1Department of Physiology and Neuroscience and Department of Biochemistry, New York University School of Medicine, New York City, New York 10016; and 2Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Mexico City 04510, Mexico
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ABSTRACT |
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Hernández-Pineda, R.,
A. Chow,
Y. Amarillo,
H. Moreno,
M. Saganich,
E. Vega-Saenz de Miera,
A. Hernández-Cruz, and
B. Rudy.
Kv3.1-Kv3.2 Channels Underlie a High-Voltage-Activating
Component of the Delayed Rectifier K+ Current in Projecting
Neurons From the Globus Pallidus.
J. Neurophysiol. 82: 1512-1528, 1999.
The globus pallidus plays central roles in
the basal ganglia circuitry involved in movement control as well as in
cognitive and emotional functions. There is therefore great interest in the anatomic and electrophysiological characterization of this nucleus.
Most pallidal neurons are GABAergic projecting cells, a large fraction
of which express the calcium binding protein parvalbumin (PV). Here we
show that PV-containing pallidal neurons coexpress Kv3.1 and Kv3.2
K+ channel proteins and that both Kv3.1 and Kv3.2
antibodies coprecipitate both channel proteins from pallidal membrane
extracts solubilized with nondenaturing detergents, suggesting that the
two channel subunits are forming heteromeric channels. Kv3.1 and Kv3.2
channels have several unusual electrophysiological properties when
expressed in heterologous expression systems and are thought to play
special roles in neuronal excitability including facilitating sustained high-frequency firing in fast-spiking neurons such as interneurons in
the cortex and the hippocampus. Electrophysiological analysis of
freshly dissociated pallidal neurons demonstrates that these cells have
a current that is nearly identical to the currents expressed by Kv3.1
and Kv3.2 proteins in heterologous expression systems, including
activation at very depolarized membrane potentials (more positive than
10 mV) and very fast deactivation rates. These results suggest that
the electrophysiological properties of native channels containing Kv3.1
and Kv3.2 proteins in pallidal neurons are not significantly affected
by factors such as associated subunits or postranslational
modifications that result in channels having different properties in
heterologous expression systems and native neurons. Most neurons in the
globus pallidus have been reported to fire sustained trains of action
potentials at high-frequency. Kv3.1-Kv3.2 voltage-gated K+
channels may play a role in helping maintain sustained high-frequency repetitive firing as they probably do in other neurons.
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INTRODUCTION |
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A large number of subunits of mammalian
K+ channels expressed in the CNS have been
identified after the cloning of the Shaker gene in
Drosophila in 1987 (reviewed in Chandy and Gutman
1995; Coetzee et al. 1999
; Jan and Jan
1997
; Pongs 1992
; Rudy et al. 1991a
). This work has revealed the existence of an
extraordinary diversity of molecular components of voltage-gated
K+ channels, predicting a functional diversity
well beyond that expected from prior functional studies (Rudy
1988
). The cloning studies have allowed enormous progress in
the understanding of the molecular mechanisms of channel function,
including the recent crystallization and high resolution structural
analysis of a K+ channel (Doyle et al.
1998
). Less progress has been obtained in understanding the
physiological significance of the molecular diversity. A major task of
future research is to identify physiological roles of the cloned
proteins, starting with the identification of native channels
containing specific types of cloned subunits.
Among the cloned subunits are the members of the Kv and KCNQ (or KQT)
families of K+ channel proteins, which are
pore-forming components of voltage-gated K+
channels (Chandy and Gutman 1995; Coetzee et al.
1999
; Jan and Jan 1997
; Pongs
1992
; Rudy et al. 1991a
). The Kv family is
divided into several subfamilies based on sequence similarities. Nearly 30 Kv proteins classified in eight subfamilies (Kv1-Kv6 and Kv8-Kv9) are known to date (Coetzee et al. 1999
).
The goal of this study was to identify the currents mediated by
channels containing proteins of the Kv3 subfamily in neurons. There are
four known Kv3 genes (Kv3.1-Kv3.4). In heterologous expression
systems, Kv3.1 and Kv3.2 proteins express tetraethylammonium (TEA)-sensitive delayed rectifier type currents, whereas Kv3.3 and
Kv3.4 proteins form transient, TEA-sensitive, A-type
K+ channels (reviewed in Vega-Saenz de
Miera et al. 1994). However, native channels may differ from
those formed by a given Kv subunit in heterologous expression systems.
Kv proteins can form heteromeric channels with novel properties with
other members of the same subfamily (Christie et al.
1990
; Covarrubias et al. 1991
; Isacoff et
al. 1990
; K. McCormack et al. 1990
;
Ruppersberg et al. 1990
; Weiser et al.
1994
). Moreover the functional characteristics of K+ channels, including those of the Kv family,
also can be modified by accessory subunits and postranslational
modifications (Barhanin et al. 1996
; Coetzee et
al. 1999
; Covarrubias et al. 1994
;
Heinemann et al. 1996
; McDonald et al.
1997
; Sanguinetti et al. 1996
; Serodio et
al. 1994
, 1996
).
We have shown previously in a study combining immunohistochemical and
electrophysiological analysis in slice preparations that drugs that
block Kv3.1 currents in heterologous expression systems blocked a
fraction of the K+ current from hippocampal
interneurons expressing Kv3.1b proteins that resembles Kv3.1 currents
in activation and deactivation properties (Du et al.
1996). However, limitations associated with voltage clamping
intact neurons in slices prevented a detailed comparison of the
properties of the putative Kv3.1-mediated currents in these cells with
the properties of Kv3.1 currents in heterologous expression systems.
Both Kv3.1 and Kv3.2 proteins are strongly expressed in neurons of the
globus pallidus (GP), suggesting that these cells might be a good
system to study the properties of native Kv3.1-Kv3.2 channels using
voltage-clamp methods (Moreno et al. 1995
; Weiser
et al. 1994
, 1995
). Moreover, methods to dissociate these
neurons from rat brain have been developed (Stefani et al. 1992
,
1995
; Surmeier et al. 1994
). Freshly dissociated
and short term cultured cells are a good system for this kind of study, allowing improved conditions for space clamp and pharmacological analysis.
We have used whole cell patch-clamp methods to analyze the
voltage-dependent K+ currents of freshly
dissociated rat GP neurons to determine whether they contain currents
with properties similar to those carried by Kv3.1-Kv3.2 channels.
Because of its central roles in movement control and perhaps also
cognitive functions, there is great interest in the anatomic and
physiological characterization of the GP (Chang et al.
1987; Chudler and Dong 1995
; DeLong 1971
,
1972
; Difiglia and Rafols 1988
; Graybiel
and Ragsdale 1979
; Hauber et al. 1998
; Kita 1992
, 1994
; Kita and Kitai 1991
,
1994
; Moriizumi and Hattori 1992
; Nambu
and Llinas 1994
; 1997
; Parent and Hazrati
1995a
,b
; Park et al. 1982
; Schneider et
al. 1985
; Stefani et al. 1992
; Surmeier
et al. 1994
). The present studies contribute novel information on the classification and cellular properties of pallidal neurons.
Previous electrophysiological analysis of the K+
currents of pallidal neurons, in the same species, revealed a low
voltage-activating fast inactivating current
(IA), a component with slower
inactivation and slow recovery from inactivation that is blocked by
micromolar concentrations of 4-AP
(IAs), and two maintained components
one blocked (IK) and one not blocked
by 10 mM TEA (Stefani et al. 1992, 1995
). None of these
components resembles Kv3 currents. These observations do not
necessarily indicate that Kv3 currents are either absent in pallidal
neurons or have properties different from those of Kv3 currents in
heterologous expression systems because the methods that are used in a
given study to isolate individual components of the total
K+ current are tailored to the goals of the
particular investigation. Therefore electrophysiological experiments
specifically designed to search for native currents with properties
similar to those of Kv3.1-Kv3.2 currents in vitro are required before
we can conclude whether or not native Kv3.1-Kv3.2 channels in pallidal
neurons have properties similar to those in heterologous expression systems.
