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The Journal of Neuroscience, December 1, 2002, 22(23):10094-10105
Delayed Rectifier K+ Currents,
IK, Are Encoded by Kv2  -Subunits and
Regulate Tonic Firing in Mammalian Sympathetic Neurons
Sacha A.
Malin and
Jeanne M.
Nerbonne
Department of Molecular Biology and Pharmacology, Washington
University School of Medicine, St. Louis, Missouri 63110
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ABSTRACT |
Previous studies have revealed the presence of four kinetically
distinct voltage-gated K+ currents,
IAf,
IAs,
IK, and
ISS, in rat superior cervical
ganglion (SCG) neurons and demonstrated that
IK and ISS are
expressed in all cells, whereas IAf and
IAs are differentially distributed. Previous
studies have also revealed the presence of distinct components of
IAf encoded by  -subunits of the Kv1 and
Kv4 subfamilies. In the experiments described here, pore mutants of
Kv2.1 (Kv2.1W365C/Y380T) and Kv2.2 (Kv2.2W373C/Y388T) that function as
Kv2 subfamily-specific dominant negatives (Kv2.1DN and Kv2.2DN) were
generated to probe the functional role(s) of Kv2 -subunits.
Expression of Kv2.1DN or Kv2.2DN in human embryonic kidney-293 cells
selectively attenuates Kv2.1- or Kv2.2-encoded K+
currents, respectively. Using the Biolistics Gene Gun, cDNA constructs encoding either Kv2.1DN or Kv2.2DN [and enhanced green fluorescent protein (EGFP)] were introduced into SCG neurons. Whole-cell
recordings from EGFP-positive Kv2.1DN or Kv2.2DN-expressing cells
revealed selective decreases in IK.
Coexpression of Kv2.1DN and Kv2.2DN eliminates
IK in most (75%) SCG cells and, in the
remaining (25%) cells, IK density is
reduced. Together with biochemical data revealing that Kv2.1 and Kv2.2
-subunits do not associate in rat SCGs, these results suggest that
Kv2.1 and Kv2.2 form distinct populations of
IK channels, and that Kv2 -subunits
underlie (most of) IK in SCG neurons.
Similar to wild-type cells, phasic, adapting, and tonic firing patterns
are evident in SCG cells expressing Kv2.1DN or Kv2.2DN, although action
potential durations in tonic cells are prolonged. Expression of Kv2.2DN
also results in membrane depolarization, suggesting that Kv2.1- and
Kv2.2-encoded IK channels play distinct
roles in regulating the excitability of SCG neurons.
Key words:
K+ channels; IK; Kv2.1; Kv2.2; Kv2.1W365C/Y380T; Kv2.2W373C/Y388T; transgenics; Gene Gun; neuronal excitability; repetitive firing patterns
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INTRODUCTION |
Voltage-gated
K+ channels are important determinants of
neuronal membrane excitability, and differences in
K+ channel expression patterns and
densities contribute to the variations in action potential waveforms
and repetitive firing patterns evident in different neuronal cell types
(Pongs, 1999 ). Electrophysiological studies have revealed that most
mammalian neurons express multiple types of voltage-gated
K+ currents with distinct time- and
voltage-dependent properties (Rudy, 1988; Storm, 1990). A large number
of voltage-gated K+ (Kv) channel
pore-forming () and a variety of Kv accessory subunits, thought to underlie these channels have now been identified (Coetzee et
al., 1999; Pongs, 1999; An et al., 2000; Kuryshev et al., 2000), and
there is presently considerable interest in defining the relationships between these subunits and neuronal voltage-gated
K+ currents.
We have shown previously that sympathetic neurons isolated from the rat
superior cervical ganglion (SCG) express four kinetically distinct
voltage-gated K+ currents: two transient
A-type currents, IAf and
IAs, a delayed rectifier current,
IK, and a steady-state current,
ISS (Malin and Nerbonne, 2000).
Although IK and
ISS are expressed in all SCG cells,
the transient currents, IAf and
IAs, are differentially distributed,
and SCG neurons were classified as type I
(IAf,
IK, ISS), type II
(IAf,
IAs,
IK,
ISS) or type III
(IK,
ISS) based on the differential
expression of these voltage-gated K+
currents (Malin and Nerbonne, 2000). In previous studies, we exploited
molecular genetic strategies to assess the roles of -subunits of the
Kv1 and Kv4 subfamilies in the generation of voltage-gated
K+ currents in rat SCG cells. Recordings
obtained from SCG neurons expressing the Kv4 subfamily-specific
dominant negative Kv4.2W362F (Barry et al., 1998) revealed that
IAf is eliminated in most type I and
all type II SCG cells (Malin and Nerbonne, 2000). Subsequent experiments revealed that expression of a Kv1 subfamily-specific dominant negative Kv1.5W461F (Li et al., 1999) eliminates
IAf in a subset of type I cells (Malin
and Nerbonne, 2001), demonstrating that there are two molecularly
distinct components of IAf. However, the molecular correlates of IK,
IAs, and
ISS in SCG cells have not been identified.
Previous studies suggest that -subunits of the Kv2 subfamily encode
neuronal delayed rectifier currents,
IK (Baranauskas et al., 1999;
Murakoshi and Trimmer, 1999; Du et al., 2000; Blaine and Ribera, 2001).
In addition, both Kv2.1 and Kv2.2 are readily detected in mammalian
hippocampal neurons, although these subunits are differentially
targeted (Hwang et al., 1992, 1993; Maletic-Savatic et al., 1995; Du et
al., 1998; Lim et al., 2000). In rat SCG neurons, Kv2.1 and Kv2.2 are
expressed at the mRNA level (Dixon and McKinnon, 1996; Pankevych et
al., 1999). In the experiments described here, Kv2 subfamily-specific
dominant negative constructs, Kv2.1W365C/Y380T (Kv2.1DN) and
Kv2.2W373C/Y388T (Kv2.2DN), were generated, characterized, and
introduced into SCG neurons to test directly the hypothesis that Kv2
-subunits underlie IK and to probe
the functional roles of Kv2-encoded K+
channels in shaping the waveforms of individual action potentials and
in regulating repetitive firing in these cells.
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MATERIALS AND METHODS |
Construction of Kv2.1W365C/Y380T and
Kv2.2W373C/Y388T. Rat Kv2.1 (obtained from R. Joho, University of
Texas Southwestern, Dallas, TX) was subcloned into the pAlter
vector (Promega, Madison, WI), and two pore mutations
(W365C/Y380T) were introduced using the Altered Sites Mutagenesis II
(pAlter) system (Promega). Codons TGG (W365) and TAC (Y380) were
mutagenized to TGC (C) and ACC (T). To aid in the detection of the
mutant construct, the FLAG epitope was added to the C terminus
of Kv2.1W365C/Y380T by PCR. The corresponding mutations (W373C/Y388T)
were also introduced into rat Kv2.2 (obtained from S. Trimmer, State
University of New York, Stonybrook, NY) in pAlter using PCR. Codons TGG
(W373) and TAC (Y388) were mutagenized to TGC (C373) and ACC (T388). The myc epitope tag was added on the C terminus of
Kv2.2W373C/Y388T by PCR, and Kv2.2W373C/Y388T-myc was cloned
into the pEF6/v5His vector (Promega). Both the Kv2.1W365C/Y380T and the
Kv2.2W373C/Y388T constructs were sequenced in their entirety to ensure
that no additional mutations were introduced.
Expression studies in human embryonic kidney-293 cells.
The functional properties of Kv2.1W365C/Y380T and
Kv2.2W373C/Y388T were examined in heterologous expression studies in
human embryonic kidney (HEK)-293 cells. HEK-293 cells were maintained
in growth medium [Eagle's Minimal Essential Medium (EMEM)
(Invitrogen, San Diego, CA), containing 10% heat-inactivated fetal
calf serum (FCS) and 100 U/ml penicillin-streptomycin], split, plated
in 35 mm tissue culture dishes, and transfected with wild-type and
mutant K+ channel -subunits [and
enhanced green fluorescent protein (EGFP)] using the calcium phosphate
method. For this purpose, cells were preincubated for 1 hr in
transfection media (EMEM with 5% serum). In most experiments, 2-3
µg of test DNA (1:2 ratio of EGFP to test constructs) and 7-8 µg
of carrier DNA (pSK; total of 10 µg of DNA) were combined in
100 µl of 0.25 M CaCl2,
and 100 µl of N,N-bis[2-hydroxyethyl]2-aminoethanesulfonic acid
(BES)-buffered saline [containing in mM: 280 NaCl, 1.5 Na2HPO4·7H2O,
50 BES (Sigma, St. Louis, MO) at pH 6.95] was then added. The
resulting solution was mixed and incubated at room temperature for 15 min before being added (drop-wise) to the cells in each 35 mm culture
dish. After 15 hr of incubation at 37°C, cells were washed in growth medium. Electrophysiological recordings were obtained from
EGFP-positive cells 12-36 hr later. In these experiments, cells were
transfected with EGFP and the following: Kv2.1, Kv2.2,
Kv2.1W365C/Y380T, Kv2.2W373C/Y388T, Kv2.1 plus Kv2.1W365C/Y380T, Kv2.1
plus Kv2.2W373C/Y388T, Kv2.2 plus Kv2.2W373C/Y388T, Kv2.2 plus
Kv2.1W365C/Y380T, Kv1.4, or Kv1.4 plus Kv2.1W365C/Y380T. To evaluate
the functional efficacy of the mutant constructs, current densities
obtained from cells transfected with the wild-type Kv-subunit (and
EGFP) constructs were compared with those from cells transfected with
wild-type and mutant Kv-subunit (and EGFP) constructs. These
experiments were performed in parallel. In most experiments, the
wild-type and mutant Kv-subunits were used at a 1:1 ratio, and the
same absolute amount of wild-type cDNA was used in control and
experimental conditions. In addition, however, some experiments were
performed with higher mutant to wild-type Kv-subunit cDNA ratios
(5:1; mutant to wild-type cDNAs) to assess the efficacy of the mutant constructs in attenuating wild-type K+
current amplitudes. In these experiments, the amount of carrier DNA
(pSK) was reduced to maintain the total amount of DNA per 35 mm dish at
10 µg.
