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The Journal of Neuroscience, August 15, 2002, 22(16):6953-6961
Two Heteromeric Kv1 Potassium Channels Differentially Regulate
Action Potential Firing
Paul D.
Dodson,
Matthew C.
Barker, and
Ian D.
Forsythe
Department of Cell Physiology and Pharmacology, University of
Leicester, Leicester, LE1 9HN, United Kingdom
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ABSTRACT |
Low-threshold voltage-gated potassium currents
(ILT) activating close to resting
membrane potentials play an important role in shaping action potential
(AP) firing patterns. In the medial nucleus of the trapezoid body
(MNTB), ILT ensures generation of single APs
during each EPSP, so that the timing and pattern of AP firing is
preserved on transmission across this relay synapse (calyx of Held).
This temporal information is critical for computation of sound location
using interaural timing and level differences. ILT currents are generated by
dendrotoxin-I-sensitive, Shaker-related K+ channels; our immunohistochemistry confirms that
MNTB neurons express Kv1.1, Kv1.2, and Kv1.6 subunits. We used
subunit-specific toxins to separate ILT into
two components, each contributing approximately one-half of the total
low-threshold current: (1) ILTS, a
tityustoxin-K -sensitive current (TsTX) (known to block Kv1.2
containing channels), and (2) ILTR,
an TsTX-resistant current. Both components were sensitive to the
Kv1.1-specific toxin dendrotoxin-K and were insensitive to
tetraethylammonium (1 mM). This pharmacological profile
excludes homomeric Kv1.1 or Kv1.2 channels and is consistent with
ILTS channels being Kv1.1/Kv1.2 heteromers,
whereas ILTR channels are probably
Kv1.1/Kv1.6 heteromers. Although they have similar kinetic properties,
ILTS is critical for generating the phenotypic single AP response of MNTB neurons. Immunohistochemistry confirms that Kv1.1 and Kv1.2 (ILTS
channels), but not Kv1.6, are concentrated in the first 20 µm of MNTB
axons. Our results show that heteromeric channels containing Kv1.2
subunits govern AP firing and suggest that their localization at the
initial segment of MNTB axons can explain their dominance of AP firing behavior.
Key words:
medial nucleus of the trapezoid body; MNTB; low-threshold
potassium currents; Kv1.1; KCNA1; Kv1.2; KCNA2; Kv1.6; KCNA6; dendrotoxin; DTX; tityustoxin; TsTX; sound localization; auditory
system; channel localization; heteromeric assembly
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INTRODUCTION |
The seven members of the
Shaker-related K+ channel
family (Kv1.1-Kv1.7) generate voltage-dependent outward currents,
which regulate action potential (AP) threshold, waveform, and pacemaker activity in nerve and muscle (Hille, 2001 ). When expressed as homomeric
channels, most have "delayed rectifier" properties (Kv1.1, Kv1.2,
Kv1.5, and Kv1.6), with others exhibiting fairly rapid inactivation
(Kv1.3, Kv1.4, and Kv1.7). They are widely expressed in axonal,
dendritic, and somatic compartments in the brain (Wang et al., 1994;
Coetzee et al., 1999), with the exception of Kv1.7 (Kalman et al.,
1998; Kashuba et al., 2001). Functional channels contain four Kv
subunits and up to four Kv subunits (Xu et al., 1998); heteromers
are common (Isacoff et al., 1990; Ruppersberg et al., 1990), but
particular subunit combinations are favored in vivo (Koch et
al., 1997; Shamotienko et al., 1997; Koschak et al., 1998; Coleman et
al., 1999; Wang et al., 1999b).
The medial nucleus of the trapezoid body (MNTB) expresses low-threshold
K+ currents
(ILT), which are blocked by
dendrotoxin-I (DTX-I), suggesting mediation by channels containing
Kv1.1, Kv1.2, and/or Kv1.6 (Brew and Forsythe, 1995). In addition,
high-threshold potassium channels, including Kv3.1, are expressed,
which minimize AP duration (Brew and Forsythe, 1995; Wang et al., 1998;
Rudy and McBain, 2001). The MNTB forms an inverting relay in the
binaural auditory pathway, in which AP timing is crucial to encoding
interaural timing and level differences associated with sound source
localization (Park et al., 1996; Trussell, 1999). The
K+ currents and specializations such as a
large synapse (calyx of Held) generating fast glutamatergic EPSCs
(which ensure minimal latency fluctuation and secure AP generation)
(Barnes-Davies and Forsythe, 1995) preserve timing by enabling MNTB
neurons to faithfully follow the pattern of presynaptic activity, even
during high-frequency transmission (up to 800 Hz) (Taschenberger and
von Gersdorff, 2000). Dendrotoxin-sensitive currents
(ID), which are similar to
ILT in the MNTB, are present in
hippocampal and cortical pyramidal neurons (Wu and Barish, 1992;
Bekkers and Delaney, 2001) in which they modulate excitability by
regulating AP firing threshold.
