
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INTRODUCTION |
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The low input resistance of
octopus cells causes synaptic responses to be brief and voltage changes
in response to synaptic current to be small. Octopus cells require the
summation of many synaptic inputs within a period of 1 ms to fire and
thus detect coincident firing among their inputs (Golding et al.
1995). In requiring the summation of many small inputs to
produce a brief but robust synaptic response, the temporal jitter in
the timing of individual auditory nerve inputs is lost in the firing of
octopus cells. In vivo octopus cells convey the timing of broadband
transients or periodicity in sounds with a temporal jitter <200 µs
(Godfrey et al. 1975
; Oertel et al. 2000
;
Rhode and Smith 1986
; Rhode et al. 1983
).
Octopus cells can fire at rates
800 action potentials/s (Rhode
and Smith 1986
). Tones typically evoke a single, sharply timed
action potential at the onset of the stimulus; in responses to tones
<800 Hz, octopus cells can fire at every cycle (Friauf and
Ostwald 1988
; Godfrey et al. 1975
; Rhode
and Smith 1986
; Smith et al. 1993
).
Octopus cells have been recognized in all mammals including humans
(Adams 1986; Hackney et al. 1990
;
Osen 1969
; Willard and Martin 1983
;
Willott and Bross 1990
). They occupy the octopus cell
area, a region that is defined by a clear border in the caudal and
dorsal part of the PVCN (Kolston et al. 1992
;
Wickesberg et al. 1994
; Willott and Bross
1990
). Their axons form one of the ascending pathways through
the brain stem. They project contralaterally through the intermediate
acoustic stria to excite neurons in the superior paraolivary nucleus
(SPN) and in the ventral nucleus of the lateral lemniscus (VNLL)
(Adams 1997
; Schofield 1995
;
Schofield and Cant 1997
; Smith et al.
1993
; Vater et al. 1997
; Warr
1969
). The influence of octopus cells on the inferior
colliculus is indirect and inhibitory as neurons in the SPN are
GABAergic (Kulesza and Berrebi 1999
) and neurons in the
VNLL that are innervated by octopus cells are glycinergic (Saint
Marie et al. 1997
). It has been suggested that these pathways
play a role in the recognition of temporal patterns in natural sounds.
Several conductances contribute to the low input resistance of octopus
cells. In the initial studies of octopus cells made with sharp
electrodes in current-clamp experiments, it was not possible to measure
the input resistance but the dramatic increase of the input resistance
in the hyperpolarizing voltage range in the presence of
Cs+ was the first clue that a
hyperpolarization-activated conductance contributed to the resting
properties of octopus cells (Golding et al. 1995).
Recordings with patch-clamp electrodes showed that octopus cells have
input resistances of ~6 M
(Bal and Oertel 2000
;
Golding et al. 1999
). The pharmacological sensitivity to 4-aminopyridine (4-AP) (Golding et al. 1999
) and to
-DTX (M. Ferragamo and D. Oertel, unpublished results) indicated
that a low-threshold K+ conductance balanced the
hyperpolarization-activated conductance at rest and played a role in
shaping the voltage changes produced by synaptic currents. To
understand the properties of these conductances, they were examined
under voltage clamp. The large amplitude and relatively depolarized
voltage range of activation allows the hyperpolarization-activated,
mixed-cation conductance, gh, to contribute to the shaping of synaptic responses and to contribute a
large steady current at the resting potential (Bal and Oertel 2000
). The present study describes K+
conductances, one of which balances
gh.
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METHODS |
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Coronal slices of the PVCN from mice (ICR strain) of between 17 and 20 postnatal days were used for these experiments. After decapitation, the head was immersed in normal physiological saline containing (in mM) 130 NaCl, 3 KCl, 1.2 KH2PO4, 2.4 CaCl2, 1.3 MgSO4, 20 NaHCO3, 3 HEPES, and 10 glucose, saturated with
95% O2-5% CO2, pH 7.4, between 25 and 31°C (Golding et al. 1995, 1999
). After
the brain was removed from the skull, it was cut coronally at the
midcollicular level. The rostral surface of the specimen was mounted on
a Teflon block with a cyanoacrylate glue (Superglue). Two slices, 180 µm thick, were then cut using an oscillating tissue slicer (Frederick
Haer, Newbrunswick, ME). These were transferred to a storage chamber
containing fresh, oxygenated saline at 33°C for
2 h. The slices
were transferred to a recording chamber of ~0.3 ml in which it was
continuously perfused at about 6 ml/min with oxygenated saline whose
temperature was kept at 33°C with a custom-made, feedback-controlled heater.
Slices were visualized with a Zeiss Axioskop with a ×63
water-immersion lens. Octopus cells were initially identified by their characteristic location within a heavily myelinated fiber bundle just
ventral and caudal to the translucent granule cell region. Illumination
under bright-field conditions with the field diaphragm nearly closed
made octopus cells appear bright among dark bundles of myelinated
fibers. On breaking in to an octopus cell, its characteristic responses
to injected current were used to confirm the visual identification
(Bal and Oertel 2000; Golding et al. 1995
,
1999
).