Isolation and in vitro maintenance of SCG
neurons. Sympathetic neurons were isolated from the SCGs of
embryonic day 21 to postnatal day 1 Long-Evans rat pups using a
procedure similar to that described previously by Chang et al. (1990).
Briefly, after anesthesia with 5% halothane, animals were decapitated, and the SCGs were removed. Ganglia were successively incubated for 30 min periods in collagenase and trypsin at room temperature, and
isolated SCG neurons were obtained by trituration and subsequent centrifugation. Dissociated SCG cells were resuspended in growth medium
(EMEM with 10% FCS, 0.14 mM
L-glutamine, 100 U/ml penicillin-streptomycin, and 0.05 mM NGF) and plated at a density of
2.5 × 104/cm2 on
glial monolayers [prepared as described by Raff et al. (1979)]. Cells
were maintained in a 95% O2/5%
CO2 37°C incubator, and the medium was
exchanged with fresh growth medium approximately every 48 hr.
Antibodies. The monoclonal anti-Kv2.1 antibody used here was
raised against residues 853-857 in the C terminus of Kv2.1 (Trimmer, 1991) and was obtained from Upstate Biochemicals, Inc. (Uppsala, NY).
The polyclonal Kv2.2-specific antibody, targeted against residues
771-788 in the C-terminal region of Kv2.2, was obtained from Dr. S. Trimmer (State University of New York, Stonybrook). A mouse monoclonal
anti-FLAG antibody (Eastman Kodak, Rochester, NY) was used to detect
Kv2.1W365C/Y380T-FLAG expression and a mouse monoclonal anti-myc
antibody (Calbiochem, La Jolla, CA) was used to detect
Kv2.2W373C/Y388T-myc in transfected SCG neurons. For
immunohistochemistry, cells were fixed in 4% paraformaldehyde for 30 min, incubated in blocking buffer (PBS containing 5% normal goat
serum, 0.02% Triton X-100, and 0.1% NaNH3) for
1 hr, and exposed to one of the Kv2 -subunit-specific primary,
anti-FLAG, or anti-MYC antibodies (1:500 dilution) at 4°C overnight.
After washing with PBS, cultures were incubated either with a
biotinylated goat anti-rabbit or donkey anti-mouse antibody (1:200
dilution; Chemicon) or with a Cy3-conjugated rabbit anti-mouse IgG
secondary antibody (Chemicon) for 1 hr at room temperature. After wash, cultures were then exposed to avidin-conjugated horseradish peroxidase (HRP) for 1 hr at room temperature (ABC kit; Vector Laboratories, Burlingame, CA) or visualized directly under epifluorescence
illumination. For detection of HRP, cultures were washed again before
incubation with 0.05% diaminobenzidine in PBS. The progress of the
reaction was monitored under the microscope, and the reactions were
quenched after ~5 min by addition of PBS.
Western blots. Extracts of HEK-293 cells transfected with
wild-type and mutant Kv2.x constructs and rat SCG (and brain) membrane preparations were prepared using methods described in detail previously (Barry et al., 1995; Pond et al., 2000). The protein content of each of
the solubilized samples and the membrane preparations was determined
using a Bio-Rad (Richmond, CA) protein assay kit with bovine serum
albumin as the standard. For Western blot analysis, equal amounts of
proteins were fractionated on 8-15% SDS-PAGE gels and transferred to
polyvinylidene difluoride (PVDF) membranes (Bio-Rad). The PVDF membrane
strips were incubated in 0.2% I-Block (Tropix) in PBS containing 0.1%
Tween 20 (blocking buffer) for 1 hr at room temperature, followed by
overnight incubation at 4°C with the monoclonal anti-Kv2.1 or the
polyclonal anti-Kv2.2 antibody at dilutions of 1:500 or 1:100,
respectively. After washing, membrane strips were incubated for 2 hr at
room temperature with alkaline phosphatase-conjugated goat anti-rabbit
or anti-mouse IgG (Tropix) diluted 1:5000 in the blocking buffer, and
bound antibodies were detected using the CPSD
chemiluminescent alkaline phosphate substrate (Tropix).
Immunoprecipitations. For each immunoprecipitation
experiment, the precipitating antibody (0.1 µg) and 50 µl of
equilibrated protein-A Sepharose beads (Sigma) were added to 100 µl
of the HEK-293 or SCG (or brain) protein preparation (prepared as
described above) and mixed (by inversion) at 4°C overnight. Eluted
proteins were fractionated by SDS-PAGE, transferred to PVDF membranes, and immunoblotted with the monoclonal anti-Kv2.1 antibody or the polyclonal anti-Kv2.1 antibody as described above. After exposure to
the appropriate (anti-mouse or anti-rabbit) secondary antibody, protein
bands were visualized by enhanced chemiluminescence as described above.
Transfection of isolated SCG neurons. In control
experiments, 1.6 µm gold beads were coated with pCMV-EGFP (Clontech,
Palo Alto, CA) and propelled (450 psi; 2 mm carrier distance) into SCG
neurons at 4 d in vitro using the Biolistics Gene Gun
(Bio-Rad). After transfections, the cultures appeared healthy, and
expression of EGFP was readily detected 24 hr later. In experiments
aimed at examining the effects of Kv2.1, Kv2.1W365C/Y380T,
Kv2.2W373C/Y388T, or Kv2.1W365C/Y380T plus Kv2.2W373C/Y388T expression
in SCG neurons, the gold particles were coated with pBK-CMV-Kv2.1 and
pCMV-EGFP (4:1 ratio); pAlter-CMV-Kv2.1W365C/Y380T and pCMV-EGFP (4:1
ratio); pEF6/v5His-EF6-Kv2.2W373C/Y388T and pCMV-EGFP (4:1 ratio); or pAlter-CMV-Kv2.1W365C/Y380T, pEF6/v5His-EF6-Kv2.2W373C/Y388T, and
pCMV-EGFP (2:2:1 ratio). In all cases, a total of 10 µg of DNA was
used in coating the beads. Because EGFP expression was used to identify
transfected neurons for subsequent electrophysiological characterization, immunohistochemical experiments using the anti-FLAG and anti-MYC antibodies were also performed to determine whether EGFP-expressing cells also expressed the test constructs.
Electrophysiological recording. Whole-cell recordings were
obtained from HEK-293 cells and from isolated SCG neurons 24-48 hr
after transfection. Experiments were performed at room temperature (22-25°C), and data were collected using an Axopatch-1B patch-clamp amplifier interfaced to a P5-120 Gateway2000 computer through a
Digidata 1200 and using the pClamp7 software package (Axon Instruments, Foster City, CA). Electrodes were fabricated from soda-lime glass (Chase 2502) with a two-stage puller, and the shanks were coated with a
silicone elastomer (Sylgard; Dow Corning, Corning, NY). Pipette
resistances were 1.5-3 M after fire-polishing. For voltage-clamp recordings, the bath solution routinely contained (in
mM): 140 NaCl, 4 KCl, 2 CaCl2, 2 MgCl2, 10 HEPES, 5 glucose, 0.001 TTX, and 0.1 CdCl2, pH 7.5, 300 mOsM. In current-clamp experiments on isolated SCG neurons, the TTX and
CdCl2 were omitted from the bath. The pipette
solution for both current- and voltage-clamp recordings contained (in
mM): 135 KCl, 10 HEPES, 5 glucose, 3 MgATP, 0.5 NaATP, 2 EGTA, and 1.1 CaCl2, pH 7.5, 300 mOsM.
Series resistances, estimated from the decays of the uncompensated
capacitative transients, were 2-5 M and were compensated
electronically by ~80-90%. Because current amplitudes were <10 nA,
the voltage errors resulting from the uncompensated series resistance
were always <10 mV and were not corrected.