In this study, we first analyzed the expression of Kv3.1 and Kv3.2
proteins in the rat GP with specific antibodies. We also determined the
developmental expression of these proteins, allowing us to select
tissue at developmental stages in which the proteins are robustly
expressed in pallidal neurons. We used pharmacological and
electrophysiological protocols on freshly dissociated neurons appropriate for the isolation of Kv3-like currents and compared these
currents to those recorded under identical conditions from mammalian
cells transfected with Kv3.1 and Kv3.2 cDNAs. The expression of Kv3
transcripts in the same cells was confirmed by single cell RT-PCR. The
studies described here have been previously presented in abstract form
(Hernández-Pineda et al. 1996; Pineda et
al. 1998
).
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METHODS |
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Antibodies to Kv3.1 and Kv3.2 proteins
Site-specific antibodies against Kv3.1b proteins were prepared
by injecting into rabbits the peptide CKESPVIAKYMPTEAVRVT coupled via
the cysteine to keyhole limpet hemocyanin (KLH). The peptide corresponds to the carboxyl terminal sequence of the Kv3.1b protein (residues 567-585) (Weiser et al. 1995). The
characterization of this antibody was described previously
(Weiser et al. 1995
). To raise antibodies against Kv3.2
proteins, rabbits were injected with the peptides: CTPDLIGGDPGDDEDLGGKR
and CTPDLIGGDPGDDEDLAAKR coupled via the cysteine to KLH (Chow et al.
1999
). The peptides correspond to a sequence present in the constant
region of the rat and mouse Kv3.2 proteins, respectively (residues
171-189 plus an N-terminal cysteine added to facilitate coupling),
before the first membrane-spanning domain in an area not conserved
among different K+ channel proteins
(Vega-Saenz de Miera et al. 1994
; see T. McCormack et al. 1990
for the rat sequence). The mouse sequence
has not been published, but it is identical to that in rat except for the substitution of glycines (186-187 by alanines). For affinity purification, the respective peptides were coupled to Sulfolink Sepharose resin (Pierce, Rockford, IL) via the cysteine residue and the
sera purified following supplier's protocols.
Immunofluorescence labeling
Male Sprague-Dawley rats (2-3 wk old) or male C57Bl6 mice (6-8
wk old), as well as Kv3.1 /
(Ho et al. 1997
) or
Kv3.2
/
(Lau et al. 1999
) mice, were anesthetized
with an injection of pentobarbital sodium (120 mg/kg ip) and perfused
transcardially with 10-20 ml Heparin (1 U/ml) in phosphate-buffered
saline (PBS: 0.06 M sodium phosphate buffer, 0.85% sodium chloride, pH
7.35) at room temperature followed by 100-200 ml of 4%
paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.4. The brain
was dissected out and blocked coronally into ~5 mm portions,
postfixed for 30 min in the same fixative at room temperature and
placed in 30% sucrose in PBS for 12-24 h at 4°C. When the tissue
had sunk in the sucrose solution, 50-µm sections were produced using
a freezing microtome and collected in PBS. The sections were washed
twice for 15 min in PBS and incubated in a blocking solution containing 10% normal goat serum (Jackson Immuno Research), 1% bovine serum albumin (Jackson Immuno Research), 0.2% cold water fish gelatin (Sigma
Chemicals), and 0.2% Triton X-100 (Sigma Chemicals) in PBS for 1 h to minimize nonspecific binding. The sections then were incubated
with primary antibody at the appropriate dilution in a working buffer
(0.1× blocking solution in PBS) for 12-24 h at 4°C. For
double-labeled sections, a primary rabbit antibody, anti-Kv3.1b or
anti-Kv3.2, and a primary mouse antibody, anti-parvalbumin (Sigma
Immunochemicals) were added simultaneously. After three 15 min washes
in PBS, secondary antibodies diluted in working buffer were applied for
15 min at room temperature. The following secondary antibodies were
used, Cy2-conjugated goat anti-mouse IgG and Cy3-conjugated goat
anti-rabbit IgG (Jackson Immuno Research). After two 15 min washes in
PBS, the sections were mounted onto glass slides and coverslipped with elvanol.
The following primary antibody concentrations were used: antibodies
against Kv3.1b (Weiser et al. 1995) at 1:50, Kv3.2 (Chow et al. 1999
) at 1:50, parvalbumin (Sigma Immunochemicals) at 1:400. Secondary fluorescent antibodies were used at 1:500. The atlas by
Paxinos and Watson (1986)
and the book edited by
Paxinos (1995)
were used as guides to identify CNS
neuronal populations and axonal projections.
Images were taken either with a Zeiss Axiophot fluorescent microscope or an Axiovert 35 M confocal microscope, with a ×40 (NA 1.3) or ×63 (NA 1.4) objective lenses. Fluorescent images were recorded using a scanning laser attachment (MRC-600 and MRC-1000, Bio-Rad Laboratories) and a krypton/argon mixed gas laser. Images were collected digitally and transferred to a graphics program (Adobe Photoshop 4.0). After brightness and contrast adjustments, the image files were printed on a Tektronix printer (Phaser 440).
Immunoblots
Rat brain membrane extracts were prepared from a P3 fraction of
tissue homogenate (Hartshorne and Catterall 1984)
solubilized for 1 h in a 2% Triton X-100 solution containing (in
mM) 50 potassium phosphate buffer, pH 7.4; 50 KCl; 2 EDTA; 1 pepstatin
A, 1 1,10-phenanthroline, 0.2 phenylmethylsulfonyl fluoride (PMSF), and
1 iodoacetamide to inhibit proteases. The suspension was spun at
100,000 g to remove nonsolubilized material, and the top
two-thirds of the supernatant used for further experiments. To prepare
membrane extracts from the GP, the nucleus was dissected from slices
prepared as described for the preparation of dissociated neurons (see
following text) and proteins solubilized as described in the preceding text.
To prepare immunoblots, 50 µg of membrane protein was mixed 1:1 with
a sample buffer [10% (vol) glycerol, 5% (vol)
b-mercaptoethanol; 60 mM Tris-HCl pH 6.8; 0.001% (weight)
bromophenol blue and 3% SDS], heated for 3 min at 80°C, and
electrophoresed in a 8% SDS polyacrylamide gel (Harlow and Lane
1988). The electrophoresed proteins were transferred onto a
nitrocellulose filter (Bio Rad). Blots were incubated with either Kv3.2
antibodies at a 1:100 dilution or Kv3.1b antibodies at 1:2000, followed
by incubation with horseradish peroxidase-linked anti-rabbit secondary
antibodies (Promega). Bound antibodies were detected using
chemilluminscence (Pierce).
Immunoprecipitation
Before immunoprecipitation, 300 µl of solubilized membranes (~400 µg protein) in 1% Triton X-100 in (in mM) 50 Tris, 150 NaCl, 1 EDTA, and 1 EGTA, pH 7.4, were precleared for 30 min at 4°C with protein A-Sepharose beads (Sigma Chemicals). After removing the beads, the extracts were incubated for 4 h at 4°C with Kv3.2 antibodies at a 1:10 dilution or Kv3.1b antibodies at 1:50 dilution. At the end of the incubation period, fresh protein A-Sepharose beads were added, and the suspension was incubated for 2-3 h at 4°C with shaking. The complexed beads were collected and washed three times in 1% Triton X-100 in (in mM) 50 Tris, 150 NaCl, 1 EDTA, and 1 EGTA, pH 7.4. Proteins then were extracted by adding an equal volume of sample buffer, heated for 3 min at 80°C and processed for immunoblotting as described in the preceding text.