All measurements of potassium currents were made under conditions in
which they could be isolated from other currents. The control solution
contained (in mM) 138 NaCl, 4.2 KCl, 2.4 CaCl2, 1.3 MgCl2, 10 HEPES, and 10 glucose, pH 7.4 when
saturated with 100% O2. The
hyperpolarization-activated current,
Ih, was blocked by the extracellular
application of 50 nM ZD7288 (Bal and Oertel 2000).
Voltage-sensitive sodium current was blocked by 1 µM tetrodotoxin (TTX) (Golding et al. 1999
). Voltage-sensitive calcium
current was blocked by 0.4 or 0.25 mM CdCl2 in
most experiments. Synaptic currents were blocked with 40 µM
6,7-dinitroquinoxaline-2,3-dione (DNQX; Tocris Cookson,
Avonmouth, UK). In experiments that concerned the
high-threshold K+ conductance, 50 nM
-dendrotoxin (
-DTX; Alomone Labs, Jerusalem, Israel) was added to
the control solution. When TEA was applied, it was substituted for
Na+. Test solutions were applied to the chamber
by redirecting the flow of liquid through a system of tubing and
valves. Unless otherwise stated, all chemicals were obtained from Sigma
(St. Louis, MO).
Pipettes were generally of low resistance (3-6 M). They were pulled
from borosilicate glass (1.2 mm OD). They were filled with a solution
that consisted of (in mM) 108 potassium gluconate, 9 HEPES, 9 EGTA, 4.5 MgCl2, 14 phosphocreatinine (tris salt), 4 ATP
(Na salt), and 0.3 GTP (tris salt); pH was adjusted to 7.4 with KOH
(Forscher and Oxford 1985
).
Current- and voltage-clamp recordings were performed with standard
whole cell patch-clamp techniques using an Axopatch-200A amplifier.
Data were low-pass-filtered at 5-10 kHz. Current and voltage records
were sampled at 10-40 kHz and were digitized on-line using a Digidata
1320 interface (Axon Instruments, Foster City, CA) and fed both to a
chart recorder and to an IBM-compatible personal computer for storage
and further analyses. Stimulus generation, data acquisition, and
off-line analysis of digitized data were done using pClamp software
(version 8.03; Axon Instruments). After the formation of
high-resistance seals (>1 G), negative pressure was applied to
obtain the whole cell configuration. Series resistance varied from 6 to15 M
. All reported results were from recordings in which
95% of
the series resistance could be compensated on-line; no corrections were
made for errors in voltage that resulted from uncompensated series
resistance. In these experiments, the actual voltage was maximally 6 mV
less positive than stated. All voltage measurements have been
compensated for a junction potential of
12 mV.
Statistical analyses were performed off-line. The results are given as means ± SD, with n being the number of cells in which the measurement was made. Significant differences between the groups were evaluated using a paired Student's t-test.
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RESULTS |
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Data reported here were obtained from recordings of 153 octopus
cells, each lasting between 20 min and 2 h. All reported
measurements were made while the hyperpolarization-activated current
was blocked by 50 nM ZD7288, voltage-sensitive
Na+ currents were blocked by with 1 µM TTX, and
excitatory synaptic currents were blocked by 40 µM DNQX. In most
experiments, 0.4 mM Cd2+ was added to block
Ca2+currents. Inhibitory synaptic currents have
not been observed in octopus cells (Gardner et al. 1999;
Golding et al. 1995
).
Definition of IKL and IKH
Depolarizing voltage steps from a holding potential of 90 mV
evoked large outward currents. The threshold for the appearance of
voltage-sensitive outward current was
70 mV. The current increased steeply in amplitude with steps to more depolarized potentials and then
decayed with relatively slow time course to a steady-state value that
was 23% of the peak (Fig.
1A). In the absence of
pharmacological blocking agents, the peak outward currents saturated
the amplifier at voltages more depolarized than
40 mV. The activation
of the outward current was rapid so that its beginning was superimposed on the capacitative current and could not be resolved reliably. Voltage
steps from
90 to
40 mV evoked currents that took 1.7 ± 0.2 ms
(n = 6) to reach half-peak amplitude. To characterize the large, voltage-sensitive outward current in octopus cells required
that the components be isolated and identified.
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Previous experiments in current clamp have shown that octopus cells
have voltage-sensitive Ca2+ currents
(Golding et al. 1999). The question arose therefore to
what extent voltage-sensitive Ca2+ currents and
Ca2+-activated K+ currents
contribute to the outward current. When external
Ca2+ was replaced with Mg2+
or 0.25 mM Cd2+ was added to the bath, the
amplitude and the shape of outward current evoked by pulses up to
40
mV did not change measurably (data not shown). Over the voltage range
90 to
40 mV under the present conditions, therefore
Ca2+ and Ca2+-activated K
currents are negligible.
Blockers of K+ channels blocked most of the
outward current and provided an initial separation and identification
of the currents. -DTX blocked a low-threshold current, leaving a
high-threshold current that was sensitive to TEA.
-DTX has been
shown to be a selective blocker for low-threshold
K+ outward currents in other types of neurons
(Brew and Forsythe 1995
; Gamkrelidze et al.
1998
; Halliwell et al. 1986
; Harvey
1997
; Owen et al. 1997
; Reid et al.