Specific toxins for Kv1 channels have been isolated from scorpion and
snake venom (Tytgat et al., 1999; Harvey, 2001). DTX-I (from
Dendroaspis polylepis polylepis) will block low-threshold K+ channels containing Kv1.1, Kv1.2, or
Kv1.6 subunits, but more specific toxins are available. Dendrotoxin-K
(DTX-K) specifically blocks Kv1.1-containing channels (Robertson et
al., 1996; Wang et al., 1999b), tityustoxin-K (TsTX) (from
Tityus serrulatus) blocks most Kv1.2-containing channels
(Matteson and Blaustein, 1997; Hopkins, 1998), and noxiustoxin (NTX)
(from Centruriodes noxius) blocks channels containing Kv1.2,
Kv1.3, or Kv1.7 (Grissmer et al., 1994; Kalman et al., 1998). Because
only one subunit is required for the toxin to bind, they block most
heteromeric channels containing at least one toxin-sensitive subunit
(Hopkins, 1998). We exploited these toxins to examine the composition
and relative importance of Kv subunits in generating
ILT channels in MNTB neurons. We find
that ILT has two components,
distinguishable by their sensitivity to tityustoxin-K, and show that
Kv1.2-containing channels are dominant in determining the unitary AP
firing of MNTB neurons.
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MATERIALS AND METHODS |
Brain slice preparation. Brain slices were prepared
using methods described previously (Barnes-Davies and Forsythe, 1995). Briefly, Lister hooded rats (8-14 d old) were killed by decapitation, and the brain was removed in ice-cold low sodium artificial CSF (aCSF) (see below). The brain was placed ventral side down and cut just posterior to the cerebellum at an angle of
~20o to the vertical, removing the
spinal cord. The hemispheres were removed with a vertical cut, and the
brainstem was mounted severed caudal surface down to the stage of a
DTK-1000 slicer (Dosaka, Kyoto, Japan). Six to eight transverse
slices 100- to 150-µm-thick were cut from the region of the brainstem
containing the MNTB. The slices were incubated at 37°C for 1 hr in
normal aCSF (see below) and then allowed to cool to room temperature.
Immunohistochemistry. Brainstems from Lister hooded rats
(postnatal day 9) were transferred from aCSF to Tissue Tek
(Sakura, Tokyo, Japan) and frozen using hexane and dry ice. Transverse cryostat sections (20 µm) were mounted on subbed slides. Sections were fixed in 2% paraformaldehyde solution for 10 min. After washing for 15 min (1% PBS), sections were permeabilized in PBS and
10% goat serum (Dako, High Wycombe, UK) and 0.5% Triton X-100 for 1 hr. Sections were washed in 1% PBS (twice for 15 min each), and
primary antibodies (Kv1.1, Kv1.2, Kv1.4, Kv1.5, and Kv1.6) were applied
at a concentration of 1:100 and then incubated overnight at 4°C.
Sections were washed in 1% PBS (six times for 10 min each), incubated
for 2 hr with a FITC secondary antibody (1:1000) at room temperature,
and then washed again in 1% PBS (twice for 15 min each).
Colocalization studies followed the immunohistochemical protocol
described previously. Kv1.1 and Kv1.2 primary antibodies (raised in
rabbit and mouse, respectively) were applied at the same dilutions.
Secondary antibodies (FITC at 1:500 and Texas Red at 1:250) were
applied together. Slides were mounted using Citifluor anti-fade
mountant. Results were visualized using a Nikon (Tokyo, Japan)
epifluorescent microscope, recorded with a Cohu (San Diego, CA) CCD
camera with no automatic gain control, and analyzed using NIH Image.
Pixel intensities of the immunoreactivity from Kv1 antibodies were
calculated by measuring the mean intensity over an area of 4800 µm2 covering the MNTB. To account for
nonspecific secondary antibody fluorescence, background
immunofluorescence was measured from control sections in which only
secondary antibody had been applied, and this was subtracted from test
data. Blocking peptide controls were used to assess nonspecific binding
of primary antibodies (see Fig. 1G). In these experiments,
blocking peptide was incubated with the primary antibody (5:1) for 1 hr
before application. No reliable Kv1.3 antibody was available, but, as
shown below, electrophysiological and pharmacological evidence
indicated that this subunit was not present. Kv1.7 expression was not
examined because this subunit is not expressed in the brain (Kalman et
al., 1998; Kashuba et al., 2001). Subcellular localization was
investigated by examining immunofluorescence using an Olympus Optical
(Tokyo, Japan) Fluoview confocal microscope (IX70) with a 60×,
numerical aperture (NA) 1.4 objective.
Electrophysiological recordings. For recording, one slice
was placed in a Peltier controlled environmental chamber mounted on the
stage of an upright E600FN microscope (Nikon) fitted with differential
interference contrast optics and a 60× (NA 1.00) water immersion
objective (Nikon). The environmental chamber was continuously perfused
with normal aCSF (~1 ml/min, 25°C). Whole-cell patch recordings
were made from visually identified MNTB neurons using an Optopatch
amplifier (Cairn, Faversham, UK). This patch-clamp amplifier has an
optically coupled head stage permitting both voltage-clamp and
current-clamp control, allowing accurate action potential measurements.
Patch pipettes with resistances of 2.5-4 M were pulled from
thick-walled borosilicate glass (GC150F-7.5; Harvard Apparatus,
Edenbridge, UK) and filled with intracellular patch solution (see
below). Series resistances were 9.3 ± 0.6 M (n = 21) and were routinely compensated by 70%. Recordings in which the
series resistances changed by more than ±2 M during the course of
an experiment were excluded from analysis. Currents were evoked by
voltage steps up to 0mV; this enabled us to investigate low-threshold
currents without introducing series resistance errors associated with
large currents. Stated voltages exclude a  7 mV junction potential.