In most experiments, voltage-gated K+
currents were evoked during 125 msec or 6 sec depolarizing voltage
steps to test potentials between  40 mV and +50 mV from a holding
potential of 90 mV. To determine the activation profiles of the
Kv2.1- and Kv2.2-encoded K+ currents in
HEK-293 cells and in rat SCG neurons under physiological conditions,
experiments were also performed in which the voltage clamp was driven
by average action potential waveform. Because SCG neurons display
variable action potential waveforms and repetitive firing patterns
(Malin and Nerbonne, 2000) and have been classified as phasic, tonic,
and adapting, the activation profiles of the currents were examined in
cells driven by each of these (action potential) phenotypes. In these
experiments, action potentials recorded from representative phasic,
tonic, and adapting cells were averaged and provided as the input to
the voltage clamp. Outward K+ currents
recorded in Kv2.1- and Kv2.2-transfected HEK cells and in isolated rat
SCG neurons in response to these action potential clamps were then
recorded and analyzed. Single action potentials and action potential
trains in isolated SCG neurons were recorded in response to brief (1.5 msec) 200-400 pA and prolonged (500 msec) 20-200 pA depolarizing
current injections.
Data analysis. Data were compiled and analyzed using pClamp7
(Axon Instruments) and Excel (Microsoft, Redmond, WA) software and are
presented as means ± SEM. The decay phases of the capacitative transients were analyzed, and only cells in which >90% of the amplitude of the capacitative transient decayed over a single exponential time course were analyzed further. The mean ± SEM input resistance and capacitance of EGFP-expressing SCG neurons were
0.34 ± 0.03 G and 32 ± 2 pF (n = 43),
respectively. Leak currents were <10 pA (at 70 mV) and were not
subtracted; leak conductances (at 70 mV) were always <0.15 nS. To
determine the amplitudes and the decay time constants of the individual
components of the total depolarization-activated outward
K+ currents in SCG neurons, the
inactivation phases of the currents recorded during prolonged (6 sec)
depolarizations were analyzed using the following equation:
y = A1e t/ 1 + A2et/2 + A3et/3 + C, where A1,
A2, and
A3 (measured in picoamperes per
picoFarad) are the amplitudes of the inactivating current components
(IAf, IAs, and
IK) that decay with time constants
 1, 2, and
3 (measured in msec), respectively, and
C is the steady-state current remaining at the end of the 6 sec depolarizations. Fits were obtained using Clampfit6, and best fits
were determined by eye (in all cases,  < 30 pA). All
current-clamp recordings were obtained from cells with overshooting
action potentials and stable resting membrane potentials negative to
40 mV. Action potential durations (APDs) were measured at 50%
(APD50) and 90% (APD90)
repolarization. Statistical significance was examined with the
Student's t test, and, where appropriate, p
values are presented below.
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RESULTS |
Generation and characterization of dominant negative
Kv2 -subunits
In preliminary studies, a single point mutation was introduced
into the pore region of Kv2.1, analogous to the approaches used to
generate the dominant negative Kv4.2 -subunit, Kv4.2W362F (Barry et
al., 1998), and the dominant negative Kv1.5 -subunit, Kv1.5W461F (Li
et al., 1999). However, heterologous expression studies revealed that
this construct produced outward K+
currents indistinguishable from those evident on expression of wild-type (rat) Kv2.1. These results are similar to those described by
Blaine and Ribera (1998) in studies with Xenopus Kv2.2.
Because a functional dominant negative construct was obtained by
introducing two mutations in the pore region of Xenopus
Kv2.2 (Blaine and Ribera, 1998), a similar strategy was used here for
(rat) Kv2.1 to generate a double pore mutant, Kv2.1W365C/Y380T (see
Materials and Methods). The Kv2.1W365C/Y380T construct, Kv2.1DN, was
epitope-tagged at the C terminus with the 8 aa FLAG tag to allow direct
detection of transgene expression.
To determine the functional properties of this construct, HEK-293 cells
were transfected with Kv2.1DN (and EGFP) alone and in combination with
Kv2.1, Kv2.2, or Kv1.4 (in a 1:1 ratio; see Materials and Methods), and
outward K+ currents, evoked in response to
125 msec depolarizing voltage steps to potentials ranging from 40 mV
to +50 mV from a holding potential of 70 mV, were recorded (Fig.
1). These experiments revealed that
mean ± SEM peak outward K+ current
densities (30 ± 7 pA/pF; n = 9) in HEK-293 cells
expressing Kv2.1DN alone (Fig. 1A) are not
significantly different from those recorded from wild-type or
mock-transfected HEK-293 cells (15 ± 6 pA/pF; n = 11). However, when the Kv2.1DN is coexpressed with wild-type Kv2.1,
outward K+ currents are attenuated
markedly compared with those recorded from cells expressing Kv2.1 (and
EGFP) alone (Fig. 1B, Table
1). Although current amplitudes are
attenuated markedly, the time- and voltage-dependent properties of the
residual currents are indistinguishable from those determined in cells
expressing Kv2.1 alone. Increasing the relative amount of the Kv2.1DN
to wild-type Kv2.1 to 5:1 completely eliminated the Kv2.1-encoded
K+ currents. Unexpectedly, however, the
currents produced on coexpression of Kv2.1DN with Kv2.2 in a 1:1 ratio
are indistinguishable from those produced by Kv2.2 alone (Fig.
1C, Table 1). Similar results were obtained when the (1:1)
ratio of the mutant construct Kv2.1DN to wild-type Kv2.2 was increased
to 5:1. Immunohistochemical experiments with the anti-FLAG antibody
(see Materials and Methods) revealed that the expression levels of the
Kv2.1DN protein were similar in HEK-293 cells expressing Kv2.1 or Kv2.2
(data not shown). Together, these results suggest that, in HEK-293
cells, Kv2.1DN does not assemble with Kv2.2 (see also below).
Additional experiments revealed that coexpression of Kv2.1DN does not
measurably affect the currents produced on expression of Kv1.4 (Fig.
1D, Table 1) or other Kv-subunits (data not
shown), revealing that the Kv2.1DN construct selectively coassembles
with Kv2.1.
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Figure 1.
Kv2.1W365C/Y380T (Kv2.1DN) specifically attenuates
Kv2.1-encoded currents in HEK cells. Whole-cell voltage-gated outward
K+ currents were recorded from HEK-293 cells
expressing Kv2.1DN alone (A), Kv2.1 or Kv2.1 plus
Kv2.1DN (B), Kv2.2 or Kv2.2 plus Kv2.1DN
(C), Kv1.4 or Kv1.4 plus Kv2.1DN
(D), and EGFP. Cells were transfected with cDNA
constructs (1:1) encoding these subunits, and currents were obtained
from EGFP-positive cells as described in Materials and Methods.
Schematic of Kv2.1DN is shown, with altered residues. A,
In cells expressing Kv2.1DN alone, outward K+
currents are indistinguishable from those in wild-type HEK-293
cells. B, In HEK-293 cells coexpressing Kv2.1DN
and Kv2.1, current densities are reduced markedly compared with those
recorded from cells expressing Kv2.1 alone (compare left
and right). C, In contrast, coexpression
of Kv2.1DN with Kv2.2 reveals outward currents indistinguishable from
those measured in cells expressing Kv2.2 alone. D,
Outward K+ currents in cells expressing Kv2.1DN and
Kv1.4 are indistinguishable from those expressing Kv1.4 alone.
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A similar strategy was exploited to generate a double pore mutant
of Kv2.2, Kv2.2W373C/Y388T, that also functions as a dominant negative
(Kv2.2DN); this construct was myc epitope-tagged at the C
terminus (see Materials and Methods). Electrophysiological experiments revealed that mean ± SEM peak outward
K+ current densities (26 ± 6 pA/pF;
n = 10) recorded from HEK-293 cells expressing Kv2.2DN
(Fig. 2A) are not
significantly different from those recorded from wild-type or
mock-transfected HEK-293 cells (15 ± 6 pA/pF; n = 11). However, coexpression of Kv2.2DN with wild-type Kv2.2 (1:1)
markedly reduced peak outward K+ current
densities compared with the currents recorded from cells expressing
Kv2.2 (and EGFP) alone (Fig. 2B, Table 1). The time- and voltage-dependent properties of the residual currents are indistinguishable from those determined in HEK-293 cells expressing Kv2.2 alone. Increasing the relative amount of the Kv2.2DN to wild-type
Kv2.2 cDNA to 5:1 eliminated the Kv2.2-encoded
K+ currents. Similar to the findings with
Kv2.1DN and Kv2.2 (Fig. 1C), coexpression of Kv2.2DN with
wild-type Kv2.1 at a 1:1 (Fig. 2C, Table 1) or 5:1 ratio
does not measurably affect Kv2.1-encoded currents. Immunohistochemical
experiments with the anti-myc antibody revealed that the expression
levels of the Kv2.2DN-myc, however, are similar in Kv2.2- and
Kv2.1-expressing HEK-293 cells (data not shown). Therefore, these
combined observations are consistent with the hypothesis advanced above
that Kv2.1 and Kv2.2 do not coassemble in HEK-293 cells (see also
below).