1999
; Southan and Robertson 2000
). Figure
1B shows that
-DTX blocked 96% of the outward current over the voltage range between about
70 and
40 mV. This experiment shows that the identical current can be isolated on the basis of its
voltage sensitivity and on the basis of its pharmacological sensitivity
to
-DTX and serves as a test for the specificity of
-DTX. The
-DTX-sensitive current had a low threshold of activation, about
70
mV, serving as the basis for our referring to this current as the
low-threshold potassium current, IKL.
Its activation was rapid, requiring on average 1.7 ms to reach the
half-peak amplitude. Over ~0.5 s, this current inactivated to
~50%. As octopus cells have resting potentials near
62 mV
(Bal and Oertel 2000
; Golding et al. 1995
,
1999
), this current is partially activated when octopus cells
are at rest. In the presence of 50 nM
-DTX, the voltage of octopus
cells could be stepped to more depolarized potentials and revealed
another outward current with a higher threshold that was sensitive to
20 mM TEA (Fig. 1D). This current had a threshold of
activation at about
40 mV and is thus termed the high-threshold potassium current, IKH (Fig. 1,
D and E). Voltage steps from
90 to
10 mV
evoked a current whose activation was partly obscured by the
capacitative current; it rose to half-peak-amplitude in 1.5 ms. This
current inactivated more slowly than the
-DTX-sensitive current.
Pharmacological experiments showed that 4-AP and TEA did not discriminate between IKL and IKH. While 5 mM 4-AP blocked IKL, it also decreased the amplitude of IKH. The effects of TEA on IKL were investigated in five octopus cells. TEA, at 10-20 mM, reduced the peak amplitude of IKL by between 30 and 40% (data not shown).
Sensitivity to blockers of Kv1 channels
The finding that IKL was
sensitive to -DTX suggested that this current was mediated through
ion channels of the Kv1 family (Harvey 1997
;
Hopkins 1998
; Hopkins et al. 1994
;
Owen et al. 1997
; Stühmer et al.
1989
). A more detailed study was therefore undertaken of the
sensitivity of IKL to
-DTX and
other toxins reported to be specific for Kv1 channels.
-DTX is
derived from the venom of the green mamba snake. Tests of the
specificity of this toxin on homomeric channels in expression systems
show that
-DTX is relatively unselective among the various Kv1
homomers. It blocks Kv1.2 channels with somewhat higher affinity than
Kv1.1, Kv1.3, and Kv1.6 channels (Dolly and Parcej 1996
;
Grissmer et al. 1994
; Harvey 1997
;
Owen et al. 1997
; Tytgat et al. 1995
).
DTX-K is derived from the venom of black mamba snakes and is a blocker
relatively more specific for Kv1.1 homomeric channels (Owen et
al. 1997
; Robertson et al. 1996
; Wang et
al. 1999a
,b
). Tityustoxin K
, derived from the venom of
scorpions, is reported to be specific for homomeric channels that
contain the Kv1.2
subunits (Hopkins 1998
;
Werkman et al. 1993
). The actions of these blockers on
heteromeric channels is less well characterized (Hopkins
1998
; Hopkins et al. 1994
; Tytgat et al.
1995
). In some combinations of
subunits, sensitivity to the
toxins seems to be conferred by a single toxin-sensitive subunit while
in other combinations of subunits sensitivity depends on all four
subunits (Hopkins 1998
).
A comparison of the sensitivity of IKL
to the less selective Kv1 channel blocker (-DTX) and the more
selective blockers of Kv1.1 (DTX-K) and Kv1.2 (tityustoxin) is shown in
Fig. 2.
-DTX (50 nM) blocked 90% of
the peak outward current (Fig. 2A); the unblocked current
had a high activation threshold (Fig. 2B), indicating that
it represented mainly IKH. 40 nM DTX-K
blocked maximally 78% and tityustoxin maximally 58% of the outward
current. The unblocked portions of the current had low activation
thresholds, indicating that each of these toxins blocked only part of
IKL (Fig. 2, D and
F). Dose-response curves (Fig. 2G) show that the concentrations of all toxins used in the experiments illustrated in
Fig. 2, A-F, are at saturating levels. Half-maximal
blocking concentrations were 5, 10, and 3 nM for
-DTX, DTX-K, and
tityustoxin, respectively. The effects of these toxins were
irreversible. These results support the conclusion that
IKL is mediated through
K+ channels of the Kv1 family. These findings
indicate that IKL in octopus cells is
generated through a heterogeneous population of Kv1 channels. About
20% lack Kv1.1 and ~45% lack Kv1.2.
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Reversal potential of IKL
The reversal potential of IKL was
measured from the tail currents obtained with a conventional
double-pulse protocol. The tail currents were evoked by repolarizing to
a range of potentials between 62 and
102 mV after a depolarizing
pulse to
50 mV for 15 ms (Fig.
3A). The reversal potential
was measured in saline that contained 10 mM TEA, 1 µM TTX, 0.25 mM
Cd2+, and 50 nM ZD7288 to suppress other
voltage-sensitive currents, IKH,
INa,
ICa, and
Ih. The amplitude of the tail current
was plotted as a function of the step potential (Fig. 3B).