Data acquisition and analysis. Data were acquired via a
CED1401 interface using Patch v6.39 software (Cambridge Electronics Design, Cambridge, UK) running on a PIII 550 computer. Data were filtered at 2 kHz and digitized at between 5 and 10 kHz. Unless stated
otherwise, current-voltage (I-V) relationships were
produced using the current amplitude measured at 10 msec into the
voltage step. Leak currents were not subtracted from the current
records, but, for presentation purposes, the leak was subtracted from
the data points in current-voltage relationships (as estimated from a
linear fit over a voltage range between 90 and 75 mV, in the presence of ZD7288 to block IH).
Half-inactivation was estimated using a fractional Boltzmann equation:
I = Imax (1 fraction)/(1 + e(V  V1/2)/K). Averaged data are
expressed as mean ± SEM.
Solutions and toxins. All chemicals were obtained from Sigma
(St. Louis, MO) unless otherwise stated. The low
Na+ aCSF used for slicing contained the
following (in mM): 250 sucrose, 2.5 KCl, 10 glucose, 1.25 NaH2PO4, 26 NaHCO3, 2 sodium pyruvate, 3 myo-inositol, 0.5 ascorbic acid, 0.1 CaCl2, and 4 MgCl2. Normal aCSF contained the following (in
mM): 125 NaCl, 2.5 KCl, 10 glucose, 1.25 NaH2PO4, 26 NaHCO3, 2 sodium pyruvate, 3 myo-inositol, 0.5 ascorbic acid, 2 CaCl2, and 1 MgCl2. For data recording, extracellular CaCl2 and MgCl2
concentrations were changed to 0.5 and 2.5 mM, respectively; to minimize calcium-activated potassium currents, ZD7288
(10 µM; Tocris Cookson, Bristol, UK) was added
to block IH. Tetrodotoxin (TTX) (1 µM; Latoxan, Valence, France) was used to block
the voltage-gated Na+ currents in
voltage-clamp experiments. External solutions were gassed with 95%
O2-5% CO2 to give a pH of
7.4. Intracellular patch solution contained the following (in
mM): 97.5 K-gluconate, 32.5 KCl, 10 HEPES, 5 EGTA, and 1 MgCl2, pH 7.2 with KOH. Toxins were applied through independent, dedicated perfusion lines to avoid contamination of control solutions. Toxins and antibodies were obtained
from the following sources: DTX-I, NTX, and Kv1.1, Kv1.2, Kv1.4, Kv1.5,
and Kv1.6 antibodies were from Alomone Labs (Jerusalem, Israel);
additional Kv1.2 and Kv1.6 antibodies were from Upstate Biotechnology
(Lake Placid, NY); TsTX was from Peptide Institute (Osaka, Japan);
CP-339,818 was from Tocris Cookson; and DTX-K was a kind gift from
Brian Robertson (Imperial College, London, UK).
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RESULTS |
Expression of Kv1 subunits in the MNTB
Immunohistochemical labeling was used to determine Kv1 subunit
expression in the MNTB. Primary antibodies for Kv1.1, Kv1.2, Kv1.4,
Kv1.5, and Kv1.6 were applied to rat brainstem sections (transverse
plane, 20 µm thick). The immunofluorescence was subtracted from
background (Fig. 1F), and average data
from three animals is plotted in the bar chart (Fig. 1G).
High levels of immunoreactivity were detected for Kv1.1, Kv1.2, and
Kv1.6, whereas only low levels were present for Kv1.4 and Kv1.5 (Fig.
1A-E). The immunoreactivity of Kv1.4 and Kv1.5 were
not significantly different from that when blocking peptide was applied
(p > 0.5; paired t test). This suggests that Kv1.4 and Kv1.5 immunoreactivity was attributable to nonspecific binding of the primary antibody. Although the
immunoreactivity measured will include membrane and cytoplasmic
staining, these data suggest that Kv1.1, Kv1.2, and Kv1.6 are expressed
in the MNTB, whereas Kv1.4 and Kv1.5 are absent.
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Figure 1.
Immunoreactivity for Kv1.1, Kv1.2, and Kv1.6
is present in the MNTB. Pixel intensities for Kv1.1 were greater than
for Kv1.2 and Kv1.6 (measured over an area of 4800 µm2). A-E, In each case, an
example low-power (10× objective) image of the MNTB is shown. The
primary antibody used is indicated in the bottom left of
each panel. F, Control image denoting background
fluorescence represents secondary antibody fluorescence with the
primary antibody substituted for 10% goat serum. G,
Mean immunofluorescence above background from three animals. Kv1.1,
Kv1.2, and Kv1.6 immunofluorescence was significantly higher than when
blocking peptide was incubated with the primary antibody
(*p < 0.05; paired t test).
Immunofluorescence for Kv1.4 and Kv1.5 was not significantly different
from that in the presence of blocking peptide, suggesting that the
measured fluorescence was attributable to nonspecific binding of these
primary antibodies. AU, Arbitrary units. Scale bar, 100 µm.