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Figure 2.
Kv2.2W373C/Y388T (Kv2.2DN) specifically attenuates
Kv2.2-encoded currents in HEK cells. Whole-cell voltage-gated outward
K+ currents were recorded from HEK-293 cells
expressing Kv2.2DN (A), Kv2.2 or Kv2.2 plus
Kv2.2DN (B), Kv2.1 or Kv2.1 plus Kv2.2DN
(C), and EGFP, as described in the legend to
Figure 1. A, Schematic of Kv2.2DN is shown, with the
altered residues indicated. In cells expressing Kv2.2DN alone, outward
K+ currents are similar to those in wild-type
cells. B, When Kv2.2DN is coexpressed with Kv2.2,
however, Kv2.2-encoded outward K+ currents are
reduced compared with cells expressing Kv2.2 alone (compare
left and right). C, In
contrast, Kv2.2DN does not affect Kv2.1-encoded currents.
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Expression of Kv2.1DN or Kv2.2DN attenuates
IK in SCG neurons
To assess the functional consequences of expression of the Kv2.x
dominant negative constructs on outward K+
currents in SCG neurons, cells were transfected with Kv2.1DN-FLAG or
Kv2.2DN-myc (and EGFP) using the Biolistics Gene Gun (see
Materials and Methods). As reported previously (Malin and Nerbonne,
2000, 2001) EGFP is readily detected in the cell bodies of SCG cells within ~15 hr of transfection (data not shown). Cells were stained with the anti-FLAG or anti-myc antibody to verify expression
of the transgenes, Kv2.1DN and Kv2.2DN, and both constructs were readily detected in all EGFP-positive cells (data not shown). Also
similar to previous findings using this methodology (Malin and
Nerbonne, 2000, 2001), all EGFP-positive cells in these cultures also
expressed the transgene.
Representative whole-cell voltage-gated outward
K+ currents recorded from three
EGFP-positive Kv2.1DN-expressing SCG cells are presented in Figure
3 (middle). As in wild-type
cells (Fig. 3, left), type I, type II, and type III
Kv2.1DN-expressing cells were readily distinguished (Table
2). Most (~70%) cells express IAf,
IK, and
ISS, and are therefore classified as
type I cells (Malin and Nerbonne, 2000). However, the density of
IK in Kv2.1DN-expressing type I cells
is significantly (p < 0.005) lower than
IK density in wild-type I cells (Table
2). Approximately 20% of the Kv2.1DN-expressing SCG cells are type II
(expressing IAf,
IAs,
IK, and
ISS), similar to the percentage
(~25%) of wild-type II cells (Fig. 3, Table 2). In type II, as in
type I, cells, Kv2.1DN expression significantly (p < 0.05) reduces
IK density (Table 2). In both type I
and type II cells, the effects of Kv2.1DN expression are specific;
neither the densities of IAf,
IAs, or
ISS nor the kinetics of
IAf or
IAs decay in these cells are
significantly different from those measured in wild-type I and II SCG
cells (Table 2). Similar to the findings in wild-type cells, ~10% of
Kv2.1DN-expressing cells are classified as type III SCG neurons,
expressing only IK and
ISS, and, interestingly, IK density in type III cells is
unaffected by Kv2.1DN expression (Table 2). Therefore, expression of
Kv2.1DN results in the selective attenuation (by ~50%) of
IK in type I and type II SCG
cells.
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Figure 3.
Expression of Kv2.1DN or Kv2.2DN reduces
IK density in SCG neurons. Whole-cell
voltage-gated outward K+ currents were recorded from
isolated SCG neurons in response to 6 sec depolarizing voltage steps to
test potentials between 10 and +50 mV from a holding potential of
90 mV. Experiments were conducted as described in Materials and
Methods, with 1 µM TTX and 100 µM
CdCl2 in the bath solution to block voltage-gated inward
Na+ and Ca2+ currents,
respectively. The records shown to the left,
middle, and right were recorded from
wild-type, Kv2.1DN-expressing, and Kv2.2DN-expressing cells,
respectively. There are distinct and stereotyped differences in the
waveforms of the currents in wild-type I, type II, and type III SCG
cells (Malin and Nerbonne, 2000). The numbers
given above the records in each column reflect the percentages of cells
studied under each experimental condition that display the type I, type
II, or type III outward K+ current phenotype.
Although the percentages of type I, type II, and type III Kv2.1DN- or
Kv2.2DN-expressing cells are not different from those in wild-type SCG
cells, expression of either Kv2.1DN (middle) or Kv2.2DN
(right) decreases the density of the slowly decaying
current, IK, in type I cells.
Expression of Kv2.1DN also decreases IK
density in type II cells (middle).
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Similar experiments were conducted on cells expressing Kv2.2DN, and
representative outward K+ currents
recorded from (3) Kv2.2DN- (and EGFP) expressing SCG neurons are shown
in Figure 3 (right). As in wild-type and Kv2.1DN-expressing SCG cells, most (~70%) Kv2.2DN-expressing cells can be classified as
type I, expressing IAf,
IK, and
ISS. In Kv2.2DN-expressing type I
cells (Fig. 3), IK density is
significantly (p < 0.005) lower than in
wild-type I SCG neurons (Table 2). The densities of
IAf and
ISS and the kinetics of
IAf and
IK decay, in contrast, are unaffected
by Kv2.2DN expression (Table 2). In ~20% of the Kv2.2DN-expressing
SCG cells, IAf,
IAs,
IK, and
ISS were evident, and these cells were
classified as type II (Malin and Nerbonne, 2000). The mean ± SEM
densities and inactivation rates of
IAf, IAs,
IK, and
ISS in Kv2.2DN-expressing type II
cells are not significantly different from those recorded in wild-type
II cells (Table 2). In contrast to the findings with Kv2.1DN, which
reduces IK density in type I and type
II cells, expression of Kv2.2DN reduces
IK density in type I cells without
affecting IK density in type II cells
(Table 2). In these experiments, two Kv2.2DN-expressing cells were
classified as type III cells (expressing
IK and
ISS). Although
IK densities in these (2) cells are
lower than in wild-type III cells (Table 2), the small number of
Kv2.2DN-expressing type III cells precluded statistical analysis.
Coexpression of Kv2.1DN and Kv2.2DN eliminates most of
IK in SCG neurons
The results of the experiments described above suggested that the
Kv2.1 and Kv2.2 -subunits form distinct populations of IK channels in SCG cells, and that
Kv2.1-encoded IK channels are expressed in both type I and type II cells, whereas Kv 2.2-encoded IK channels are expressed only in type
I SCG cells (Table 2). To explore this hypothesis further, currents
were recorded from cells expressing both Kv2.1DN and Kv2.2DN (and
EGFP). In these experiments, the relative amounts of the Kv2.1DN and
Kv2.2DN cDNAs were the same, and each was one-half of the amounts of
each used in the single transfection experiments (Fig. 3) to avoid any
possible complications attributable to gene dosage effects. As is
evident in the records shown in Figure 4,
the combined expression of Kv2.1DN and Kv2.2DN eliminates
IK in most cells. In 12 of 16 (75%)
of the cells examined, only IAf and
ISS were detected; these cells were
classified as type I cells lacking IK
(Table 2). The remaining four (of 16; 25%) cells are classified as
type II cells, expressing IAf,
IAs,
IK, and
ISS, although the mean ± SEM
IK density is reduced markedly in
these cells, compared with wild-type II cells (Table 2). However, the
mean ± SEM IK density in type II
cells expressing both Kv2.1DN and Kv2.2DN is not significantly
different from the IK density in
Kv2.1DN-expressing cells, consistent with the hypothesis that Kv2.1 but
not Kv2.2 contributes to IK in type II
cells. Importantly, in both type I and type II cells,
IAf,
IAs, and
ISS are unaffected by the combined
expression of Kv2.1DN and Kv2.2DN, indicating that Kv2 -subunits do
not contribute to these currents (Table 2). In these experiments, no
type III cells (expressing IK and ISS) were detected, an observation
that likely reflects the small number (16) of cells studied.
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Figure 4.
Coexpression of Kv2.1DN and Kv2.2DN eliminates
IK in most SCG neurons. Isolated SCG neurons
were transfected with both Kv2.1DN and Kv2.2DN (and EGFP) using the
Biolistics Gene Gun (as described in Materials and Methods), and
outward K+ currents were recorded from EGFP-positive
cells as described in the legend to Figure 3. Two distinct current
waveforms were evident in these recordings: most (75%) cells were
found to express only IAf and
ISS and therefore are type I cells lacking
IK; the remaining cells (25%)
express IAf,
IAs,
IK, and
ISS and are classified as type II cells with
reduced IK density (Table 2). The densities
of IAf,
IAs, and
ISS in Kv2.1DN plus Kv2.2DN-expressing type
I and type II cells are indistinguishable from those measured in
wild-type I and II cells (Table 2).