Culture of chinese hamster ovary (CHO) cells
CHO cells were cultured in -MEM (pH 7.4, GIBCO BRL,
Gaithersburg, MD) supplemented with 10% of fetal bovine serum (FBS,
GIBCO BRL) in the presence of penicillin and streptomycin at 37°C in a 95% O2 with 5% of CO2
atmosphere in 100-mm-diam culture dishes (Costar, Cambridge, MA). When
the cells were confluent, the monolayer was incubated with trypsin-EDTA
(GIBCO BRL) for
1 min, and the cells resuspended in
-MEM
containing 10% of FBS and plated at a 1/5 dilution in new 100 mm
dishes. The medium was changed every 2 days, and the cells passed every
4 days.
Functional expression of Kv3.1 and Kv3.2 potassium channels in CHO cells
To study Kv3.2 currents, we used the Kv3.2a stably transfected
cell line previously described (Moreno et al. 1995). To
study Kv3.1 currents, wild-type CHO cells were transiently transfected with Kv3.1b cDNA. After reaching 90% confluence in 100 mm dishes, CHO
cells were trypsinized and resuspended in 2 ml of
-MEM with 10%
FBS. One milliliter of the cell suspension was diluted and plated in 30 mm dishes at 40% confluence. Two to 4 h later, when the cells had
attached to the bottom of the dish, they were washed two times with
-MEM without serum and transfected with Lipofectamine (Life
Technologies, Gaithersburg, MD) following the manufacturer's protocols. To identify transfected cells, they were cotransfected with
a second plasmid containing the cDNA encoding the reporter protein
Green Fluorescent Protein (GFP, Life Technologies, Gaithersburg, MD).
Transfected cells were detected by the emission of green fluorescence
(520 nm) under epifluorescence with 488 nm excitation light. Typically,
electrophysiological recordings were carried out 1-2 days after transfection.
Dissociation of neurons from the GP
Young Sprague-Dawley rats 10-16 days of age were used. The brain was quickly removed and submerged in ice-cold normal extracellular solution (NES) containing (in mM) 130 NaCl, 3 KCl, 2 MgCl2, 1 NaHCO3, 0.5 NaH2PO4, 5 HEPES, 2 CaCl2, and 5 glucose, pH 7.4. The solution was gassed with 95% O2-5% CO2 for 15 min before starting the dissociation and then continuously during the procedure. Once the cerebellum had been removed, the brain hemispheres were separated along the midline and cut in the parasagittal plane with a vibratome (Campden Instruments, London, UK) in 400 µm slices. The slices were collected and maintained in ice-cold NES solution. The GP was identified by visual inspection under a stereomicroscope (see Fig. 7). The GP from four to seven slices was dissected out and subjected to enzymatic digestion for 25-35 min (depending on the age of the animal) at 37°C in NES with Pronase (Sigma Chemicals, St. Louis, MO). The tissue then was washed three times in NES without calcium and triturated mechanically by means of glass Pasteur pipettes with tips of decreasing diameters in a final volume of 2 ml. An aliquot (~500 µl) of the cell suspension was seeded in a recording chamber (RC-13, Warner Instruments, Hamden, CT) and mounted on the plate of an inverted microscope for electrophysiological recording. The cells were perfused continuously (~1 ml/min) with NES at room temperature.
Electrophysiological analysis
Electrophysiological recordings used voltage clamp under the
whole cell configuration of the patch-clamp technique (Hamill et
al. 1981). The same extracellular and intracellular solutions were used for recordings with CHO cells and pallidal neurons. Extracellular solution (NES) was supplemented with 1 µM tetrodotoxin (TTX; Alomone, Jerusalem, Israel) and 200 µM of
CdCl2 (Sigma Chemical) to block voltage-dependent
sodium and calcium currents, respectively. By inhibiting calcium
influx, CdCl2 also limited the activation of
calcium-activated potassium currents. Potassium channel blockers (TEA,
Sigma; 4-AP, Aldrich; charybdotoxin and dendrotoxin, Alomone) were
dissolved in NES with TTX and CdCl2. All the
experiments were carried out at room temperature (21-23°C).
The recording patch electrodes were made with borosilicate glass
capillary tubes (GC120F-10, Warner Instrument) filled with an
intracellular solution containing (in mM) 106 KH2PO4, 2 MgCl2, 10 HEPES-K, 10 BAPTA-K (Molecular Probes,
Eugene, OR), 2 ATP-Mg, and 0.5 GTP (Sigma Chemicals); pH 7.35 with
NaOH. The use of a high concentration of the fast calcium chelator
bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid
(BAPTA) was aimed at further limiting the activation of
calcium-activated membrane conductances. The typical resistances of the
electrodes when filled with this solution varied between 2.5 and 3.0 M. The patch pipette Ag-Cl wire was connected to the input of an Axopatch-1D or Axoptach 200A amplifier (Axon Instruments, Foster City,
CA). To generate voltage clamp protocols and for data acquisition and
analysis, we used the pClamp software (Axon Instruments). Large neurons
with short processes (<40 µm) were selected for recording. Series
resistance was estimated from the time constant of the capacity
transient and ranged between 5 and 10 M
in the cells used for
analysis. The series resistance was compensated (70-80%) and
monitored throughout the course of the experiment. If the series
resistance could not be compensated or if it changed by >20%, the
cell was discarded. Input capacitance of the cells included in this
study ranged between 15 and 33 pF and the time constant of the capacity
current transient between 0.18 and 0.31 ms. Drugs were applied locally
by means of a puffer pipette with a relatively wide tip (10 µm), made
of a pulled borosilicate glass capillary and placed with a second
micromanipulator to a distance of 100-200 µm of the cell. The
application was made either by gravity or pressure pulses (10 psi)
controlled with a picospritzer device (General Valve).
Single-cell reverse transcriptase-polymerase chain reaction (single-cell RT-PCR)
The presence of Kv3s mRNAs in dissociated GP cells was
determined by single-cell RT-PCR. This was performed as described by Vega-Saenz de Miera et al. (1997) with the following
modifications: the solutions were prepared in double-distilled
RNase-free water from Sigma. The content of individual cells was
collected from the recording chamber using gentle suction with a wide
tip glass micropipette filled with 3 µl of a solution containing 150 mM KCl, 30 mM Tris-HCl, pH 8.3, and 10 U of Promega RNase inhibitor. The tip of the pipette was broken inside a 500-µl Eppendorf tube, and
its contents emptied. The tubes were placed in dry ice-ethanol bath to
freeze the contents and then stored at
70°C until use.
For reverse transcription, 17 µl of a solution containing 1.18 mM dNTP, 3.82 mM MgCl2, 50 U of Moloney Murine Leukemia Virus RNase H minus reverse transcriptase (Gibco-BRL), 4.4 µM random hexamer primer (Pharmacia), 20 U/ml of RNase inhibitor, 30 mM KCl, and 6 mM Tris-HCl pH 8.3, were added to the Eppendorf tube containing the neuron's contents. Mineral oil (50 µl) were laid on top of the aqueous solution and the tubes incubated successively at 25°C for 5 min, 37°C for 15 min, and 42°C for 15 min. The tubes then were heated at 94°C for 5 min and then cooled to 0°C until used for PCR amplification.