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DTX-I blocks the low-threshold current
Voltage-gated currents were evoked by voltage steps
ranging from 90 to 0 mV in 5 mV increments (but for the purposes of
clarity, only alternate traces are shown in the figures). Each test
step was preceded by a 750 msec prepulse to 100mV (Fig.
2E) to remove resting
voltage-dependent inactivation or a prepulse to 30 mV (Fig.
2E, dashed line) to induce inactivation
(Brew and Forsythe, 1995; Wang et al., 1998). Outward potassium
currents began to activate at potentials more positive than 70 mV
(Fig. 2A,C), activated rapidly,
peaked at 10 msec (measured on a step to 30 mV), and then partially
inactivated. The magnitude of the outward currents evoked by steps to
45 and 0 mV was 0.60 ± 0.05 and 4.8 ± 0.4 nA,
respectively (n = 21). Previously, Brew and Forsythe (1995) demonstrated that DTX-I, which specifically blocks channels containing Kv1.1, Kv1.2, or Kv1.6 (IC50 of 3 nM for homomers) (Hopkins, 1998), blocks the
low-threshold current in MNTB neurons. In our experiments, 100 nM DTX-I blocked 82 ± 5% of the current measured at 45 mV (n = 4) (Fig.
2C,D,F). The
DTX-I-insensitive current at 45 mV is presumably the high-threshold
current, which begins to activate at these potentials (Brew and
Forsythe, 1995; Macica and Kaczmarek, 2001). DTX-I block of the
low-threshold current can be clearly seen in the first four
traces after the prepulse to 30 mV (Fig.
2B,D).
ILT exhibits some partial inactivation (Brew and Forsythe, 1995), seen in Figure 2, compare A with
C.
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Figure 2.
DTX-I, a potent blocker of Kv1.1, Kv1.2,
and Kv1.6, blocks the low-threshold component
(ILT) of outward currents in MNTB
neurons. Traces show I-V relationships
for the same neuron after a 750 msec prepulse (PP) to
either 100 mV (A, C) or 30 mV
(B, D). Voltage protocols are show in
E. At the end of each protocol, the cell returned to a
holding potential of 70 mV; current traces are aligned to the current
at this potential. A, Control responses after a 100 mV
prepulse. B, A 30 mV prepulse inactivates the
transient portion of the outward current. C, Currents
after a 100 mV prepulse in the presence of 100 nM DTX-I.
DTX-I blocks the low-threshold current, so that outward current is now
seen only at potentials positive to 50 mV. D, Currents
after a 30 mV prepulse in the presence of 100 nM DTX-I.
E, Voltage protocols applied. Test steps (200 msec) to
potentials between 90 and 5 mV were preceded by prepulses to 100
mV (solid line) or 30 mV (dashed line).
F, I-V relationships for
A-D, with the linear leak subtracted. Data is presented
from one neuron, with similar results observed in three neurons.
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Inactivation of ILT
ILT in MNTB neurons has a
partially inactivating component (Fig. 2). We investigated the
half-inactivation potential of ILT by
applying 750 msec prepulses to potentials between 100 and 40mV,
followed by a test step to 40mV, as shown in Figure
3A. The peak current during
the test step was measured and plotted as a partial Boltzmann (Fig.
3B). Half-inactivation of
ILT was determined empirically to be
at approximately 50 mV, but estimation of the true
V1/2 was not possible because
ILT inactivates only partially (40%)
and overlaps with activation of high-threshold currents (Fig.
3B).
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Figure 3.
Half-inactivation of the low-threshold current.
A, The current response to a test step at 40 mV, after
750 msec prepulses to potentials ranging from 100 to 40 mV.
B, I/Imax is
plotted against prepulse potential. The inactivation of the
low-threshold current was well fit by a fractional Boltzmann, with a
V1/2 of 63 ± 3 mV, a
K value of 7.9 ± 0.8 mV, and a fraction of
0.40 ± 0.01 (n = 4). The Boltzmann fit is to
potentials from 100 to 50 mV (filled
squares). At potentials more positive than 50 mV, the
high-threshold current begins to activate (open
squares).
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Block of low-threshold current by subunit-specific toxins
To further characterize the low-threshold current and to provide
clues to the channel subunit composition, we used toxins specific for
one or two Kv1 subunits. DTX-K (100 nM), which blocks channels containing Kv1.1 subunits (IC50 of 2.5 nM for homomers) (Robertson et al., 1996; Wang et al.,
1999a), blocked 90 ± 4% of the low-threshold current in MNTB
neurons (measured at 45 mV; n = 4) (Fig.
4A,D).
NTX blocks channels containing Kv1.2, Kv1.3, or Kv1.7
(Kd of 2 nM for
homomers) (Grissmer et al., 1994; Kalman et al., 1998). Noxiustoxin
(100 nM) only partially blocked the low-threshold
current (Fig. 4B), blocking 48 ± 5% of the
current at 45 mV (n = 3) (Fig.