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Expression of Kv2 -subunits in rat SCG neurons
Together, the simplest interpretation of the results presented
above is that Kv2.1- and Kv2.2-encoded K+
channels underlie IK in type I cells,
whereas only Kv2.1 (and not Kv2.2) channels contribute to
IK in type II cells. These findings suggest that there is an additional component of
IK in type II SCG neurons that is not
encoded by Kv2 -subunits. In addition, the combined observations in
Kv2.1DN-, Kv2.2DN-, and Kv2.1DN plus Kv2.2DN-expressing SCG cells
suggest that the Kv2.1 and Kv2.2 -subunits do not associate to form
heteromultimeric K+ channels in SCG
neurons. Therefore, these results are consistent with the
electrophysiological findings in HEK-293 cells suggesting that Kv2.1
and Kv2.2 do not coassemble (Figs. 1, 2). Consequently, subsequent
experiments were focused on examining Kv2.1 and Kv2.2 expression in
HEK-293 cells and in SCG neurons and on determining whether Kv2.1 and
Kv2.2 are associated in situ.
Biochemical experiments were performed on extracts of Kv2.1 and
Kv2.2-transfected HEK-293 cells and on fractionated SCG neuronal membranes using specific anti-Kv2.1 and anti-Kv2.2 antibodies (see
Materials and Methods). As illustrated in Figure
5A, the anti-Kv2.1 antibody
detects a single band at ~114 kDa in HEK-293 cells transfected with
Kv2.1 and Kv2.2. Similarly, in blots probed using the anti-Kv2.2
antibody, a single protein band at ~90 kDa was identified (Fig.
5A). Western blot analysis also revealed robust expression
of Kv2.1 and Kv2.2 in lysates of rat SCG (Fig. 5A).
Importantly, both the anti-Kv2.1 and the anti-Kv2.2 antibodies can be
used to immunoprecipitate proteins (against which each of these
antibodies was targeted) from homogenates of HEK-293 cells transfected
with both the Kv2.1 and Kv2.2 cDNAs (Fig. 5B). In contrast,
however, Kv2.2 does not coimmunoprecipitate with the anti-Kv2.1
antibody, and Kv2.1 does not precipitate with the anti Kv2.2 antibody
(Fig. 5B). When the immunoprecipitation was performed with
the anti-Kv2.1 antibody, the Kv2.2 (nonprecipitating) protein was
identified in the supernatant (Fig. 5B, lanes S). In addition, when the anti Kv2.2 antibody was used in the
immunoprecipitation, the Kv2.1 (nonprecipitating) protein was readily
detected in the supernatants (Fig. 5B, lanes S).
Similar results were obtained in experiments performed on lysates of
rat SCG. The anti-Kv2.1 antibody precipitates the Kv2.1 protein but not
the Kv2.2 protein expressed in SCG neurons (Fig. 5C).
Similarly, after immunoprecipitations with the anti-Kv2.2 antibody, the
Kv2.2 protein is found in the pellet (Fig. 5C, lane
P), whereas the Kv2.1 protein is in the supernatant (Fig.
5C, lane S). Together, these data suggest that Kv2.1 and Kv2.2 do not associate either in HEK-293 cells or in SCG
neurons but rather preferentially form monomeric (Kv2.1 or Kv2.2)
voltage-gated K+ channels.
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Figure 5.
Kv2.1 and Kv2.2 are expressed in rat SCGs but do
not appear to associate. A, Lysates of control and
transfected HEK-293 cells and of isolated rat SCGs were fractionated in
SDS-PAGE gels and immunoblotted (IB) with the monoclonal
anti-Kv2.1 (left) and the polyclonal anti-Kv2.2
(right) antibodies. B, C, Lysates
prepared from transfected HEK-293 cells (B) and
rat SCG neurons (C) were immunoprecipitated
(IP) with either the monoclonal anti-Kv2.1 or the
polyclonal anti-Kv2.2 antibody, fractionated, and immunoblotted with
the same antibodies. Although both the anti-Kv2.1 and anti-Kv2.2
antibodies reliably immunoprecipitate the proteins against which each
of these antibodies were generated, the Kv2.1 and Kv2.2 -subunits do
not coimmunoprecipitate from lysates prepared from Kv2.1- and
Kv2.2-transfected HEK-293 cells or rat SCGs. After immunoprecipitations
with the anti-Kv2.1 antibody, the Kv2.2 protein is evident in the
supernatants (lanes S) but not in the pellet
(lanes P). Similarly, the Kv2.1 protein is found in the
supernatant (S) after
immunoprecipitation with the anti-Kv2.2 antibody. Closed
arrows indicate Kv2.1; open arrows indicate
Kv3.2.
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Kv2.1-encoded IK channels regulate
action potential durations in tonic SCG cells
In wild-type rat SCG neurons, three distinct repetitive firing
patterns, phasic, adapting, and tonic, are observed in response to
prolonged (6 sec) depolarizing current injections (Malin and Nerbonne,
2000). These studies revealed that ~45% of the cells are phasic,
firing one or two action potentials, and then become refractory in
response to prolonged (6 sec) current injections. Importantly, phasic
cells do not fire additional action potentials in response to larger
current injections. In contrast, adapting cells (~30%) fire trains
of action potentials in response to low amplitude current injections.
However, when the amplitude of the current injection is increased, the
firing rate adapts and these cells become refractory. Adapting cells
were further distinguished (from phasic and tonic cells) by increased
input resistances and decreased current thresholds for action potential
generation. Approximately 25% of wild-type SCG cells fire trains of
action potentials in response to depolarizing current injections, and the firing frequency increases with the amplitude of the injected current [i.e., no refractoriness is evident in these (tonic) cells over the range of injected currents examined]. Tonic cells were also
distinguished from phasic and adapting cells by briefer action potential durations (Malin and Nerbonne, 2000).
In initial experiments focused on exploring the hypothesis that
Kv2.x-encoded IK channels might play a
role in shaping action potential waveforms in SCG neurons,
Kv2.x-expressing HEK-293 cells and isolated SCG neurons were held at
48 mV (the mean resting membrane potential of rat SCG neurons), and a
voltage-clamp paradigm that simulates action potentials typically
recorded in phasic, adapting, and tonic SCG neurons was presented. The
action potential clamp paradigms are illustrated in Figure
6 (bottom). As illustrated at
the top of Figure 6, Kv2.1-encoded K+
currents in HEK-293 cells are activated similarly using the phasic, tonic, and adapting action potential clamp waveforms. The outward K+ currents peak ~1 msec after the peak
of the action potential at current densities of ~25-40 pA/pF (Table
3). This is ~20% of the current
activated in these cells by a step depolarization to +50 mV (289 ± 60 pA/pF; see Table 1). Therefore, substantial currents can be
evoked by single neuronal action potential waveforms when Kv2.x
subunits are expressed in HEK-293 cells. These findings are consistent
with the results observed using standard voltage-step protocols, which
reveal activation thresholds between 40 mV and 30 mV for Kv2.x
channels in HEK cells (Figs. 1, 2).
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Figure 6.
Activation of Kv2-encoded K+
currents during action potentials in SCG neurons. To explore directly
the activation of Kv2.x-encoded K+ currents during
action potential waveforms in SCG neurons, cells were held at the
typical resting membrane potential of SCG neurons (~48 mV; see Table
3), and outward K+ currents activated by typical
phasic, adapting, and tonic action potential waveforms were recorded.
The voltage-clamp paradigms are illustrated at the
bottom. Representative outward current waveforms in
Kv2.1-expressing HEK-293 cells driven by the phasic adapting and tonic
action potential waveforms are illustrated at the top.
Representative outward K+ current waveforms in
isolated SCG neurons (activated using the action potential
voltage-clamp paradigms shown below the record) are
presented at the bottom. Action potential clamp
recordings from wild-type (solid line),
Kv2.1DN-expressing (short dashed line), and
Kv2.2DN-expressing (long dashed line) SCG cells are
superimposed for comparison purposes.
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To probe directly the activation of Kv2.x channels in SCG cells,
similar experiments were performed in SCG cells using the action
potential clamp paradigms (Fig. 6). Peak outward
K+ current densities measured in SCG cells
with the action potential clamp paradigm are 50-60 pA/pF (Table 3).
Comparison of these values with current densities measured using the
conventional step depolarization paradigm (Fig. 3) reveals that peak
current densities activated during the action potential clamp are
~25% of the mean peak current densities measured at +50 mV in
response to step depolarizations from 70 mV (Fig. 3, Table 2). In
addition, in SCG cells expressing Kv2.1DN or Kv2.2DN, peak outward
K+ currents activated by phasic, tonic,
and adapting action potential clamp waveforms are reduced significantly
compared with those in wild-type cells (Table 3). Representative
voltage-clamp records are illustrated in the middle of Figure 6. As
might be expected from the data in HEK-293 cells, these analyses
suggest that the contribution of the Kv2.x-encoded
K+ currents is greater at the time
corresponding to 50% rather than 90% repolarization. Together, these
observations suggest that there is substantial outward
K+ current through Kv2-encoded
K+ channels in SCG neurons during action potentials, and
also that blocking Kv2-encoded K+ currents
likely would impact action potential durations. Subsequent experiments
were aimed at testing this hypothesis directly.