At a normal potassium concentration of 4.2 mM, the current reversed at
75.6 ± 3.8 mV (n = 20). The dependence of the
reversal potential on the extracellular K+
concentration was determined by repeating the experiment in saline in
which KCl was substituted for equimolar NaCl to give final K+ concentrations of 17 and 34 mM. Figure
3C shows that the reversal potential of
IKL roughly obeys the Nernst
relationship for K+, confirming the conclusion
that IKL is largely carried by
K+.
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The measurement of the reversal potential of
IKL deviated somewhat from the
theoretical potassium equilibrium potential. The I-V
relationship in Fig. 3B deviates from linearity over the
voltage range negative to the reversal potential. Also the dependence of the reversal potential on the extracellular K+
concentration deviated from the Nernst relationship at negative potentials; at 4.2 mM
[K+]o the measured
reversal potential was 76 mV, whereas the equilibrium potential for
K+ was
85 mV. There are several possible
explanations and interpretations for these deviations. First, the
magnitude of the tail current was measured 0.7 ms after the onset of
the second step to avoid possible artifacts from the transient
capacitative current, but over such a time period the tail currents can
be distorted. A voltage dependence of the decay of tail currents can
cause a relative underestimation of the tail currents in the negative
voltage range where they decline most rapidly. Second, it is also
possible that the outward current might have been contaminated with
Ih, a current that is strongly
activated at hyperpolarized voltages and that may not have been blocked
completely by ZD7288 (Bal and Oertel 2000
). Third, it is
likely that not all parts of the octopus cell were equally well clamped
under all conditions. Fourth, it is possible that
IKL is not absolutely specific for
K+. Fifth, similar deviations in measurements of
the reversal potential in avian auditory neurons have been attributed
to the extracellular accumulation of K+
(Rathouz and Trussell 1998
). Several of these
possibilities were assessed in separate experiments.
The reversal potential was measured in recordings in the
cell-attached configuration from cell bodies using similar voltage protocols (Fig. 4). In these experiments,
the intracellular content was not disturbed, currents are not affected
by imperfections in the space-clamp, and there is unlikely to be any
extracellular accumulation of K+. Recordings were
made with pipettes that contained extracellular saline with 50 nM
ZD7288, 1 µM TTX, 10 mM TEA, and 0.25 mM Cd2+.
At the end of the recordings, the resting potential was measured in the
whole cell mode. The mean reversal potential measured in these
experiments was 77 ± 3 mV (n = 3), a value that
corresponded closely to the reversal potential measured in the
whole-cell configuration. The nonlinear I-V relationship of
the tail currents under these conditions indicates that neither
space-clamping nor K+ accumulation were the cause
of the distortions of the measurements under whole cell conditions.
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The possibility that space-clamp artifacts or K+
accumulation affected measurements of the reversal potential of
IKL was tested further by measuring
the reversal potential of outward currents in outside-out patches. In
these measurements, the reversal potential under normal conditions was
found to be 74 ± 2 mV (n = 3, data not shown),
a value close to that measured in whole cell recordings and deviating
from the equilibrium potential of
85 mV.
The most likely explanation for the deviation from theoretical behavior
is that the presence of some residual
Ih contaminates measurements of the
reversal potential at negative potentials. Ih in octopus cells has a threshold of
activation between 35 and
40 mV and is strongly activated at more
hyperpolarized potentials (Bal and Oertel 2000
). The
current is small near its reversal potential,
38 mV, where deviations
of the reversal potential of IKL from
EK are small, and it is large at more
hyperpolarized potentials where the driving force is larger and the
conductance is more strongly activated. If 50 nM ZD7288 blocked 91% of
a current measured to be between 1 and 2 nA at rest, the resting
gh would be expected to contribute
between ~0.1 and 0.2 nA at the resting potential and between 0.2 and
0.4 nA at
110 mV.
Voltage sensitivity of the activation of IKL
The potassium currents measured in whole cell recordings of
octopus cells are large, requiring additional manipulations to measure
the voltage sensitivity of gKL over
the physiological range. Steps from 90 to
35 mV often evoked
currents that exceeded 20 nA, not only exceeding the limits of the
amplifier but also subjecting the measured currents to distortion by
the series resistance. To overcome these technical problems, low
concentrations of
-DTX were used to reduce peak currents to workable
levels. In these experiments, IKL was
evoked with steps to
45 mV, then between 5 and 8 nM
-DTX was
applied. The ratio of the peak current in the presence and absence of
-DTX indicated what fraction of IKL was blocked by the low concentration of
-DTX and determined the correction factor of gKL in the
following measurement. The difference between currents measured in low
and high (60 nM) concentrations of
-DTX could then be used to reveal
the voltage sensitivity of the unblocked portion of
gKL. The results of these experiments are illustrated in Fig. 5. In the
presence of 8 nM
-DTX, the outward current evoked by a step to
45
mV was reduced by 76.5% (not shown) and outward currents evoked by
depolarizing steps to 0 mV could be measured (Fig. 5A).