For PCR amplification, 75 µl of a solution containing 50 mM KCl, 0.85 mM MgCl2, 1.7 µM of the sense and antisense external primers, and 10 mM Tris-HCl, pH 8.3 were added to the tubes containing the reverse-transcribed DNA. The tubes were heated for 5 min at 94°C. While the tubes were at 94°C, 5 µl of a solution containing 2.5 units of Perkin Elmer Taq polimerase, 50 mM KCl, and 10 mM Tris-HCl pH 8.3 were introduced through the oil. The tubes then were subjected to 35 cycles of denaturalization at 94°C for 1 min, annealing at 48°C for 1 min, and extension at 72°C, for 1 min followed by one final incubation at 72°C for 7 min. One microliter of a 1:1 dilution of the previous reaction was used as template for the second PCR reaction in a new PCR tube. 94 µl of a solution containing 2.63 mM MgCl2, 210 µM dNTPs, 50 mM KCl, 10 mM Tris-HCl pH 8.3, and 160 nM of the specific primers were added to these tubes. Oil (50 µl) was laid on top and the tubes heated and Taq polimerase added as described in the preceding text. The tubes were subjected to 35 cycles of PCR amplification (1 min each of denaturalization, annealing and extension at 94, 55, and 72°C, respectively) followed by a last extension period at 72°C for 7 min. The PCR products were analyzed by electrophoresis in 2% agarose gels.
The external degenerate primers, designed to amplify all Kv3 sequences, from Drosophila to man, used in the first PCR reaction, had the following sequences: sense primer, CTC GAA TTC I TT(C/T) TG(C/T) (C/T)TN (A/G)A(A/G) ACN CA; antisense primer, CTCGAATTC GGA (A/G)TA (A/G)TA CAT N(C/G)C (G/A)AA (G/A)TT. The sequence for the specific primers was: Kv3.1, sense primer: CGC TTC AAC CCC ATC GTG AAC (position 1801-1821 in Accession No. M68880); antisense primer: GTG TGT GTG TTC GCT GGC GCT (2290-2310). The size of the expected product is 532bp. Kv3.2, sense primer CC AGC GCT GTT CTC CAG TAT (882-901 in No. M34052); antisense primer: C AAT GGG GAT GTT TTT GAA CTG (1358-1337). The size of the expected product is 477 bp. Kv3.4, sense primer: CCT GAT ACG TTG GAC TTT GTC (1312-1333 in No. X62841), antisense primer: ATT GCC CCG TGG GTC AGA T (1629-1647). The size of the expected product is 336 bp.
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RESULTS |
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Expression of Kv3.1 and Kv3.2 proteins in the rat GP
Kv3 mRNAs are not expressed at significant levels before birth
(Goldman-Wohl et al. 1994; Perney et al.
1992
; Vega-Saenz de Miera et al. 1994
; reviewed
in Rudy et al. 1999
). Immunoblots using antibodies
against Kv3.1 (Weiser et al. 1995
) and Kv3.2 (Chow et
al. 1999
) were used to study the postnatal developmental expression of
Kv3.1 and Kv3.2 proteins in membrane extracts from the GP (Fig.
1B). Membranes were prepared
from GP dissected in the same fashion as the tissue used to prepare
dissociated neurons (Fig. 7D). The levels of both proteins
increased significantly after postnatal days 6-8, although it appears
that the expression of Kv3.1 develops somewhat faster than the
expression of Kv3.2. Maximum levels of Kv3.1 protein were seen around
p15 (Fig. 1B, lanes 1-4), whereas for Kv3.2, maximum levels
were not achieved until p20 (Fig. 1B, lanes 5-7). Both
proteins were not detectable earlier than p6-7. Similar results were
obtained in three separate experiments with each antibody.
|
The bands observed with Kv3.1 or Kv3.2 antibodies are not seen when the
immunoblots are reacted with antibodies preincubated with an excess of
the Kv3.1 (Weiser et al. 1995) or Kv3.2 peptides (Fig.
1A, lane 2) used to prepare the antisera. Also, no bands are
detected in immunoblots treated with the preimmune sera derived from
the rabbit used to raise the Kv3.2 antibodies (Fig. 1A, lane 3). Moreover, the Kv3.2 protein is absent in membranes derived from
Kv3.2
/
mice (Fig. 1A, lane 5) but is present in
membranes from Kv3.1
/
mice (Fig. 1A, lane 4), whereas
the reverse is seen with Kv3.1 antibodies (Fig. 1A, lanes 6 and 7).
Immunohistochemistry was used to study the cellular and subcellular localization of Kv3.1 and Kv3.2 proteins in the rat GP (Fig. 2). Antibodies against Kv3.1 produced strong staining of the somatic membrane and the cytoplasm immediately beneath the membrane of neurons located throughout the GP (Fig. 2A). Fewer stained cells were seen in the most lateral border of the nucleus. The same section shown in Fig. 2A was stained with antibodies against parvalbumin (Fig. 2B). Most neurons positive for Kv3.1 also are stained for parvalbumin and vice versa. As in other neurons expressing Kv3.1, there is little staining of the dendrites of pallidal neurons. There was a faint staining of the pallidal neuropile (Fig. 2A). Higher magnification images from another experiment confirmed that Kv3.1b proteins and parvalbumin are expressed in the same pallidal neurons (Fig. 2, C-E), although parvalbumin staining tends to occupy most of the cytoplasm, whereas Kv3.1b staining is stronger in the proximity of the membrane. Antibodies against Kv3.2 also were expressed in parvalbumin-containing neurons (Fig. 2, F-H), although Kv3.2 antibodies produced a somewhat stronger staining of the neuropile than the antibodies against Kv3.1. Background staining is observed when the sections are treated with antibodies preincubated with the corresponding immunogenic peptide (data not shown). Kv3.1 and Kv3.2 antibodies also stain the GP in mouse (Fig. 3, A and D). Kv3.1 or Kv3.2 staining is absent in tissue derived from the corresponding knockout mice (Fig. 3), confirming the specificity of both antibodies for immunohistochemistry.
|
|
The immunohistochemical data (Fig. 2) suggest that both Kv3.1 and Kv3.2
are co-expressed in the same pallidal neurons, the projecting,
parvalbumin-containing neurons, which are the main neuronal population
in the GP (Hontanilla et al. 1994; Kita
1994
; Rajakumar et al. 1994a
,b
; Riedel et
al. 1998
). Kv3 proteins form heteromultimeric channels in vitro
with other Kv3 proteins but not with proteins of other subfamilies
(K. McCormack et al. 1990
; Vega-Saenz de Miera et
al. 1994
; Weiser et al. 1994
). This is similar
to what has been observed with other Kv proteins (Coetzee et al.
1999
). Because both Kv3.1 and Kv3.2 proteins are expressed in
the same pallidal neurons, it is likely that both proteins are part of
the same heteromeric channels. To test this hypothesis, we used
immunoprecipitation assays from pallidal membranes solubilized with
nondenaturing detergents (Sheng et al. 1993
; Wang
et al. 1993
). As shown in Fig. 1C, antibodies
against Kv3.1 or Kv3.2 proteins immunoprecipitate both Kv3.1 and Kv3.2
proteins. Thus immunoblots with Kv3.1 antibodies stain Kv3.1 proteins
immunoprecipitated with Kv3.1 (Fig. 1C, lane 1) or Kv3.2
antibodies (Fig. 1C, lane 2); and immunoblots with Kv3.2
antibodies identify Kv3.2 proteins in Kv3.1 (Fig. 1C, lane
6) and Kv3.2 (Fig. 1C, lane 5) immunoprecipitates. No
channel protein is detected when the immunoprecipitation is done with
antibodies preincubated with the corresponding immunogenic peptide
(Fig. 1C, lanes 3 and 4 and 7 and 8). The
coimmunoprecipitation studies demonstrate that heteromeric complexes of
both types of channel proteins exist in pallidal membranes. Taken
together the data suggest that parvalbumin-containing projecting
pallidal neurons have heteromeric channels containing both Kv3.1 and
Kv3.2 subunits.