4B,D). TsTX (100 nM) blocks channels containing Kv1.2
(IC50 of 0.55 nM for
homomers) (Hopkins, 1998) and blocked 48 ± 4% of the current at
45 mV (n = 7) (Fig. 4C,D). The
toxin block data suggests that there are at least two components to the
low-threshold current. One component is blocked by TsTX and NTX and
therefore presumably contains at least one Kv1.2 subunit; we termed
this component ILTS, for a
low-threshold tityustoxin-sensitive current. DTX-K blocks both
components of the low-threshold current, suggesting that each contains
at least one Kv1.1 subunit. The current blocked by DTX-K but resistant
to TsTX we termed ILTR, for
low-threshold tityustoxin-resistant current. To investigate whether
Kv1.3 subunits contribute to ILTS, we
used CP-339,818, which blocks Kv1.3 and Kv1.4
(IC50 of 0.23 µM for
Kv1.3 homomers) (Nguyen et al., 1996; Jager et al., 1998). CP-339,818
(1 µM) had no effect on the current measured at
45 mV (n = 3; data not shown), suggesting that
ILTS channels do not contain Kv1.3
subunits.
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Figure 4.
Action of subunit-specific toxins on
ILT. DTX-K, a potent blocker of Kv1.1,
blocks all of the low-threshold current, whereas TsTX (blocks Kv1.2)
and NTX (blocks Kv1.2-, Kv1.3-, or Kv1.7-containing channels) only
block half of ILT. Example data are shown
for three different cells. The respective inset traces
are the current activated by a step to 45 mV after a 750 msec
prepulse to 100 mV. The top trace is a control, and
the bottom trace is the current in 100 nM
toxin. I-V relationships are shown after linear leak
subtraction, but example traces are not leak subtracted.
A, DTX-K blocks 90 ± 4% of the current at 45 mV
(n = 4). The high-threshold current, seen in the
presence of DTX-K (open symbols), activates at
approximately 45 mV. B, NTX blocks 48 ± 5% of
the current at 45 mV (n = 3). C,
TsTX blocks 48 ± 4% of the current at 45 mV
(n = 7). D, Bar chart of the
percentage of the current at 45 mV blocked by 100 nM
DTX-K (n = 4), NTX (n = 3), and
TsTX (n = 7).
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ILTS is of dominant
functional relevance
MNTB neurons characteristically fire a single action potential in
response to sustained current injection or synaptic stimulation (Brew
and Forsythe, 1995). The low-threshold potassium current is crucial in
maintaining this response because, when it is blocked, a train of APs
is fired. In control conditions, single APs were evoked in each of six
cells by current injections in 50 pA
increments over a range of 0-350 pA (Fig. 5A, Table
1). The threshold for AP firing was
reached with current injection above 100 pA in all cells, but,
in some neurons, threshold was as high as 200 pA (n = 6). AP characteristics were measured from six neurons during a 200 pA
current injection from a membrane potential of 70 mV (Table
1). Each AP was followed by a shallow afterhyperpolarization with an
undershoot of 8.0 ± 0.6 mV (n = 6) (Fig.
5C). In the presence of 100 nM DTX-K,
which blocks both low-threshold currents (ILTS and
ILTR), MNTB neurons fired trains of
APs (Fig. 5A); the mean number of APs fired during a 200 msec step of 200 pA was 18 ± 4 (n = 3).
Interestingly, 100 nM TsTX, which blocks only ILTS, also caused the MNTB neuron to
fire trains of APs at a similar rate (Fig. 5B). The mean
number of APs fired during a 200 msec step of 200 pA in TsTX was
18 ± 4 (n = 3). Although in the example record in
Figure 5A fewer APs were seen in DTX-K, on average, the
number of APs was the same in TsTX and DTX-K. DTX-K or TsTX had no
effect on the waveform of the first AP compared with control APs (Table
1). Subsequent APs in the presence of DTX-K had the same
characteristics as those in TsTX, suggesting that
ILTR has little effect on these
parameters. Because multiple APs were not generated under control
conditions, we were not able to assess whether
ILTS affects the waveform of
subsequent APs. In TsTX and DTX-K, firing threshold was lower than
control (Table 1), being reached with current injection of <50 pA in
some neurons and achieved in all cells with injection of 100 pA
(n = 6).
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Figure 5.
DTX-K and TsTX both cause MNTB neurons to fire
multiple action potentials. A, Characteristic single
action potential in response to sustained depolarization in control
conditions. In 100 nM DTX-K, which blocks the
low-threshold current, the same cell fires multiple action potentials.
B, In 100 nM TsTX, which blocks one
component of the low-threshold current
(ILTS), MNTB neurons also fire
multiple action potentials. In this example, more APs are fired in TsTX
than in the example shown for DTX-K. However, the mean number of APs
fired was 18 ± 4 for both DTX-K and TsTX (during a 200 msec step
of 200 pA; n = 3 for each toxin). Bottom
trace shows current step time course for A and
B. C, An action potential showing points
of measurement for quantification presented in Results and Table
1. Representative traces are shown, although similar results were
observed in three neurons.
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Because ILTS accounts for half of the
low-threshold current, it is important to determine whether the effect
of TsTX on AP firing is a specific consequence of the
ILTS component or merely attributable
to a general block of half the low-threshold current. To distinguish
between these possibilities, we compared the AP response after
half-inactivation of all low-threshold K+
currents with the response in TsTX.
ILTR and
ILTS will be equally and maximally
inactivated (by 40% of their peak amplitude) (Fig. 3) at a membrane
potential of 50 mV.
The effect of half-inactivation of low-threshold currents on AP firing
was investigated in current clamp by depolarizing from a membrane
potential of 50 mV (as opposed to 70 mV for control data). The mean
number of action potentials observed in response to a 350 pA current
injection from 50 mV was 6 ± 2 (n = 6) compared with 26 ± 5 (n = 3) with TsTX from 70 mV (Fig.