The phasic, adapting, and tonic firing patterns seen in wild-type SCG
cells are also evident in recordings from cells expressing Kv2.1DN, and
the selective attenuation of the Kv2.1-encoded
IK does not affect the distribution of
firing patterns (Fig. 7). The expression
of Kv2.1DN also does not affect the mean ± SEM input resistances,
resting potentials, action potential amplitudes, or the current
thresholds for action potential generation in phasic, adapting, or
tonic SCG cells (Table 4). As in
wild-type SCG cells, Kv2.1DN-expressing adapting cells are readily
distinguished from phasic and tonic cells by increased input
resistances and lower current thresholds for action potential
generation (Table 4). In tonic cells expressing the Kv2.1DN, however,
the mean ± SEM action potential duration at 90% repolarization
is significantly (p < 0.02) longer than in
wild-type tonic cells (Fig. 7B, Table 4). Interestingly,
although action potential durations in Kv2.1DN-expressing tonic cells
are prolonged, tonic firing is not eliminated, and the firing
frequencies of tonic cells are unchanged. Therefore, brief action
potentials, regulated by Kv2.1-encoded
IK, are not the sole determinant of
tonic firing.
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Figure 7.
Phasic, adapting, and tonic firing patterns in SCG
neurons expressing Kv2.1DN. SCG neurons were transfected with Kv2.1DN
(and EGFP), and action potentials and repetitive firing patterns were
recorded in response to brief or prolonged depolarizing current
injections, as described in Materials and Methods. A,
Current-clamp recordings from three representative Kv2.1DN-expressing
cells are shown. In each cell, single action potentials were elicited
by 1.5 msec current injections (left), and repetitive
firing patterns were recorded in response to 100 pA
(middle) or 200 pA (right) 500 msec
current injections. Based on the response(s) to the 500 msec current
injections, cells were classified as phasic (top),
adapting (middle), or tonic (bottom)
(Table 4). Although Kv2.1DN expression does not affect the
distribution of firing patterns in SCG neurons, action potentials in
tonic cells expressing Kv2.1DN are prolonged
(B). Action potential durations in phasic and
adapting cells (B), in contrast, are unaffected
by Kv2.1DN expression (Table 4). wt, Wild type.
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Kv2.2-encoded IK channels regulate
action potential threshold and repolarization
To explore the functional role of Kv2.2-encoded
IK channels, current-clamp recordings
were obtained from SCG cells expressing Kv2.2DN (and EGFP). Similar to
the findings with Kv2.1DN, all three firing patterns are observed in
Kv2.2DN-expressing cells (Fig. 8). Also,
similar to the findings with Kv2.1DN, action potential durations at
90% repolarization in tonic cells expressing Kv2.2 are significantly
(p < 0.02) longer than in tonic wild-type cells (Table 4). However, in contrast to the findings with Kv2.1DN, expression of Kv2.2DN results in an increase in the percentage of
adapting cells and a decrease in the percentage of phasic cells (Fig.
8, Table 4). Elimination of Kv2.2-encoded
IK channels also has marked effects on
the excitability of SCG neurons. In adapting and tonic cells expressing
Kv2.2DN, for example, resting membrane potentials are depolarized
significantly (p < 0.001) compared with resting
membrane potentials in wild-type adapting and tonic SCG cells (Table
4). In addition, the current thresholds for action potential generation
are reduced significantly in phasic (p < 0.002)
and in tonic (p < 0.02) firing SCG cells
expressing Kv2.2DN. However, input resistances and action potential
amplitude are unaffected by Kv2.2DN expression (Table 4). Together,
these results suggest that, unlike Kv2.1-encoded
IK channels, Kv2.2-encoded IK channels play a role in setting
resting potentials and in regulating action potential generation.
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Figure 8.
Expression of Kv2.2DN in SCG neurons increases the
number of adapting cells. In SCG neurons transfected with Kv2.2DN (and
EGFP), action potentials and repetitive firing patterns were recorded,
and cells were classified as phasic (top), adapting
(middle), or tonic (bottom), as described
in the legend to Figure 7. Similar to the results obtained with
Kv2.1DN, expression of Kv2.2DN prolongs action potential durations in
tonic cells (B). In addition, Kv2.2DN expression
alters the distribution of firing patterns: the percentage of phasic
cells is reduced (by ~40%), and the percentage of adapting cells is
increased (Table 4). wt, Wild type.
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Elimination of IK prolongs action
potentials and reduces tonic firing
The voltage-clamp experiments detailed above revealed that
expression of either Kv2.1DN or Kv2.2DN reduces
IK density in type I SCG cells by
~50%, whereas only Kv2.1DN expression attenuates IK in type II cells. In addition,
coexpression of Kv2.1DN and Kv2.2DN eliminates
IK in all type I cells and reduces
IK density in type II cells. Together
with the biochemical data presented (Fig. 5), these results suggest
that there are two distinct populations of Kv2.x-encoded
IK channels, and that these channels
are differentially expressed: Kv2.1-encoded
IK channels in both type I and type II cells and Kv2.2-encoded IK channels in
type I cells. To determine the effects of removing both populations of
Kv2 -subunit-encoded IK channels,
action potentials and repetitive firing patterns were recorded from SCG
neurons expressing both Kv2.1DN and Kv2.2DN. Recordings from
representative cells are shown in Figure
9. Several of the effects of Kv2.1DN and
Kv2.2DN coexpression appear to be the sum of the effects of Kv2.1DN or
Kv2.2DN expression alone. As in Kv2.2DN-expressing cells, for example,
the mean ± SEM current threshold for action potential generation
is decreased in cells expressing both dominant negative constructs. In
addition, resting membrane potentials are depolarized in these cells
compared with wild-type cells (Table 4). Interestingly, action
potential durations in adapting (p < 0.02) and
phasic (p < 0.004) Kv2.1DN plus
Kv2.2DN-expressing cells are prolonged significantly, whereas
expression of either Kv2.1DN or Kv2.2DN alone does not affect phasic
and adapting action potential durations (Table 4). The most prominent
effect of coexpression of both Kv2 dominant negative constructs,
however, is the reduction of tonic cell number from 25% of wild-type
SCG neurons to 8% of Kv2.1DN plus Kv2.2DN-expressing neurons. These
observations are consistent with a role for
IK in action potential repolarization and in the regulation of tonic firing.
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Figure 9.
Coexpression of Kv2.1DN and Kv2.2DN markedly
reduces tonic firing. Isolated SCG neurons were transfected with
Kv2.1DN, Kv2.2DN, and EGFP. Action potentials and repetitive firing
patterns were recorded, and cells were classified as phasic
(top), adapting (middle), or tonic
(bottom), as described in the legend to Figure 7. In
contrast to the results obtained with expression of Kv2.1DN or Kv2.1DN
alone (Figs. 7, 8), expression of both dominant negative constructs
markedly decreases the percentage of tonic firing cells (Table
4).
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Expression of wild-type Kv2.1 -subunits increases
tonic firing
The results of the experiments described here suggest that
Kv2-encoded IK channels play a role in
determining tonic firing. To explore this hypothesis further, action
potentials and repetitive firing patterns were recorded from SCG cells
transfected with wild-type Kv2.1 and EGFP (Fig.
10). These experiments revealed that
the membrane properties of Kv2.1-transfected and wild-type phasic,
adapting, and tonic SCG cells are indistinguishable (see on-line Table,
available at www.jneurosci.org). However, expression of Kv2.1 markedly
reduces the number of phasic (~25%) and adapting (~25%) cells and
increases the number of tonic cells (50%). In addition, mean ± SEM action potential durations at 50 and 90% repolarization in
Kv2.1-expressing tonic cells are significantly (p < 0.02) briefer than in phasic and adapting
cells.
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Figure 10.
Expression of Kv2.1 in SCG neurons increases
tonic firing. Action potentials and repetitive firing patterns,
recorded as described in the legend to Figure 7, were obtained from
isolated SCG neurons 24 hr after transfection with wild-type Kv2.1 and
EGFP. As in wild-type cells, the phasic (top), adapting
(middle), and tonic (bottom) firing
patterns were seen in recordings from cells transfected with Kv2.1.
However, the percentage of tonic cells is higher and the percentages of
adapting and phasic cells are lower in Kv2.1-expressing cells than in
either wild-type, Kv2.1DN-expressing (Fig. 7), or Kv2.2DN-expressing
(Fig. 8) SCG cells (Table 4).