Kv3.1 and Kv3.2 currents in transfected CHO cells
To compare the properties of the currents recorded from CHO cells
transfected with Kv3.1 or Kv3.2 cDNAs with putative Kv3 currents in
freshly dissociated pallidal neurons, all cells were recorded with
identical intra- and extracellular solutions. CHO cells transfected
with cDNAs encoding Kv3.2a or Kv3.1b proteins had large delayed
rectifier-type voltage-dependent K+ currents
(Fig. 4) that resembled the currents
observed in Xenopus oocytes injected with Kv3.2a or Kv3.1b
cRNAs (reviewed in Vega-Saenz de Miera et al. 1994).
Both Kv3.2a or Kv3.1b currents (see Fig. 4, A and
C, respectively) start activating when the membrane is depolarized to potentials more positive than
10 mV and rise
relatively fast (as compared with other voltage-gated
K+ channels) (see Coetzee et al.
1999
), with a similar time course, to a maximum level that is
maintained for the duration of the pulses used in this experiment. A
slow inactivation becomes evident with pulses of longer duration (data
not shown). Untransfected CHO cells had negligible outward currents
under the same pulse protocols (<100 pA for the largest
depolarizations).
|
Preliminary studies with pallidal neurons showed that a large
proportion of the outward current in these cells could be suppressed by
holding the membrane at depolarized potentials. This could be a useful
strategy to eliminate a fraction of the potassium currents if Kv3
currents are not affected by such treatment. We therefore tested the
effect of varying the holding potential on Kv3.1 and Kv3.2 currents
expressed in isolation. Figure 4 compares the currents produced by a
series of voltage steps applied from a holding potential of 80 or
40 mV in the Kv3.1- and Kv3.2-transfected CHO cells. As seen in these
representative examples, the currents generated by the depolarizing
test pulses are very similar in magnitude and kinetics whether the cell
is held at
80 or
40 mV. Changing the holding potential to
40 mV
suppressed Kv3.1 and Kv3.2 currents by only 9.3 ± 0.1%
(mean ± SE; n = 4) and 9.8 ± 0.6%
(n = 4), respectively, with no effect on current kinetics.
Kv3.1 and Kv3.2 currents in CHO cells also have a similar voltage
dependence, as observed in Xenopus oocytes (reviewed in Vega-Saenz de Miera et al. 1994). Figure 4, E
and F, shows the normalized conductance
(G/Gmax) as a function of
voltage, obtained from several cells expressing Kv3.2 or Kv3.1
currents, respectively. The data were fitted to Boltzmann functions of
the form G/Gmax = 1/[1
exp(Vm
V1/2)/k]. From these fits, we
derived a midpoint of activation of
V1/2 = 12.1 ± 1.26 mV
(n = 6) for Kv3.2 currents and 18.1 ± 1.01 mV
(n = 6) for Kv3.1 and a steepness parameter k of 8.4 ± 0.25 mV for Kv3.2 and 11.0 ± 0.2 mV
for Kv3.1. Kv3 currents are unusual in requiring very depolarized
potentials to start activating. However, the midpoints of the
conductance-voltage relationships of Kv3 channels are not that
different from those of other voltage-dependent
K+ currents, reflecting a steep voltage
dependence. This distinguishes mammalian Kv3 currents from the currents
expressed by the Drosophila Shaw protein, which also starts
activating at high voltages but has a very weak voltage dependence,
producing a midpoint of activation above +70 mV (Johnstone et
al. 1997
; Smith-Maxwell et al. 1998
).
Another unusual feature of Kv3 currents is the fast rate of
deactivation on repolarization, first described for Kv3.1 currents expressed in NIH-3T3 and L929 cells by Grissmer et al.
(1994). These authors found that Kv3.1 currents deactivated
~10 times faster than several other cloned mammalian voltage-gated
K+ channels when compared at the same membrane
potentials. Since then, many new voltage-gated channels have been
identified in mammals; however, only one of them, Kv1.7, a nonneuronal
member of the Kv1 family, deactivates fast (closing rates are ~3
times the deactivation rates of Kv3.1 channels) (Coetzee et al.
1999
). Kv3.1 currents also deactivate extremely fast in CHO
cells under our recording conditions (Fig.
5). Kv3.2 currents deactivate at rates
somewhat slower than Kv3.1 but still significantly faster than
K+ currents from channels of other subfamilies
(Fig. 5).
|
At present there are no specific blockers for Kv3.1 and Kv3.2 channels.
However, in Xenopus oocytes all Kv3 currents are blocked by
low concentrations of TEA or 4-aminopyridine (4-AP) (reviewed in
Vega-Saenz de Miera et al. 1994). Kv3.1 and Kv3.2
currents in CHO cells, under our recording conditions, were also very
sensitive to these channel blockers. TEA dose-response curves for Kv3.1 and Kv3.2 currents are shown in Fig. 6.
From these curves, we derived IC50s of 0.28 mM
(n = 4) for Kv3.2 and 0.38 mM (n = 4) for Kv3.1 currents. We also have confirmed that as in other
experimental systems (Grissmer et al. 1994
;
Vega-Saenz de Miera et al. 1994
), Kv3.1 and Kv3.2
currents in CHO cells are not affected by dendrotoxin or charybdotoxin
at concentrations of
1 µM (data not shown). The effects of 4-AP on
these currents were not examined in detail. Preliminary experiments
discarded 4-AP as a useful tool to distinguish native currents carried
by channels of the Kv3 subfamily in pallidal neurons because the drug
blocks, also at low concentrations, a component of the outward current
that also is blocked by dendrotoxin.
|
The currents obtained when both Kv3.1 and Kv3.2 proteins are
co-expressed in Xenopus oocytes (Vega-Saenz de Miera
et al. 1994; Weiser et al. 1994
) or CHO cells
(data not shown) are similar to those obtained in cells expressing only
one of the two subunits. This is not surprising given the similarities
between Kv3.1 and Kv3.2 currents and is consistent with the observation
that heteromultimeric Kv channels have properties intermediate between
those of the corresponding homomultimeric channels (Christie et
al. 1990
; Isacoff et al. 1990
; K. McCormack et al. 1990
; Ruppersberg et al. 1990
; Weiser et al. 1994
). The properties of Kv3.1 and Kv3.2
currents in CHO cells are summarized in Table
1.
|
K+ currents in acutely dissociated neurons from the rat GP
As a product of the enzymatic dissociation of the GP, we typically
found two morphological subtypes of neurons (Fig.
7), similar to those observed in previous
studies of dissociated pallidal neurons (Stefani et al. 1992,
1995
; Surmeier et al. 1994
). We also found
astrocytes and a population of small cells (data not shown). All our
records were obtained from the two main types shown in Fig. 7. Most
neurons (type A, Fig. 7, A and B) had
bipolar-fusiform or triangular shape and were similar in size and
morphological appearance to pallidal GABAergic projecting neurons
(Park et al. 1982
; Surmeier et al. 1994
).
The second type, much less frequently encountered (type B, Fig.
7C), consisted of multipolar cells that were distinguished
mainly by having somas significantly larger than those of type A cells.
These cells may correspond to the large cholinergic neurons that lie
along the medial border of the GP (Surmeier et al.
1994
), but no evidence of this was obtained in this study.
|
It was also possible to group the dissociated GP neurons according to
the amount and kinetics of the transient currents observed when the
cells were depolarized from a holding potential of 80 mV. Figure
8 shows records from three different
neurons at two holding potentials. The cell shown in A has
very little low-voltage activating A-type current, whereas this current
is large in the cells shown in C and E. In
addition, the cell shown in E has a substantial amount of a
slowly inactivating A-type current similar to the
IAs described in pallidal neurons by
Stefani et al. (1995)
. The records in Fig. 8 also show
that a holding potential of
40 mV (Fig. 8, B, D, and
F) inactivates not only the transient currents but also a
substantial portion of the sustained current. We did not observe a
clear correlation between the phenotype of the currents at a holding
potential of
80 mV and the cell's morphology.
|
The experiments described next, aimed at searching for Kv3.1- and Kv3.2-like currents in dissociated pallidal neurons, asked whether in these cells there is a component of the potassium current whose kinetics, voltage dependence, and pharmacology resembles those of the currents recorded under the same experimental conditions in CHO cells expressing Kv3.1 or Kv3.2 proteins. The results will be presented in two parts each comprising results obtained from one of two distinct subpopulations of GP neurons, distinguishable by their morphology and electrophysiological characteristics: type A and type B cells. As indicated in METHODS, identical intra- and extracellular solutions to those used to record currents in CHO cells were used in pallidal neuron recordings.