6A). The number of APs
generated for a given current step is plotted in Figure 6A for three conditions: control (APs evoked from
70 mV); after half-inactivation (at a membrane potential of 50 mV);
and after application of TsTX (from 70 mV). The same data are also
plotted as the number of APs against the absolute membrane potential in Figure 6B to demonstrate that the difference in AP
firing is not attributable to changes in membrane conductance. For
current steps over 250 pA, there are significantly more APs in TsTX
compared with half-inactivating ILT
(at a membrane potential of 50mV) as shown in Figure 6, A
and C (p < 0.05; unpaired
t test). These data show that half-block of
ILT by TsTX gives a different
functional response than half-block of
ILT through inactivation. Because TsTX
is specifically blocking only those channels contributing to
ILTS, this strongly implies that
ILTS has the dominant functional impact on AP firing in MNTB neurons. The fact that
ILTS is 40% inactivated at 50mV explains
why more than one AP can be generated from this potential (Fig. 6).
Depolarizing the membrane to 50 mV will result in some sodium channel
inactivation; however, this is not responsible for the differences in
AP frequency. At the same absolute membrane potential (when sodium
channels will be equally inactivated), more APs are evoked in the
presence of TsTX than during partial inactivation of
ILT (from 50 mV) (Fig. 6B). In addition, at a membrane potential of 50 mV
in the presence of TsTX, APs are continuously fired (even in the
absence of current injection), suggesting that sodium channel
inactivation is not sufficient to prevent repetitive firing at this
potential. These data demonstrate that the difference in AP firing in
the presence of TsTX at 70 mV and in control at 50 mV is not simply
attributable to sodium channel inactivation.
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Figure 6.
Multiple AP firing in TsTX is not attributable to
half-block of ILT. A, The
number of APs generated in response to 200 msec current injections.
More APs were fired when ILT was
half-blocked with TsTX (open circles;
n = 3) than when half-inactivated at a membrane
potential of 50 mV (filled diamonds;
n = 6). In control conditions, only one AP was
fired throughout the range tested (filled
circles; n = 6). B, The same
data are replotted as AP number against absolute potential. This was
defined as the potential at the end of the current step and was
equivalent to the threshold potential at high frequencies.
C, Representative records from a current-clamped neuron
depolarized from 50 mV in control aCSF and from 70 mV in 100 nM TsTX.
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|
Kv1.2-containing channels are located in axons of MNTB neurons
Although ILTS and
ILTR contribute equally to the
low-threshold current, the physiology shows that
ILTS is of most functional significance in suppression of AP firing. Because the two components have similar kinetic properties, one possible explanation is that channels containing Kv1.2 are preferentially localized to an area concerned with spike initiation. To determine the subcellular location
of ILTS, we used immunohistochemical
labeling of Kv1.2 viewed on a confocal microscope (Fig.
7A). Kv1.2 cytoplasmic
labeling was present in the soma but there was little enhancement at
the plasma membrane. The highest Kv1.2 labeling was in the initial 20 µm of the axon (Fig. 7A, arrow). To determine
whether Kv1.1 was expressed in the same region as Kv1.2, we used double
labeling (Fig. 7B-D). Immunoreactivity for Kv1.1 and Kv1.2
was detected in the somatic cytoplasm and concentrated in the initial
20 µm of the axon (arrows). The high degree of Kv1.1
overlap with Kv1.2 supports the idea that
ILTS channels are Kv1.1/Kv1.2
heteromers. To investigate the subcellular location of
ILTR, we examined Kv1.6 labeling (Fig.
7E). Somatic Kv1.6 immunofluorescence was detected, but no
axonal Kv1.6 staining was seen in the MNTB, suggesting that
ILTR channels do not reside in the
initial portion of the axon. The distribution of ion channels has
important consequences for neuronal excitability, integration, and
action potential firing. Although multiple sites of AP generation can
exist in some neurons (Luscher and Larkum, 1998), the initial segment
of an axon is of general importance for spike generation (Stuart and
Sakmann, 1994). Association of ILTS
but not ILTR with this region may
explain the dominance of ILTS in
regulating AP firing pattern in MNTB neurons.
View larger version (100K):
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|
Figure 7.
Kv1.1 and Kv1.2 are highly expressed in the axons
of MNTB neurons. Somatic and axonal immunoreactivity can be seen for
Kv1.1 and Kv1.2. Somatic but not axonal immunoreactivity can be seen
for Kv1.6. A, Kv1.2 immunofluorescence with an
FITC-conjugated secondary antibody. Cytoplasmic staining was observed
in the soma, with no obvious additional staining at the plasma
membrane. The initial 20 µm of the axon has the highest level of
immunoreactivity (arrows). A Z-series projection of 20 images through an MNTB neuron with a step size of 0.175 µm.