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DISCUSSION |
Two distinct components of IK encoded by
Kv2.1 and Kv2.2 in SCG neurons
The studies described here were designed to probe the role of Kv2
-subunits in the generation of voltage-gated
K+ currents in SCG neurons. Biochemical
experiments revealed that both Kv2.1 and Kv2.2 are expressed in SCG
neurons, and that these -subunits do not appear to associate in
these cells or in HEK-293 cells (Fig. 5). To explore the functional
roles of Kv2 -subunits, pore mutants of Kv2.1 (Kv2.1DN) and Kv 2.2 (Kv2.2DN), designed to function as dominant negatives, were
constructed. Heterologous coexpression studies in HEK-293 cells
revealed that the dominant negative effects of Kv2.1DN and Kv2.2DN are
specific for Kv2.1 and Kv2.2 channels, respectively (Figs. 2, 3, Table
1). The simplest interpretation of these observations is that Kv2.1 and
Kv2.2 -subunits do not coassemble, at least in HEK-293 cells (Table
1). When expressed in SCG cells, Kv2.1DN selectively attenuates
IK in type I (expressing
IAf,
IK,
ISS) and type II (expressing
IAf,
IAs, IK,
ISS) cells;
IAf,
IAs, and
ISS are unaffected by Kv2.1DN
expression (Table 2). Expression of Kv2.2DN, in contrast, reduces
IK only in type I cells (Table 2).
The experiments here also revealed that the effects of Kv2.1DN
and Kv2.2DN are additive. Coexpression of Kv2.1DN and Kv2.2DN, for
example, eliminates IK in type I SCG
cells (Fig. 4, Table 2). Importantly, in these experiments, the total
amount of cDNA used in the transfections was the same as with Kv2.1DN
or Kv2.2DN alone. The additivity of the effects of Kv2.1DN and Kv2.2DN,
therefore, does not simply reflect a dose-dependent attenuation of the
current. Rather, the experimental observations suggest that Kv2.1DN and Kv2.2DN affect different populations of
IK channels, consistent with the
hypothesis that Kv2.1 and Kv2.2 do not coassemble in type I SCG
neurons. The results presented here also reveal that Kv2-encoded
K+ channels underlie
IK in all type I SCG neurons. In type
II cells, in contrast, IK is not
eliminated with Kv2.1DN and Kv2.2DN coexpression. This might reflect
the slow turnover rate of some Kv2.x-encoded K+ channels in type II SCG neurons.
Alternatively, it is also possible that the expression levels of the
dominant negative constructs were too low in these experiments to
effectively eliminate IK in type II
cells. Both of these hypotheses seem unlikely, however, in light of the
results obtained in type I cells and the similarities in the reductions
in IK in Kv2.1DN- and Kv2.1DN plus
Kv2.2DN-expressing type II cells. It seems more likely that only a
subset of IK channels in type II cells
are encoded by Kv2 (Kv2.1) -subunits, and that the residual
IK channels reflect the contribution
of another Kv-subunit subfamily. Nevertheless, additional
experiments aimed at determining the molecular identity of the
individual IK channels in type II cells will be necessary to test this hypothesis directly.
Kv2.1- and Kv2.2-encoded IK channels
are differentially expressed in type I and type II SCG cells:
Kv2.1-encoded IK is expressed in both
type I and type II cells, whereas Kv2.2-encoded
IK channels are only evident in type I
cells (Table 2). Although the numbers and the percentages of type III
cells are small, the experiments completed to date suggest that
IK density is reduced in type III cells expressing Kv2.2DN but not in cells expressing Kv2.1DN (Table 2).
No type III cells were seen in recordings from cells coexpressing Kv2.1DN and Kv2.2DN, an observation that likely reflects the small number (n = 16) of cells studied in these experiments.
IK density regulates action potential
repolarization and tonic firing
Expression of either Kv2.1DN or Kv2.2DN selectively increases
action potential durations in tonic cells, suggesting that
IK channels contribute to action
potential repolarization in these cells (Table 4). Although expression
studies in Xenopus oocytes suggest relatively depolarized
(~0 mV) thresholds for activation (Fink et al., 1996), Kv2.x-encoded
K+ channels in mammalian cell lines
activate at more depolarized (approximately 30 to 40 mV) potentials
(Figs. 1, 2) (Murakoshi et al., 1997). In addition, action potential
clamp experiments reveal substantial activation (~20% maximal) of
Kv2.x-encoded currents in HEK-293 cells during a single (SCG) action
potential (Fig. 6). Furthermore, expression of Kv2.1DN or Kv2.2DN
markedly reduces the currents elicited during action potentials in SCG cells (Fig. 6). These results suggest an important role for
Kv2.x-encoded K+ channels in action
potential repolarization in SCG neurons and suggest that these
channels, like other K+ channels, are
differentially modified by expression environment (Peterson and
Nerbonne, 1999). Interestingly, a similar conclusion was reached
independently by Du et al. (2002) in studies focused on the functioning
of Kv2.1-encoded currents in hippocampal neurons.
Although IK shapes individual action
potential waveforms in tonic cells, reductions in
IK density do not affect the
percentage of cells that fire tonically (Table 4), revealing that brief action potentials are not the sole determinant of tonic firing. However, coexpression of Kv2.1DN and Kv2.2DN and the resulting reductions (in type II cells) or elimination (in type I cells) of
IK markedly reduce the percentage of
tonic firing cells (Fig. 9). Although action potential repolarization
in phasic and adapting cells is not affected by removal of either
Kv2.1- or Kv2.2-encoded IK,
elimination of both Kv2.1- and Kv2.2-encoded
IK significantly (p < 0.001) increases action potential
durations in these cells (Table 4). Together, therefore, these
observations suggest that IK channels
contribute to action potential repolarization in phasic, adapting, and
tonic SCG cells, and that total IK
density is a critical determinant of the tonic firing pattern.
Consistent with this hypothesis, expression of Kv2.1 increases
IK density and the percentage of tonic
cells (see Fig. 10 and on-line Table, available at
www.jneurosci.org).
Kv2.2-encoded IK regulates
neuronal excitability
Although both Kv2.1- and Kv2.2-encoded
IK channels play roles in action
potential repolarization in SCG neurons, only Kv2.2-encoded IK channels appear to contribute to
the regulation of (resting) membrane excitability and to affect action
potential firing. Expression of Kv2.2DN, for example, depolarizes all
SCG cells and reduces the current thresholds for action potential
generation in phasic and tonic cells (Table 4). Thus, unlike
Kv2.1-encoded channels, Kv2.2-encoded
IK channels contribute to setting the
resting membrane potentials of SCG neurons and function to regulate
membrane excitability. Interestingly, the properties of Kv2.1- and
Kv2.2-encoded K+ channels are very similar
in heterologous expression systems (Figs. 1, 2), suggesting that there
are cell-type-specific regulatory pathways that differentially modulate
Kv2.1- and Kv2.2-encoded K+ channels. The
functional distinctions between the Kv2.1- and Kv2.2-encoded
IK channels revealed in the
experiments reported here suggest that differential regulation of these
two IK components, either by
post-translational modification (Summers and Gelband, 1998; Colbert and
Pan, 1999; Gelband et al., 1999; Zhu et al., 1999) or by coassembly
with regulatory subunits (Chiara et al., 1999; Kerschensteiner and
Stocker, 1999), would have dramatically different effects on SCG cell
membrane excitability. In addition, because Kv2.1- and Kv2.2-encoded
IK channels are differentially expressed in type I, type II, and type III SCG cells, differential modulation of Kv2.x-encoded IK
channels would be expected to regulate neuronal excitability in a
cell-type-specific manner. Additional experiments aimed at testing this
hypothesis directly will clearly be of interest.
Previous studies have shown that the current thresholds for action
potential generation are lower in adapting than in phasic or tonic
cells, which appears to reflect, at least in part, the reduced density
of the fast transient current IAf in
adapting cells (Malin and Nerbonne, 2000, 2001). The observations
presented here that expression of Kv2.2DN reduces action potential
thresholds and increases adapting cell number (Table 4) suggest a
similar role of Kv2.2-encoded IK
channels. Furthermore, these results suggest that the adapting
phenotype is correlated with low current thresholds for action
potential generation, and that these low threshold values in adapting
cells arise from reduced density of Kv2.2-encoded
IK (present study), as well as
IAf (Malin and Nerbonne, 2000, 2001),
in these neurons.
| |
FOOTNOTES |
Received July 24, 2002; revised Sept. 10, 2002; accepted Sept. 10, 2002.
Financial support was provided by the National Science Foundation
(predoctoral fellowship to S.A.M.) and the National Institutes of
Health (NS-30676).
Correspondence should be addressed to Jeanne M. Nerbonne, Washington
University Medical School, 660 South Euclid, Box 8103, St. Louis, MO
63110. E-mail: jnerbonn{at}pcg.wustl.edu.
| |
REFERENCES |
-
An WF,
Bowlby MR,
Betty M,
Cao J,
Ling H-P,
Mendoza G,
Hinson JW,
Mattson KI,
Strassle BW,
Trimmer JS,
Rhodes KJ
(2000)
Modulation of A-type potassium channels by a family of calcium sensors.
Nature
403:553-556[Medline].