IKL rises steeply in the voltage range
near the resting potential as both the conductance and the driving
force increase (Fig. 5B). The voltage sensitivity of
gKL was derived from the relationship
gKL = IKL/(Vm
EK). The total
gKL was derived by applying a
correction factor based on the measurement of the proportion of
IKL blocked by a low concentration of
-DTX (Fig. 5C).
|
The voltage sensitivity of gKL in a
group of octopus cells is presented in Fig. 5D. The
conductance, gKL, has a threshold near
70 mV, rises steeply in the voltage range near the resting potential
and reaches a maximum ~0 mV. The sigmoidal relationship can be fit by
a Boltzmann function. This analysis indicates that gKL is half-activated at
45 ± 6 mV (n = 12) and has a slope factor of 9 ± 2 mV
(n = 12). The mean maximum
gKL for measured in 12 octopus cells
was 514 ± 135 nS.
Inactivation of IKL
The decline in IKL in the
presence of continuing depolarization indicates that
IKL inactivates. The decay of current
associated with inactivation in Fig. 1A can best be
described by double exponentials of roughly equal weight with time
constants in the range of 200 and 20 ms. Knowing that
gKL is activated at rest in the steady state and that this activation is functionally significant, the rate
and voltage dependence of inactivation was studied in 11 neurons. After
the membrane was conditioned for 1 s to holding potentials between
40 and
105 mV in 5-mV steps, the neuron was depolarized to a fixed
test potential,
45 mV (Fig.
6A). The normalized peak
current amplitudes were plotted as a function of holding membrane
potentials (Fig. 6B). In octopus cells,
gKL does not inactivate completely
even at very depolarized potentials. The voltage dependence of
gKL was fit by a Boltzmann function
with V1/2 and k values are
64 ± 3 and 5 ± 0.6 mV, respectively (n = 11). Near the resting potential,
62 mV, ~40% of the
gKL is inactivated; inactivation was
maximal at voltages more depolarized than
50 mV.
|
Measurement of activation and inactivation allows an estimate to be
made of the current that is mediated through gKL
at rest. At 62 mV, gKL would be
expected to have a peak activation of ~70 nS. Inactivation would be
expected to reduce this conductance by 40% to 42 nS. At the resting
potential and under normal ionic conditions, therefore
gKL is expected to mediate a current
of ~1 nA.
Recovery from inactivation of IKL
Recovery from inactivation of IKL
depended on the duration and voltage of the recovery period.
Inactivation was assayed with a test pulse that followed a conditioning
pulse after various intervals. Figure
7A illustrates the results of
one such experiment. The cell was held at 100 mV to remove
inactivation of IKL completely (Fig.
6B). A depolarizing pulse to
45 mV was used initially to activate and inactivate IKL. A similar
test pulse after intervals that varied between 10 and 2,000 ms at
100
mV was used to assess inactivation. Recovery proceeded with an
exponential time course whose time constant was 118 ± 14 ms
(n = 4) with a complete recovery after ~300 ms (Fig.
7B). The time constant of recovery depended on the voltage
of the recovery period, ranging between about 100 ms at
100-mV
holding potentials and 150 ms near the resting potential (Fig.
7C).
|
Voltage sensitivity of IKH
The high-threshold outward current,
IKH, was defined by its insensitivity
to -DTX and its sensitivity to 20 mM TEA. Figure 8 illustrates the properties of this
current. Using a bathing saline that contained 50 nM
-DTX, 1 µM
TTX, 0.25 mM Cd2+ and 50 nM ZD7288 to suppress
IKL,
INa,
ICa and
Ih,
-DTX-insensitive outward
currents were evoked by voltage steps from a holding potential of
90
mV (Fig. 8A, left). In the presence of 20 mM TEA,
those outward currents were reduced (Fig. 8A,
middle) (experiments that are not illustrated showed that
~30% of the remaining outward current could be blocked by 5 mM
4-AP). The difference between these families of currents revealed the
TEA sensitive,
-DTX-insensitive current that we define as
IKH. As the block of
IKH by 20 mM TEA is probably not
complete, the amplitude of IKH is
underestimated with this method of separating currents. This current
has a threshold of activation near
40 mV (Fig. 8B). The
voltage sensitivity of the underlying conductance was examined by
converting current to conductance using Ohm's Law and plotting
conductance as a function of voltage (Fig. 8C). The
relationship was sigmoidal, having a threshold for activation at about
40 mV and saturating at around +30 mV. The peak
gKL as a function of voltage was
described by a Boltzmann function with
V1/2 at
16 ± 5 mV
(n = 7) and a slope factor of 10 ± 4 mV
(n = 7). The maximum conductance,
gKHmax was 116 ± 27 nS
(n = 7). Long depolarizing pulses reveal that
IKH inactivated by ~85% (Fig.
8D).
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Reversal potential of IKH
The reversal potential of IKH was
measured with a similar voltage protocol as that described for
IKL (Fig.
9A). In control saline, the
tail current for IKH reversed at
76 ± 4 mV (n = 6; Fig. 9B), a value
within 1 mV of the reversal potential measured for
IKL. The finding that the deviation
from the equilibrium potential for K+ of both
IKL and
IKH are similar argues that a common
factor influences both. The results are consistent with
Ih affecting measurements of the
reversal potential when the reversal potential is in the hyperpolarizing voltage range.
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Inactivation of IKH
In the presence of 50 nM -DTX, a long voltage step to variable
voltages activated IKH. The decay of
current with continued depolarization shows that
IKH inactivates. Inactivation of
IKH was slow (Fig. 8D); its
decay followed a double exponential time course with a dominant (85%)
time constant in the range of 500 ms and a second in the range of 25 ms.