B, Kv1.1 immunofluorescence using an FITC-conjugated
secondary antibody. A Z-series projection of five images through an
MNTB neuron with a step size of 0.675 µm. C, Kv1.2
immunofluorescence using a Texas Red-conjugated secondary antibody. A
Z-series projection as in B. D,
Colocalization of Kv1.1 (green) and Kv1.2
(red). The axon in B-D is 1.3 µm in
diameter, in contrast to axons giving rise to calyces, which are 1.5-2
µm in diameter (Forsythe, 1994). E, Kv1.6
immunofluorescence from a different section from that in
B-D. Somatic but not axonal staining was observed
(n = 4 animals). Scale bars, 20 µm (the same
scale was used for B-E). Kv1.2 and Kv1.6 antibodies
(Upstate Biotechnology) and a Kv1.1 antibody (Alomone Labs) were used
because these had the lowest background staining.
|
|
| |
DISCUSSION |
We used immunohistochemistry, pharmacology, and electrophysiology
to investigate low-threshold potassium currents
(ILT) in MNTB neurons. The data
indicate that Kv1.1, Kv1.2, and Kv1.6 subunits are strongly expressed
in the MNTB. These subunits assemble as heteromeric channels to produce
two pharmacologically distinct currents, each of which contributes
approximately one-half of the low-threshold current. One component,
named ILTS, is a low-threshold tityustoxin-sensitive current, and the other
(ILTR) is a low-threshold tityustoxin-resistant current. The Kv1.2-containing channels that generate ILTS have the dominant
function in determining the physiology of action potential firing in
the MNTB.
Our data allow us to address two issues: first, the functional
relevance of multiple low-voltage-activated currents; and second, the
subunit composition of native Kv1 channels. A pharmacological approach
has advantages over the use of transgenic knock-out animals to study
physiological function, because we are examining native channels in the
absence of compensation. Subunit-specific toxins were used to
investigate the composition of ILT
channels. DTX-K and TsTX bind to specific Kv1 subunits and are thought
to plug the channel pore (Ellis et al., 2001; Harvey, 2001). In
heteromeric channels containing Kv1.1 and Kv1.2 subunits, only one
toxin-sensitive subunit need be present for the toxin to block the
channel (Hopkins, 1998). More complex binding occurs for
Kv1.2/Kv1.4-containing channels, but we have no evidence for Kv1.4 in
rat MNTB (from immunohistochemical, pharmacological, and
electrophysiological data). Our data can also exclude the involvement
of Kv1.3 and Kv1.5 subunits in ILT.
Previous immunohistochemical studies have shown that Kv1.1 and Kv1.2
are expressed in brainstem nuclei in the mouse (Wang et al., 1994;
Grigg et al., 2000), and our data confirm this in the rat.
We subdivided the low-threshold current in MNTB neurons into two
components, ILTS and
ILTR. The simplest interpretation of the subunit composition of the channels underlying
ILTS is that they are Kv1.1/Kv1.2
heteromers, whereas those of ILTR are
Kv1.1/Kv1.6 heteromers. The arguments for this conclusion are developed
below. First, there is no evidence for homomeric Kv1.1 or Kv1.2
channels: homomeric Kv1.1 channels are blocked by 1 mM tetraethylammonium (TEA) (Hopkins, 1998), and
we saw no effect of TEA on ILT (data not shown) (Brew and Forsythe, 1995). Similarly,
ILT is blocked by the Kv1.1-specific
toxin DTX-K; this excludes homomeric Kv1.2 channels, because they would
be resistant to DTX-K. Second, both components contain Kv1.1 subunits,
because ILT is blocked by DTX-K. Third, ILTR does not contain Kv1.2 or
Kv1.3, because it is resistant to TsTX, NTX, and CP-339,818. Because
Kv1.6 is the only other Kv1 subunit expressed in the MNTB, the simplest
interpretation is that ILTR is a
Kv1.1/Kv1.6 heteromer and ILTS is a
Kv1.1/Kv1.2 heteromer.
Evidence from studies of Kv subunits in expression systems suggests
that Kv subunits associate first as dimers and then two dimers
associate to form a tetramer (Tu and Deutsch, 1999). If the dimer
association were of general application for all Kv1 channels, then our
data are compatible with a very simple hypothesis: ILTR is made up of two Kv1.1/Kv1.6
dimers, whereas ILTS is formed from
Kv1.1/Kv1.2 dimers. ILTS might include
one dimer of Kv1.1/Kv1.6 or Kv1.2/Kv1.6, but the immunohistochemistry
suggests that Kv1.6 is not associated with
ILTS (Fig. 7). One caveat is that our
methods are relatively insensitive to currents generated by subunit
combinations, contributing <10% of the total current. Hence, it is
conceivable that some ILT channels
could include other Kv1 subunits. A possible explanation for the
formation of heteromeric over homomeric channels is that particular
channel subunits may have a higher affinity for different subunits,
analogous to that proposed for AMPA receptors (Mansour et al., 2001).
An alternative explanation is that some of the subunits present in the
heteromeric channels increase the efficacy of endoplasmic reticulum
(ER) export and trafficking, with homomeric channels being
confined to the ER (Manganas and Trimmer, 2000).
Immunoprecipitation studies have shown that Kv1.1 and Kv1.2 are
expressed in the brain as heteromers and that these heteromers can
contain Kv1.6. Such heteromers can also contain Kv1.3 and Kv1.4
subunits (Koch et al., 1997; Shamotienko et al., 1997; Coleman et al.,
1999), but we have no evidence for the involvement of these subunits in
ILT in MNTB neurons. In addition,
Kv1.1 homomers have not been found in the brain using
immunoprecipitation (Shamotienko et al., 1997; Koschak et al., 1998;
Coleman et al., 1999). These findings are consistent with our proposal
that ILTS is a Kv1.1/Kv1.2 heteromer
and ILTR is a Kv1.1/Kv1.6 heteromer,
although the relatively small amount of tissue in the rat MNTB limits
the application of immunoprecipitation to confirm this.