Type A GP neurons
This group was composed of cells with fusiform/bipolar and
triangular/multipolar somata (such as those shown in Fig. 7,
A and B). Typical records obtained from a cell
with these morphological characteristics are shown in Fig.
9. Figure 9A shows a family of
currents obtained during depolarizing pulses from a holding potential
of 40 mV, and Fig. 9B illustrates the currents obtained in
the same cell after the application of 1 mM TEA to the external solution. The studies with Kv3.1 and Kv3.2 currents in heterologous expression systems indicate that such a concentration of TEA should eliminate an important fraction (~80%) of the current generated by
Kv3 channels. A holding potential of
40 mV was used to reduce the
components of the total K+ current and facilitate
the isolation of Kv3-like currents, which are not significantly
affected by holding the membrane at this potential (see Fig. 4).
|
The currents recorded under control conditions
(IGPc) begin to activate at about 20
mV and have characteristics of delayed rectifying currents. The
TEA-resistant current component (IGP,
R) also possesses characteristics of a sustained current of
the delayed rectifier-type, although its activation kinetics is slower
than that of IGPc. Figure
9C shows the current component sensitive to TEA
(IGP, TEA), obtained by digital
subtraction of the current traces shown in B from the
corresponding traces in A. In type A cells,
IGP, TEA is also a sustained current
of the delayed rectifier type that represents in this cell ~50% of
the total current from a holding potential
(VH) of
40 mV. Typically the
activation kinetics of IGP, TEA was
faster than that of IGP, R (compare
Fig. 9, B and C). It is also apparent from the
records shown in Fig. 9, B and C, that the
TEA-resistant and -sensitive components of the current in this cell
also differ in their rate of deactivation at
40 mV. The deactivation
of IGP, R is slow as compared with the
rate of deactivation of IGP, TEA
(
deact = 25.64 ± 3.38 ms (n = 9) for
IGP, R and 2.72 ± 0.24 ms
(n = 6) for I GP,
TEA). While the proportion of IGP,
R and IGP, TEA varied
among type A cells (IGP, TEA ranged
between 30 and 85% of total current at a
VH of
40 mV, with a mean of 54%),
the kinetic features illustrated here were reproducibly observed.
Figure 9D shows the normalized conductance
(G/Gmax) for
IGP, TEA. This component of the
current starts activating between 10 and
20 mV. The continuous line
represents the fit of the data to a Boltzmann equation with a
V1/2 and k of 16.94 mV and 10.59 mV
1, respectively (n = 6).
These data show that the component of the outward current from type A
cells that is blocked by 1 mM TEA resembles in voltage dependence as
well as in activation and deactivation kinetics the currents carried by
Kv3.1 and Kv3.2 channels.
To explore this conclusion further, we compared more closely the kinetics of activation and deactivation of IGP, TEA with the kinetics of Kv3.1 and Kv3.2 currents in CHO cells. Figure 10 shows current records obtained with identical pulse protocols in CHO cells transfected with Kv3.1 and Kv3.2 cDNAs (A and B, respectively) and IGP, TEA in a type A GP neuron (C). In D-G, we have scaled and superimposed the first 150 ms of the current traces in A-C, at four different voltages. It is clear from this comparison that the TEA-sensitive component of the K+ current in type A GP neurons has activation kinetics that closely resembles the activation kinetics of Kv3.1 and Kv3.2 currents in CHO cells.
|
IGP, TEA also resembles Kv3.1 and
Kv3.2 currents in deactivation kinetics. Figure
11 examines the kinetics of the tail
current recorded under control conditions in a type A GP neuron. The
tail current that results from repolarizing the membrane potential from
+40 to 40 mV (Fig. 11A) was best fitted by the sum of two exponentials, suggesting that the deactivation process of this current
includes two components with fast and slow time constants (
1 = 1.78 ms and
2 = 21.8 ms, respectively). In contrast, the tail current of the
TEA-insensitive component (Fig. 11B) and the TEA-sensitive
component (Fig. 11C) can be fitted to a single exponential with time constants of 28.8 and 2.27 ms, respectively. These two values
resemble the time constants of the two components seen in the total
current. The time constant of deactivation of the TEA-sensitive current
from a number of experiments (n = 4) is plotted against
the repolarizing membrane potential in Fig. 11D. IGP, TEA deactivates very fast, at rates
similar (and roughly intermediary) to those of Kv3.1 and Kv3.2 currents
in CHO cells. A summary of the comparison of the properties of
IGP, TEA in type A GP neurons with the
properties of Kv3.1 and Kv3.2 currents is shown in Table 1.
|
Type B GP neurons
The characteristics of the currents obtained from GP neurons
described earlier were typical of the majority of the cells studied. However, we found that in a small group of cells, 1 mM TEA blocked a
fast inactivating current. Many of these cells (type B) had a distinct
morphology characterized by large multipolar somas with about four to
five dendrites (such as the cell illustrated in Fig. 7C).
Typical records from one of these cells are shown in Fig.
12. Depolarizing pulses from a holding
potential of 40 mV produced currents of the delayed rectifier type
similar to those seen in type A cells (Fig. 12A), although
the kinetics of activation of these currents was faster than that of
the currents recorded in type A GP neurons (rise time between 10 and
90% at +40 mV of 14.0 ± 1.2 ms, n = 3 for type B
cells and 25 ± 5 ms, n = 6 for type A cells). The
predominant time constant of deactivation of the currents in these
cells is slow (
= 25.86 ± 1.17 ms after a pulse to +40
mV; n = 4). Application of 1 mM TEA inhibited
~10-15% of the current (Fig. 12B). The TEA-resistant
current had slower activation kinetics than the control (10-90% rise
time: 22.4 ± 2.5 ms, n = 3). The TEA-sensitive
component (IGP, TEA), obtained by
digital subtraction (Fig. 12C), was composed predominantly
of a current that activates very rapidly starting at voltages more positive than
10 mV (time to peak at +40 mV of 3.6 ± 0.7 ms, n = 4) and presented marked and fast inactivation that
could be fitted to a single exponential function. Figure 12E
plots the time constant of inactivation of the transient current versus
the potential during the pulse for this cell. Clearly the rate of
inactivation is dependent on the voltage, becoming faster as the
depolarization increases.
|
The TEA-sensitive component in these cells also includes a sustained
component. The ratio of sustained to transient component varied from
cell to cell. It is small in the cell illustrated in Fig. 12 but
represented up to ~40% of the current in some type B neurons. The
TEA-sensitive current in these cells started to activate at very
depolarized potentials (~10 mV) as was the case of the
IGP, TEA in type A cells and Kv3.1 and
Kv3.2 in CHO cells. The normalized peak conductance
(G/Gmax) as a function of
voltage for the fast transient component of several type B neurons is shown in Fig. 12D. The experimental data were fit to a
Boltzmann function with a V1/2 and
k of 15.47 mV ± 0.5 and 14.34 ± 1.0 mV
1, respectively; n = 4).