Figure 10 shows an experiment in which variable voltage steps of 1.5 s were applied; inactivation did not reach a steady state over this time period but octopus cells tended not to tolerate longer depolarizations. Inactivation was assayed with a subsequent voltage step to +45 mV. The peaks of responses to the test pulse varied as a function of the level of the previous depolarization. A plot of the conductance, derived from the peak tail currents, as a function of the previous voltage step shows a sigmoidal shape that can be fit with a Boltzmann function. The V1/2 and slope factor values are 54 ± 6 and 10 ± 2 (n = 6) mV, respectively. The fact that inactivation did not reach steady state means that the maximal inactivation was underestimated; Fig. 10B shows maximal inactivation to be 75% whereas it can reach 85%.
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Contribution of K+ currents to membrane excitability
To gain an appreciation for the biological consequences of these
conductances on octopus cells, the effects of -DTX, 4-AP and TEA on
firing properties of octopus cells were examined in current-clamp
experiments. The voltage-clamp experiments show that the application of
50 nM
-DTX blocked IKL almost
completely. When
-DTX was applied in current clamp, octopus cells
depolarized and became more excitable (Fig.
11). The input resistance increased from 6.2 ± 2.5 to 20 ± 6 M
(n = 10),
resulting in an increase in the membrane time constant from 0.22 ± 0.04 (n = 5) to 1.9 ± 1 ms (n = 5; Fig. 11B). In the presence of
-DTX, repetitive slow
action potentials could be evoked with depolarizing current. The peaks
of the first in the train of action potentials occurred later and
varied more than under control conditions (Fig. 11C). The
latencies from the onset of the current pulse to the peaks of action
potentials evoked with depolarizing currents of 1 nA were shifted from
0.58 ± 0.08 (n = 10) to 1.8 ± 0.8 ms
(n = 9) by
-DTX; latencies of action potentials in
responses to current pulses of 5 nA changed from 0.35 ± 0.08 (n = 10) to 0.69 ± 0.3 ms (n = 9).
-DTX also caused a 6.2 ± 2.5 mV depolarizing shift of the
resting potential. The subsequent application of 20 mM TEA, which was
demonstrated in voltage-clamp experiments to block IKH, eliminated the repetitive firing
and allowed octopus cells to be depolarized to a plateau potential that
lay positive to 0 mV that was presumably maintained by
voltage-sensitive Na+ and
Ca2+ currents. These experiments show that
gKL contributes to the setting of the
resting potential and of the input resistance and time constant of the
resting octopus cell. It also shapes the peaks of action potentials and
decreases their temporal jitter. As expected, the application of TEA
affected only the most depolarized electrophysiological responses,
preventing the repolarization of large action potentials (Fig.
11C).
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DISCUSSION |
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This study provides a description of voltage-sensitive
K+ currents, IKL
and IKH, and their underlying
conductances, gKL and gKH, in octopus cells of the ventral
cochlear nucleus. These currents are both identified as
K+ currents by their reversal potentials. The
mamba snake toxin, -DTX, blocks 90% of the
K+ current that is activated at potentials more
negative than
45 mV but leaves a K+ current
that was activated at more positive potentials. This toxin was used to
separate IKL from
IKH in the voltage range where both
are activated so that these currents could be studied in isolation. The
sensitivity of IKL to the dendrotoxins
and to tityustoxin indicates that IKL
is probably mediated through voltage-sensitive K+
channels of the Kv1 family. The high- and low-threshold
K+ conductances differ in their voltage
sensitivity. The threshold of gKL lay
at
70 mV, and activation was maximal at voltages positive to 0 mV.
This conductance is activated over the entire physiological voltage
range of octopus cells. It contributes both to setting the resting
potential near
62 mV and to the shaping of responses to synaptic
activation through the auditory nerve (Golding et al.
1995
). In contrast, the threshold of
gKH is at
40 mV and it is maximally
activated at +30 mV. This conductance affects only the largest,
suprathreshold events. It does not affect the resting properties of
octopus cells and it does not contribute to the shaping of small
synaptic potentials. The biophysical specializations of octopus cells
are generated with K+ conductances whose
biophysical and pharmacological properties are conventional but which
are represented in octopus cells in unusually large quantities.
IKL and IKH are K+ currents
Measurements of the reversal potentials for both
IKL and
IKH deviated from the theoretical
equilibrium potential, EK, when the
external concentration of K+ was low, raising the
question whether these currents are always carried by
K+. Under control conditions, both
IKL and
IKH reversed at about 76 mV when
EK was
84.5 mV. Similar deviations
have been documented in bushy cells of the mammalian cochlear nucleus
(Manis and Marx 1991
) and their avian homologs
(Rathouz and Trussell 1998
). When the extracellular
K+ concentration was raised, shifting
EK to more depolarized levels, the
reversal potential of gKL matched
EK. The finding that a depolarized reversal potential for IKL was
measured also in cell-attached and outside-out configurations indicates
that the deviation from the theoretical
EK was not produced by space-clamp
errors. Several observations indicate that the deviation from
EK arose from the contamination of
K+ currents with
Ih. First, the deviation was similar
for both IKL and
IKH, indicating that a common factor
affected both measurements. Second, the deviation was observed in the
voltage range where Ih was activated,
and it was not observed outside that voltage range. Third, 50 nM ZD7288
leaves 7% of Ih unblocked (Bal
and Oertel 2000
); in cells with an unusually large
gh whose voltage range of activation
is unusually depolarized, the remaining current is substantial and can
account for the deviation. Fourth, deviation was similar in whole cell
cell-attached and outside-out patch configurations, arguing against
mechanisms that involve depletion or accumulation. We conclude that
both IKL and
IKH are K+ currents.