-
Baranauskas G,
Tkatch T,
Surmeier JD
(1999)
Delayed rectifier currents in rat globus pallidus neurons are attributable to Kv2.1 and Kv3.1/3.2 K+ channels.
J Neurosci
19:6394-6404[Abstract/Free Full Text].
-
Barry DM,
Trimmer JS,
Merlie JP,
Nerbonne JM
(1995)
Differential expression of voltage-gated K+ channel subunits in adult rat heart. Relation to functional K+ channels.
Circ Res
77:361-369[Abstract/Free Full Text].
-
Barry DM,
Xu H,
Schuessler RB,
Nerbonne JM
(1998)
Functional knock-out of the transient outward current, long-QT syndrome, and cardiac remodeling in mice expressing a dominant-negative Kv4 -subunit.
Circ Res
83:560-567[Abstract/Free Full Text].
-
Blaine JT,
Ribera AB
(1998)
Heteromultimeric potassium channels formed by members of the Kv2 subfamily.
J Neurosci
18:9585-9593[Abstract/Free Full Text].
-
Blaine JT,
Ribera AB
(2001)
Kv2 channels form delayed-rectifier potassium channels in situ.
J Neurosci
21:1473-1480[Abstract/Free Full Text].
-
Chang JY,
Martin DP,
Johnson Jr EM
(1990)
Interferon suppresses sympathetic neuronal cell death caused by nerve growth factor deprivation.
J Neurochem
55:436-445[Web of Science][Medline].
-
Chiara MD,
Monje F,
Castellano A,
Lopez-Barneo J
(1999)
A small domain in the N-terminus of the regulatory -subunit Kv2.3 modulates Kv2.1 potassium channel gating.
J Neurosci
19:6865-6873[Abstract/Free Full Text].
-
Coetzee WA,
Amarillo Y,
Chiu J,
Chow A,
Lau D,
McCormack T,
Moreno H,
Nadal MS,
Ozaita A,
Pountney D,
Saganich M,
Vega-Saenz de Meira E,
Rudy B
(1999)
Molecular diversity of K+ channels.
Ann NY Acad Sci
868:233-285[Web of Science][Medline].
-
Colbert CM,
Pan E
(1999)
Arachadonic acid alters the availability of transient and sustained dendritic K+ channels in hippocampal CA1 pyramidal neurons.
J Neurosci
19:8163-8171[Abstract/Free Full Text].
-
Dixon JE,
McKinnon D
(1996)
Potassium channel mRNA expression in prevertebral and paravertebral sympathetic neurons.
Eur J Neurosci
8:183-191[Web of Science][Medline].
-
Du J,
Tao-Cheng J-H,
Zerfas P,
McBain CJ
(1998)
The K+ channel, Kv2.1, is apposed to astrocytic processes and is associated with inhibitory post-synaptic membranes in hippocampal and cortical principal neurons and inhibitory interneurons.
Neuroscience
84:37-48[Web of Science][Medline].
-
Du J,
Haak LL,
Phillips-Tansey E,
Russell JT,
McBain CJ
(2000)
Frequency-dependent regulation of rat hippocampal somato-dendritic excitability by the K+ channel subunit Kv2.1.
J Physiol (Lond)
522:19-31[Abstract/Free Full Text].
-
Fink M,
Duprat F,
Lesage F,
Heurteaux C,
Romey G,
Barhanin J,
Lazdunski ML
(1996)
A new K+ channel
 subunit to specifically enhance Kv2.2 (CDRK) expression.
J Biol Chem
271:26341-26348[Abstract/Free Full Text]. -
Gelband CH,
Warth JD,
Mason HS,
Zhu M,
Moore JM,
Kenyon JL,
Horowitz B,
Sumners C
(1999)
Angiotensin II type 1 receptor-mediated inhibition of K+ channel subunit Kv2.2 in brain stem and hypothalamic neurons.
Circ Res
84:352-359[Abstract/Free Full Text].
-
Hwang PM,
Glatt CE,
Bredt DS,
Yellen G,
Snyder SH
(1992)
A novel K+ channel with unique localizations in mammalian brain: molecular cloning and characterization.
Neuron
8:473[Web of Science][Medline]:481.
-
Hwang PM,
Fotuhi M,
Bredt DS,
Cunningham AM,
Snyder SH
(1993)
Contrasting immunohistochemical localizations in rat brain of two novel K+ channels of the Shab subfamily.
J Neurosci
13:1569-1576[Abstract].
-
Kerschensteiner D,
Stocker M
(1999)
Heteromeric assembly of Kv2.1 with Kv9.3: effect on the state dependence of inactivation.
Biophys J
77:248-257[Web of Science][Medline].
-
Kuryshev YA,
Gudz TI,
Brown AM,
Wible BA
(2000)
KChAP as a chaperone for specific K+ channels.
Am J Physiol
278:C931-C941[Web of Science].
-
Li H,
Guo W,
Nerbonne JM
(1999)
Functional consequences of cardiac-specific expression of wild-type or mutant (dominant negative) Kv1.5 -subunits in mouse ventricular myocytes.
Circulation
102:2-261[Free Full Text].
-
Lim ST,
Antonucci DE,
Scannevin RH,
Trimmer JS
(2000)
A novel targeting signal for proximal clustering of the Kv2.1 K+ channel in hippocampal neurons.
Neuron
25:385-397[Web of Science][Medline].
-
Maletic-Savatic M,
Lenn NJ,
Trimmer JS
(1995)
Differential spatiotemporal expression of K+ channel polypeptides in rat hippocampal neurons developing in situ and in vitro.
J Neurosci
15:3840-3851[Abstract].
-
Malin SA,
Nerbonne JM
(2000)
Elimination of the fast transient in superior cervical ganglion neurons with expression of Kv4.2W362F: molecular dissection of IA.
J Neurosci
20:5191-5199[Abstract/Free Full Text].
-
Malin SA,
Nerbonne JM
(2001)
Molecular heterogeneity of the voltage-gated fast transient outward K+ current, IAf, in mammalian neurons.
J Neurosci
21:8004-8014[Abstract/Free Full Text].
-
Murakoshi H,
Trimmer JS
(1999)
Identification of the Kv2.1 K+ channel as a major component of the delayed rectifier K+ current in rat hippocampal neurons.
J Neurosci
19:1728-1735[Abstract/Free Full Text].
-
Murakoshi H,
Shi G,
Scannevin RH,
Trimmer JS
(1997)
Phosphorylation of the Kv2.1 K+ channel alters voltage dependent activation.
Mol Pharmacol
52:821-828[Abstract/Free Full Text].
-
Pankevych H,
Kristufek D,
Huck S
(1999)
Perinatal and postnatal regulation of Shaker-related genes in rat superior cervical ganglion.
Soc Neurosci Abstr
25:739.1.
-
Petersen KR,
Nerbonne JM
(1999)
Expression environment determines K+ current properties: Kv1 and Kv4 subunit-induced K+ currents in mammalian cell lines and cardiac myocytes.
Pflügers Arch
437:381-392[Web of Science][Medline].
-
Pond AL,
Scheve BK,
Benedict AT,
Petrecca K,
Van Wagoner DR,
Shrier A,
Nerbonne JM
(2000)
Expression of distinct ERG proteins in rat, mouse, and human heart: relation to functional IKr channels.
J Biol Chem
275:5997-6006[Abstract/Free Full Text].
-
Pongs O
(1999)
Voltage-gated potassium channels: from hyperexcitability to excitement.
FEBS Lett
452:31-35[Web of Science][Medline].
-
Raff MC,
Fields KL,
Hakomori S-I,
Mirsky R,
Pruss RM,
Winter J
(1979)
Cell-type-specific markers for distinguishing and studying neurons and the major classes of glial cells in culture.
Brain Res
174:283-308[Web of Science][Medline].
-
Rudy B
(1988)
Diversity and ubiquity of K channels.
Neuroscience
24:729-749[Web of Science][Medline].
-
Storm JF
(1990)
Potassium currents in hippocampal pyramidal cells.
Prog Brain Res
83:161-187[Web of Science][Medline].
-
Summers C,
Gelband CH
(1998)
Neuronal ion channel signaling pathways: modulation by angiotensin II.
Cell Signal
10:303-311[Web of Science][Medline].
-
Trimmer JS
(1991)
Immunological detection and characterization of a delayed rectifier K+ channel polypeptide in rat brain.
Proc Natl Acad Sci USA
88:10764-10768[Abstract/Free Full Text].
-
Zhu M,
Gelband CH,
Posner P,
Sumners C
(1999)
Angiotensin II decreases neuronal delayed rectifier potassium current: role of calcium/calmodulin-dependent proteins kinase II.
J Neurophysiol
82:1560-1568[Abstract/Free Full Text].
Copyright © 2002 Society for Neuroscience 0270-6474/02/222310094-12$05.00/0
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|
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|
|
|
|
|
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|
|
|
|
|
|
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[Full Text]
[PDF]
|
|
|
|
|
|
|
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[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
TOP
ABSTRACT
INTRODUCTION