The transient currents recorded from type B neurons of the GP resemble
the currents expressed by Kv3.4 proteins (Table 1). These are proteins
of the Kv3 subfamily that express fast activating and inactivating
currents resembling Kv3.1 and Kv3.2 in voltage dependence and
pharmacology (Rudy et al. 1991b, 1999
; Schroter et al. 1991
; Vega-Saenz de Miera et al. 1994
).
This result was surprising at first because in situ hybridization
studies reported that Kv3.4 was only expressed at very low levels in a
scattered pattern in the GP (Weiser et al. 1994
).
Because there are no antibodies available against Kv3.4 proteins, we used single-cell RT-PCR to investigate whether type B cells in the GP contain Kv3.4 transcripts. Single-stranded cDNA synthesized from the mRNA obtained from the cytoplasm of several freshly dissociated type A and type B GP neurons was used as template for two rounds of PCR amplification. In the first round, we used a pair of primers designed to amplify the products of all Kv3 genes. This was followed by amplification using internal primers designed to amplify specifically the products of Kv3.1, Kv3.2, and Kv3.4 genes (see details in METHODS). The amplified products obtained when cDNA for each Kv3 gene was used as template are shown in Fig. 13A. Each product has a different molecular weight facilitating the identification of the transcript. Figure 13, B-D, shows the amplified bands obtained with several type A and type B cells. These studies showed that Kv3.2 transcripts are found in most type A (81%; n = 17) and some type B cells (64%; n = 8), Kv3.1 is found mainly in type A cells (85%), and Kv3.4 only in type B cells.
|
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DISCUSSION |
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Key properties of Kv3.1 and Kv3.2 currents expressed in mammalian cell lines
Both Kv3.1 and Kv3.2 cDNAs result in the expression of similar
currents in CHO cells that resemble the currents expressed in
Xenopus oocytes (reviewed in Vega-Saenz de Miera et
al. 1994). These currents have several properties (see Table 1)
that distinguish them from those of other delayed rectifier
K+ channels known (Coetzee et al.
1999
). One property is an activation voltage range that is more
positive than that of other heterologously expressed voltage-gated
K+ channels, besides those of the Kv3 subfamily.
The channels with the nearest activation voltage (Kv2.1 and Kv2.2)
start activating at 10-20 mV more negative potentials. Although Kv3
channel opening starts at high potentials (more positive than
10 mV),
the probability of channel opening increases steeply with voltage, and
>80% of the channels are opened at +30 mV. The currents deactivate
very fast, at rates that are
7-10 times faster (when compared at the same voltage) than those of other known mammalian voltage-gated K+ channels, except for Kv1.7 a nonneuronal
member of the Kv1 subfamily (deactivation rates are 2-3 times slower
than Kv3.1 or Kv3.2) (Coetzee et al. 1999
;
Grissmer et al. 1994
). The rate of rise of the currents
is relatively fast; faster than many other voltage-gated K+ channels (e.g., Kv2.x; Kv1.2) but slower than
that of other voltage-gated K+ channels such as
several Kv1 channels like Kv1.1 and Kv1.5 (Coetzee et al.
1999
). In contrast to other delayed rectifiers, Kv3.1 and Kv3.2
currents are not significantly inactivated by depolarizing prepulses
(see Fig. 4) and do not show cumulative inactivation (Grissmer
et al. 1994
; unpublished observations). These distinctive properties are likely to endow neurons with special
electrophysiological properties (see following text).
Kv3.1-Kv3.2 channels in pallidal neurons
The studies described here provide strong evidence that in
pallidal neurons, Kv3.1-Kv3.2 proteins in heteromultimeric complexes form K+ channels mediating a
high-voltage-activating component of the delayed rectifier current that
closely resembles the currents expressed by these proteins in
heterologous expression systems. The immunohistochemical studies
demonstrated that there is expression of both Kv3.1 and Kv3.2 proteins
in the GP and that both proteins are colocalized in the same cell type,
the triangular and bipolar parvalbumin-containing (PV+) neurons, which
constitute the major neuronal population in the rodent GP (see
following text). Moreover, antibodies against Kv3.1 or Kv3.2 proteins
coprecipitate both subunits, strongly suggesting that the proteins
exist in heteromeric complexes. It remains to be shown, however, that
the functional channels are heteromultimeric. This will be a difficult
task unless major, not yet detected, differences between homomultimeric
and heteromultimeric channels are discovered. We also have demonstrated that in acutely dissociated pallidal neurons having triangular or
bipolar shapes (type A), shown to express Kv3.1 and Kv3.2 transcripts by single cell RT-PCR, a component of the current not inactivated when
the cell is held at 40 mV and blocked by 1 mM TEA, has properties that closely resemble those of Kv3.1 and Kv3.2 channels in CHO cells
(see Table 1).
The concentration of TEA used to isolate the current in pallidal
neurons (1 mM) blocks >80% of Kv3.1 and Kv3.2 currents in heterologous expression systems. This concentration of TEA produces significant inhibition of only a few other known
K+ channels. These include the large conductance
Ca2+-activated K+ channels
containing proteins of the slo family
(Kd 80-330 µM) and Kv1.1 channels
(Kd ~ 0.5 mM) (Coetzee et al.
1999). These channel types are unlikely to contribute to the
current isolated from the pallidal neurons in these studies. In our
experiments, the activation of Ca2+-activated
K+ channels was suppressed by using
Cd2+ in the extracellular solution and by using
BAPTA in the intracellular solution. In the pallidal area Kv1.1
proteins apparently are expressed somatically only in the ventral
pallidum (Wang et al. 1994
). Moreover, Kv1.1 channels
also are blocked by dendrotoxin (Kd ~ 10-20 nM) (Coetzee et al. 1999
). In pallidal
neurons, this toxin blocked a small inactivating current component
(~10-15% of total outward current) resembling a D current
(Wu and Barish 1992
), which was suppressed significantly
by holding the cell at
40 mV. Dendrotoxin also blocks other channels
of the Kv1 subfamily that are not highly sensitive to TEA
(Coetzee et al. 1999
) and may mediate the D-like current
in pallidal neurons.
This study confirms and extends the observations of Du et al.
(1996), who showed that hippocampal interneurons expressing Kv3.1 proteins had a current that showed resemblance to Kv3.1 currents
in heterologous expression systems. Also the l-type current in T lymphocytes was shown to be very similar to Kv3.1 currents in
Xenopus oocytes when recordings in the two preparations were obtained with the same solutions (Grissmer et al. 1992
).
Kv3.1-like currents also have been described in auditory neurons
(Wang et al. 1998
).
It appears from these results that the properties of native channels
containing Kv3.1 and Kv3.2 proteins are not significantly affected by
factors such as associated subunits or postranslational modifications
as might be the case for other cloned subunits, at least in the cells
studied until now. It is therefore valid to ask why were Kv3.1- and
Kv3.2-like currents not described in the many neuronal populations
expressing these proteins prior to the cloning studies? Most likely the
Kv3 channel-mediated current was buried in other components of the
K+ current. This emphasizes the suggestion made
in the introduction that experimental conditions and methods to isolate
individual components of the K+ current tailored
to search for specific current components are required before it is
possible to determine whether native currents resemble those in
heterologous expression systems. In the specific case of pallidal
neurons, the Kv3.1-Kv3.2 current isolated in our study was most likely
buried in the delayed rectifier component (Ik) isolated with 10 mM TEA by
Steffani et al. (1992, 1995
).
Physiological significance
The GP in rodents consist of a main neuronal mass homologous to
the external segment of the GP in primates and often is referred to
simply as the GP. A smaller nuclear group situated at a certain distance and embedded among the fiber bundles of the internal capsule
usually called entopeduncular nucleus (not included in most of our
dissociations) is thought to correspond to the internal segment of the
GP in primates (Heimer et al. 1995). The GP proper contains several neuronal populations, the majority of which are medium
to large neurons (20-40 µm in length along their longer axis) with a
fusiform (bipolar) or triangular shape. There are also small neurons
(12-16 µm in length), which may correspond to the small dissociated
cells that were excluded from the present study, and a few scattered
large multipolar cells located mainly in the medial border in rat,
which may correspond to cholinergic neurons (Difiglia et al.