Activation and inactivation of IKL at rest
The observations that gKL is
activated at the resting potential and that the maximum
gKL is large indicate that
gKL contributes substantially to
shaping synaptic responses. This conductance had an activation
threshold near 70 mV and was half-maximally activated at
45 mV. At
rest,
62 mV, a substantial proportion of the mean maximum peak
conductance is 514 ± 135 nS, ~70 nS, is activated. However,
some of that conductance, ~40%, will be inactivated, leaving a
resting gKL of 42 nS and a resting
IKL of ~1 nA. This resting
K+ outward current is roughly balanced by an
inward current, Ih, which has been
estimated to be on average 1.4 nA (between 0.9 and 2.1 nA) (Bal
and Oertel 2000
; Oertel et al. 2000
).
High- and low-threshold K+ currents in auditory brain stem nuclei
The K+ conductances in octopus cells bear a
striking resemblance to conductances in other auditory neurons. In
cochlear nuclear bushy cells and their avian homologs as well as in
neurons of the medial nucleus of the trapezoid body, low- and
high-threshold K+ conductances have been
described (Brew and Forsythe 1995; Manis and Marx
1991
; Rathouz and Trussell 1998
; Reyes et
al. 1994
). While the methods for separation of currents varied,
the low-threshold conductances were sensitive to dendrotoxins
(Brew and Forsythe 1995
; Rathouz and Trussell
1998
). In octopus cells, the maximum gKL, 514 ± 135, is at least six
times larger than in any other auditory or nonauditory neurons in which
it has been measured. Estimates of the maximum
gKL in other auditory neurons are as follows: isolated bushy cells, 15 nS (Manis and Marx
1991
); avian nucleus magnocellularis, 68 nS (Rathouz and
Trussell 1998
); and neurons of the medial nucleus of the
trapezoid body, 10 nS (Brew and Forsythe 1995
).
High-threshold conductances in octopus cells were also larger than in
other cells, but the difference was smaller. Comparison of magnitudes
of gKH is made difficult by the
differences in techniques for defining the high-threshold conductance.
In octopus cells, the maximum gKH
averaged 116 nS. In neurons of the medial nucleus of the trapezoid
body, maximum gKH is roughly 32 nS (Brew and Forsythe 1995
). In avian homologs of
bushy cells, it was 44 nS (Rathouz and Trussell 1998
).
Roughly the total maximal gK in
octopus cells is at least eight times that in other neurons in auditory
brain stem (Brew and Forsythe 1995
; Manis and
Marx 1991
; Rathouz and Trussell 1998
). The
maximal total gK is 10-100 times
larger in octopus cells than in nonauditory neurons in the CNS (e.g.,
Bardoni and Belluzzi 1993
; Schofield and Ikeda
1989
; Wu and Barish 1992
).
Low-threshold K+ conductances that are sensitive
to -DTX and that belong to the Kv1 family are widespread in the
nervous system. They have been shown to play a role in the hippocampus
(Halliwell et al. 1986
) and in central vestibular
neurons (Gamkrelidze et al. 1998
). They have been shown
to regulate excitability in not only cell bodies but also in axons and
axon terminals (Reid et al. 1999
; Southan and
Robertson 2000
; Zhou et al. 1999
).
IKL is mediated through K+ channels of the Kv1 family
Native channels of the Kv1 family are generally thought to
comprise four and four
subunits. Combinations of
subunits form functional homomeric as well as heteromeric channels in vitro, even in the absence of
subunits, indicating that the
subunits form the pore of the channel and control its gating. The
subunits of Kv1 channels in mammalian cells affect the expression and modulate the biophysical properties of the channels (Coleman et al.
1999
). Kv
subunits accelerate inactivation and cause a
hyperpolarizing shift in the activation curve of Kv1 channels
(Accili et al. 1997
; Heinemann et al.
1996
; Rettig et al. 1994
; Uebele et al.
1998
).
A group of peptide toxins has recently been developed and characterized
that has proven to be specific for channels of the Kv1 family. These
peptide toxins include -DTX, its close homologue DTX-I, DTX-K,
DTX, and tityustoxin. When tested on homomeric channels in expression
systems,
-DTX and DTX-I potently block Kv1.2 channels, and with
lower potency, they also block Kv1.1, Kv1.3, and Kv1.6 channels
(Dolly and Parcej 1996
; Grissmer et al.
1994
; Harvey 1997
; Hopkins 1998
;
Owen et al. 1997
; Tytgat et al. 1995
).
DTX-K specifically blocks Kv1.1 homomers and has little effect on other
Kv1 channels (Robertson et al. 1996
; Wang et al.
1999a
,b
).