Low-threshold currents maintain MNTB action potential
firing pattern
MNTB neurons fire a single action potential in response to the
giant EPSP produced as a result of synaptic stimulation. A rapid EPSP
is mediated by AMPA receptors, but there is also a slower NMDA
receptor-mediated component (Forsythe and Barnes-Davies, 1993). During
high-frequency firing, this slow component would cause multiple action
potentials to be fired (Brew and Forsythe, 1995). However,
low-threshold potassium currents (ILT)
in MNTB neurons oppose the depolarization from the slow component and hence preserve the single AP response.
ILT in MNTB neurons acts to both keep
the membrane potential below threshold during small depolarizations and
bring the membrane back below threshold after a single AP. Like DTX-I
and DTX-K, TsTX (which blocks ILTS
alone) caused multiple APs in response to sustained current injection (Fig. 5). This response was not reproduced by nonspecific partial inactivation of both low-threshold K+
currents, suggesting that ILTS has a
specific function in regulating AP firing in MNTB neurons. Because
ILTS and
ILTR have similar properties, it seems
unlikely that this differential function is the result of kinetic
differences. One explanation for the dominance of
ILTS in determining AP firing pattern
would be that ILTS is preferentially
localized to the spike initiation region. The immunohistochemistry
confirmed that Kv1.2 (and therefore
ILTS) is highly expressed in the
initial region of MNTB axons (Fig. 7A,C, arrows), although
cytoplasmic staining of both Kv1.1 and Kv1.2 makes it difficult to
determine differential location of these subunits in the somatic
membrane. Somatic but not axonal immunoreactivity was observed for
Kv1.6 (Fig. 7E), suggesting that channels underlying
ILTR are not located in the initial
region of the axon. Differential distribution of
K+ channels has been observed in rat
spinal cord neurons, with delayed rectifier channels being concentrated
in axons and dendrites and at low levels on the soma (Wolff et al.,
1998). Trafficking to the cell surface is enhanced by association with
some Kv subunits (Manganas and Trimmer, 2000), and, in cultured
hippocampal neurons, coexpression with Kv2 results in axonal
targeting of Kv1.2 (Campomanes et al., 2002). Immunoprecipitation
studies have found that Kv2.1 subunits are expressed in neurons with
channels containing Kv1.2 (Shamotienko et al., 1997; Coleman et al.,
1999). Other proteins such as Caspr and Caspr2 have also been
implicated in localizing Kv1.1 and Kv1.2 to jutaparanodal regions of
myelinated axons (Poliak et al., 1999). It is conceivable that Kv2
or Caspr proteins may preferentially associate with
ILTS and contribute to the
differential localization of ILTS and
ILTR. If Kv1.1 subunits, which
confer N-type inactivation, were also present, this could account for the partial inactivation of ILT (Fig.
2A).
The role of ILTR is not clear, but it
would certainly bolster the low-threshold conductance during large
depolarizations and would also support differential modulation by
second messengers. For instance, PKC inhibits Kv1.1 (Boland and
Jackson, 1999), whereas PKA increases Kv1.1 and Kv1.2 currents (Huang
et al., 1994; Levin et al., 1995; Jonas and Kaczmarek, 1996).
Phosphorylation by tyrosine kinases suppresses Kv1.2 current (Lev et
al., 1995; Holmes et al., 1996). However, the effect of phosphorylation
on heteromeric channels such as ILTS
and ILTR may differ from homomeric channels.
Similar DTX-sensitive currents in other neurons
Several DTX-sensitive low-threshold currents similar to
ILT have been found in other neurons
in rat, mouse, and avian brain. These currents are often found in
neurons with similar firing properties and are responsible for
maintaining the single AP response. Such currents are present in
mammalian bushy cells of the cochlear nucleus (Manis and Marx, 1991),
the nucleus magnocellularis (the avian homolog of the bushy cells)
(Rathouz and Trussell, 1998), octopus cells of the cochlear nucleus
(Bal and Oertel, 2001), neocortical pyramidal neurons (Bekkers and
Delaney, 2001), embryonic central vestibular neurons (Gamkrelidze et
al., 1998), in basket cell terminals (Southan and Robertson, 2000), and
ID in hippocampal neurons (Wu and
Barish, 1992). These currents are likely to be mediated by Kv1
heteromers similar to ILTR and
ILTS.
| |
FOOTNOTES |
Received April 15, 2002; revised May 17, 2002; accepted May 21, 2002.
This work was supported by the Wellcome Trust and the Medical Research
Council. P.D.D. is a Wellcome Trust Prize PhD student. We thank Drs.
Margaret Barnes-Davies, Brian Billups, and Brian Robertson for
critically reading this manuscript.
Correspondence should be addressed to Prof. I. D. Forsythe,
Department of Cell Physiology and Pharmacology, University of Leicester, P.O. Box 138, Leicester, LE1 9HN, UK. E-mail: idf{at}le.ac.uk.
| |
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ABSTRACT
INTRODUCTION