1982
; Fox et al. 1974
; Heimer et al. 1995
; Iawhori and Mizuno 1981
; Kita 1992
,
1994
; Kita and Kitai 1994
; Millhouse
1986
; Moriizumi and Hattori 1992
; Park et
al. 1982
; Tkatch et al. 1998
). Most pallidal
neurons, including the triangular and fusiform cells, are GABAergic.
Many of these cells stain for parvalbumin, which labels about
two-thirds of projecting pallidal neurons (Hontanilla et al.
1994
; Kita 1994
; Rajakumar et al.
1994a
,b
; Riedel et al. 1998
). According to our
immunohistochemical studies, Kv3.1 and Kv3.2 are present in the PV+
neurons (Fig. 2). Furthermore the morphology of the majority of the
dissociated cells identified as type A in this study corresponds to the
morphology of the PV+ cells in situ. Together with the results from the
single cell RT-PCR, this suggests that the neurons expressing Kv3.1- and Kv3.2-like currents correspond to the projecting GABAergic, PV+,
neurons. The ventral pallidum, which may have contaminated some of our
dissociations, contains similar GABAergic and cholinergic neurons
(Heimer et al. 1995
).
The Kv3.1-Kv3.2 component of the delayed rectifier current in pallidal
neurons represents a significant component of the total K+ current (~50% of the current when the cell
was depolarized from a holding potential of 40 mV) and is therefore
likely to contribute to the firing properties of these cells. Because
Kv3.1-Kv3.2 channels are not opened until the membrane potential is
depolarized beyond
10 mV, it has been suggested that these channels
are activated late in the action potential and, when present in
sufficient amounts, influence action potential repolarization. Thus
Kv3.1-Kv3.2 channels would help dictate action potential duration
without competing much with the Na+ current in
generating the rising phase of an action potential and influencing
firing threshold, in contrast to K+ channels that
are activated earlier during a spike (Lenz et al. 1994
;
Moreno et al. 1995
; Rudy et al. 1999
;
Sekirnjack et al. 1997
; Vega-Saenz de Miera et
al. 1994
; Wang et al. 1998
; Weiser et al.
1995
). These arguments, supported by computer modeling (A. Erisir, D. Lau, B. Rudy, and C. S. Leonard, unpublished data), suggest that high-voltage-activating K+ channels
would modulate firing properties more selectively than K+ channels that activate at more negative voltages.
Many of the neuronal populations expressing Kv3.1 and Kv3.2 channels
fire trains of brief action potentials at high rates (Erisir et
al. 1998; Martina et al. 1998
; Massengill
et al. 1997
; Perney et al. 1992
; Rudy et
al. 1992
; Sekirnjack et al. 1997
; Wang et
al. 1998
; Weiser et al. 1994
, 1995
), such as
fast-spiking interneurons in the cortex and the hippocampus.
Kv3.1-Kv3.2 channels may help maintain high firing rates by keeping
action potentials short, reducing Na+ channel
inactivation, and facilitating fast recovery of
Na+ channels from inactivation by hyperpolarizing
the cell following the spike. Their fast deactivation on repolarization
will quickly eliminate the increase in K+
conductance, and therefore these channels will contribute little to
increasing the refractory period. K+ channels
that are open at lower potentials or do not deactivate as fast could
repolarize the spike but at the same time also limit firing frequency
by contributing to the refractory period. In fact pharmacological
treatments that suppress Kv3 currents impair fast spiking in
neocortical neurons, but blockade of other K+
currents actually increases firing frequency (Erisir et al. 1998
).
Although there has not been a study combining immunohistochemistry and
electrophysiology of pallidal neurons, it is likely that the PV+
pallidal neurons correspond to the repetitive firing group of cells
recorded in an in vivo intracellular study in rats by Kita and
Kitai (1991) because they both represent the largest cell
population and they have similar morphologies (Kita
1994
; Kita and Kitai 1991
, 1994
). These cells,
which probably correspond to the type II neurons observed in
intracellular recordings from guinea pig slices (Nambu and
Llinas 1994
, 1997
), show fast repetitive firing (
200 Hz) with
weak accommodation when depolarized.
It is possible that the role of Kv3.1-Kv3.2 channels in PV+ pallidal neurons is similar to their proposed role in cortical interneurons to facilitate sustained high firing rates. The firing frequency of the repetitive firing cells in the GP is not as high or sustained as that of fast-spiking cortical neurons. Analysis of the currents in the latter cells shows that they have a significantly higher proportion of Kv3-like currents than pallidal neurons and lack subthreshold-activating A-type currents (A. Erisir, D. Lau, B. Rudy, C. S. Leonard, unpublished data). These differences in channel composition may explain the differences in spike frequency adaptation of the two cell types. The resting potential of pallidal neurons will change the contribution of Kv3 currents to the total current, it is therefore also possible that the firing frequency or adaptation of PV+ pallidal neurons will depend on the resting potential.
Expression and role of channels containing Kv3.4 proteins in a small subpopulation of pallidal neurons
The most surprising result of this study was the finding of fast,
transient, high-voltage-activating, TEA-sensitive currents in a small
subpopulation of the dissociated cells. The currents resemble those
expressed by Kv3.4 subunits in heterologous expression systems (Table
1) (see also Vega-Saenz de Miera et al. 1994). The
hypothesis that these transient currents in GP neurons are mediated by
channels containing Kv3.4 proteins is supported by the findings from
single-cell RT-PCR, which showed that Kv3.4 transcripts are present
only in the cells having the high-voltage activating transient current.
We did not expect to find Kv3.4 channels in pallidal neurons because
Weiser et al. (1994)
reported very weak expression of
Kv3.4 transcripts in the GP (in situ hybridization signals for these
mRNAs were reported as "weakly above background"). However,
Weiser et al. (1994)
cautioned in their paper that low expression of Kv3.4 transcripts in neurons expressing other Kv3 proteins could be of physiological significance because Kv3.4 subunits
can form heteromultimeric channels with other Kv3 proteins resulting in
a large amplification of the transient current. Type B pallidal neurons
might be an example of the situation predicted in this paper.
Nevertheless relative to the other outward currents, the Kv3.4-like current in type B pallidal neurons contributes such a small proportion of the total current that one could be tempted to suggest it could play little role in the excitability of these cells. However, although the Kv3.4-like current contributes little current, it produces a large effect on the rise time of the total current. The currents remaining after 1 mM TEA are not very different in magnitude from the original current; however, they are clearly much slower (see Fig. 12). This suggests a new role for Kv3.4-like currents: to accelerate the rate of rise of the repolarizing currents. Further studies of type B pallidal neurons or other cells expressing Kv3.4 channels will allow future tests of this hypothesis.
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ACKNOWLEDGMENTS |
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This research was supported by National Institute of Neurological Disorder and Stroke Grants NS-30989 and NS-35215 and National Science Foundation Grant IBN 9209523 to B. Rudy. A. Hernández-Cruz was awarded Grant 2366PN from the National Council for Science and Technology (CONACyT). R. Hernández-Pineda was awarded a Fundacion UNAM fellowship, a Ph.D. fellowship from CONACyT, and the Direccion General de Asuntos del Personal Academico, UNAM.
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FOOTNOTES |
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Address for reprint requests: B. Rudy, Dept. of Physiology and Neuroscience, New York University School of Medicine, 550 First Ave., New York City, NY 10016.
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 19 October 1998; accepted in final form 29 March 1999.
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REFERENCES |
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