DTX blocks Kv1.1 channels with high potency (Hopkins 1998
). Tityustoxin has been shown specifically
to block homomeric Kv1.2 channels (Hopkins 1998
;
Werkman et al. 1993
). In general these toxins also block
heteromers that contain a single subunit for which they are specific
(Hopkins 1998
; Hopkins et al. 1994
;
Wang et al. 1999a
). There are exceptions, however; the tityustoxin block of Kv1.2/Kv1.4 heteromers does not fit this model
(Hopkins 1998
).
It is likely that Kv1 channels exist as heteromers in octopus cells.
Manganas and Trimmer (2000) suggest that Kv1.1 and Kv1.2
subunits require coassembly with another member of the Kv1 family, one possibility being Kv1.4, to be expressed at the surface of mammalian cells. Biochemical and immunohistochemical studies confirm this conclusion and indicate that Kv1.1
subunits form
heteromultimers with either Kv1.4 or Kv1.2 or both (Coleman et
al. 1999
; Koch et al. 1997
; Shamotienko
et al. 1997
). Coexpression of Kv1 subunits in
Xenopus oocytes resulted in the formation of
heterotetrameric channels with pharmacological and biophysical
properties distinct from those of corresponding homotetrameric channels
subunit (Hopkins 1998
; Hopkins et al.
1994
; Isacoff et al. 1990
; Ruppersberg et al. 1990
; Sheng et al. 1993
; Tytgat et
al. 1995
; Wang et al. 1993
).
None of the homomeric channel currents of Kv1.1, Kv1.2, and Kv1.4 in
vitro expression systems has identical pharmacological and biophysical
properties to IKL in octopus cells.
Instead, IKL in octopus cells combines
the features of homomeric channels. The low threshold of
IKL resembles that of Kv1.1 homomeric
channels (Grissmer et al. 1994; Hopkins et al.
1994
; Stühmer et al. 1989
). The rapid
inactivation resembles that of Kv1.4 homomeric channels (Dolly
and Parcej 1996
; Stühmer et al. 1989
). The
properties of inactivation resemble those of Kv1.1 and Kv1.2 channels
(Hopkins et al. 1994
).
IKL in octopus cells is likely to be
generated through a heterogeneous population of heteromeric channels
that contain Kv1.1 and/or Kv1.2 as well as Kv1.4. These subunits are
strongly expressed in octopus cells. In situ hybridization shows the
expression of mRNA for Kv1.1 and Kv1.2 (Grigg et al.
2000). Immunohistochemical evidence has also revealed the
presence of Kv1.1 and Kv1.2 (Wang et al. 1994
) and Kv1.4
(Fonseca et al. 1998
) proteins in octopus cells. The
differential sensitivity of IKL to
-DTX, DTX-K, and tityustoxin suggests that the population of
K+ channels that mediates
IKL is heterogeneous. DTX-K, specific for Kv1.1 channels (Dolly and Parcej 1996
; Wang
et al. 1999a
), blocks maximally 70% of
IKL. Tityustoxin blocks maximally
~60% of IKL, indicating that 40%
of channels lack Kv1.2 subunits (Hopkins 1998
;
Werkman et al. 1993
). Assuming that a single Kv1.1 or
Kv1.2 subunit renders channels sensitive to DTX-K and tityustoxin,
respectively, an assumption known not to apply to all combinations of
subunits (Hopkins 1998
), then ~30% of channels lack
Kv1.1 subunits and ~40% lack Kv1.2 subunits.
Octopus cells have subunits. Fonseca et al. (1998)
have observed that the subunits, Kv
1 and Kv
2, are abundantly
expressed in octopus cells.
subunits may account partially for the
rapid inactivation and hyperpolarized activation threshold of
IKL.
Molecular basis for IKH is unknown
IKH is characterized by its
high-threshold, 40 mV, by its insensitivity to
-DTX, and by its
sensitivity to 20 mM TEA. It is not possible to attribute this current
to a particular family of K+ channels. Ion
channels in the Kv3, or Shaw, family generally have
high-thresholds and vary in inactivation. Octopus cells have been
examined for the presence of mRNA for the Kv3.1 subunit. While the
present study shows that gKH is larger
in octopus cells than in bushy cells, both mRNA and protein is present
at lower levels in octopus than in other cochlear nuclear cells
including bushy cells (Grigg et al. 2000
; Perney
and Kaczmarek 1997
).
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ACKNOWLEDGMENTS |
---|
We owe particular thanks to the staff of Alomone Labs, who were exceptionally helpful in advising us and supplying us with dendrotoxins and tityustoxin. Dr. K. Fujino made thoughtful comments about the manuscript for which we are very grateful. This work benefited from discussions with Drs. S. Gardner and K. Fujino about practical and theoretical details. We also thank I. Siggelkow for help in keeping us supplied with solutions.
This work was supported by National Institute on Deafness and Other Communication Disorders Grant DC-00176.
Present address of R. Bal: Dept. of Physiology, Faculty of Veterinary Medicine, Mustafa Kemal University, Antakya/Hatay, Turkey.
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FOOTNOTES |
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Address for reprint requests: D. Oertel, Dept. of Physiology, University of Wisconsin Medical School, 1300 University Ave., Madison, WI 53706 (E-mail: oertel{at}physiology.wisc.edu).
Received 7 March 2001; accepted in final form 2 July 2001.
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REFERENCES |
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