Volume 16, Number 16,
Issue of August 15, 1996
pp. 4846-4860
Copyright ©1996 Society for Neuroscience
Voltage-Gated K+ Channel 
Subunits: Expression and
Distribution of Kv1 and Kv2 in Adult Rat Brain
Kenneth J. Rhodes1,
Michael M. Monaghan1,
Nestor X. Barrezueta1,
Stanley Nawoschik1,
Zewditu Bekele-Arcuri2,
Maria F. Matos2,
Kensuke Nakahira2,
Lee E. Schechter1, and
James S. Trimmer2
1 CNS Disorders, Wyeth-Ayerst Research, Princeton, New
Jersey 08543, and 2 Department of Biochemistry and Cell
Biology and Institute for Cell and Developmental Biology, State
University of New York, Stony Brook, New York 11794
ABSTRACT
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- DISCUSSION
- FOOTNOTES
- REFERENCES
ABSTRACT 
Recent cloning of K+ channel 
subunits
revealed that these cytoplasmic polypeptides can dramatically alter the
kinetics of current inactivation and promote efficient glycosylation
and surface expression of the channel-forming 
subunits. Here, we
examined the expression, distribution, and association of two of these
subunits, Kv1 and Kv2, in adult rat brain. In situ
hybridization using cRNA probes revealed that these -subunit genes
are heterogeneously expressed, with high densities of Kv1 mRNA in
the striatum, CA1 subfield of the hippocampus, and cerebellar Purkinje
cells, and high densities of Kv2 mRNA in the cerebral cortex,
cerebellum, and brainstem. Immunohistochemical staining using
subunit-specific monoclonal and affinity-purified polyclonal antibodies
revealed that the Kv1 and Kv2 polypeptides frequently co-localize
and are concentrated in neuronal perikarya, dendrites, and terminal
fields, and in the juxtaparanodal region of myelinated axons.
Immunoblot and reciprocal co-immunoprecipitation analyses indicated
that Kv2 is the major subunit present in rat brain membranes,
and that most K+ channel complexes containing
Kv1 also contain Kv2. Taken together, these data suggest
that Kv2 is a component of almost all K+
channel complexes containing Kv1 subunits, and that individual
channels may contain two or more biochemically and functionally
distinct -subunit polypeptides.
Key words:
ion channel;
central nervous system;
auxiliary subunit;
striatum;
immunoprecipitation;
immunohistochemistry
INTRODUCTION
Voltage-gated K+ channels
are composed of pore-forming subunits and associated cytoplasmic
-subunit polypeptides (Pongs, 1995
). These channels are critical for
action potential conduction and neurotransmitter release, and are
essential to the control of neuronal excitability (Hille, 1992).
Expression of -subunit mRNAs in heterologous cells gives rise to
tetrameric channel complexes with electrophysiological characteristics
similar to A-type or delayed-rectifier channels (for review, see Pongs,
1992). The large number of -subunit genes, their ability to assemble
into heteromultimers (Ruppersberg et al., 1990; Sheng et al., 1993;
Wang et al., 1993; Scott et al., 1994a; Rhodes et al., 1995), and their
heterogeneous expression in mammalian brain undoubtedly contributes to
the diversity of voltage-gated K+ channels
in situ (Stühmer et al., 1989; Jan and Jan, 1990).
However, the recent discovery of auxiliary subunits associated with ion
channels in general (for review, see Isom et al., 1994), and with
voltage-gated K+ channels in particular (Parcej
et al., 1992; Rettig et al., 1994; Scott et al., 1994b), revealed that
their electrophysiological and biochemical properties can be
dramatically affected by the presence of subunits (Rettig et al.,
1994; Majumder et al., 1995; Morales et al., 1995; Shi et al., 1996).
This association of subunits with K+ channels
not only increases the potential for diversity, it also indicates that
the functional properties of individual channels are governed by the
specific combination of and subunits present in the channel
complex.
The subunits of voltage-gated K+ channels
were identified as 38-41 kDa polypeptides associated with the
dendrotoxin acceptor purified from bovine brain (Parcej and Dolly,
1989, 1992; Scott et al., 1994a,b), and as a 38 kDa polypeptide in
immunoprecipitated rat brain K+ channel complexes
(Trimmer, 1991). Subsequent cloning of cDNAs encoding a bovine subunit (Scott et al., 1994b); the related Kv1, Kv2, and Kv3
in rat brain (Rettig et al., 1994; Heinemann et al., 1995); and closely
related subunits in Drosophila (Chouinard et al., 1995),
ferret (Morales et al., 1995), and human tissues (England et al.,
1995a,b; Majumder et al., 1995; McCormack et al., 1995) indicated that
these subunits are highly conserved and that some can modulate the
rate of inactivation of certain subunits (Rettig et al., 1994;
England et al., 1995a,b; Heinemann et al., 1995; Majumder et al., 1995;
McCormack et al., 1995; Morales et al., 1995).
We recently reported that Kv1 and Kv2 associate with subunits
early in their biosynthesis and exert chaperone-like effects on the subunits, promoting their efficient glycosylation and stable expression
in the plasma membrane (Shi et al., 1996). In addition, we reported
that Kv1 and Kv2 associate with all members of the
Shaker-related (Kv1) -subunit subfamily upon
co-expression in transfected mammalian cells (Nakahira et al., 1996).
Here, we used riboprobes and antibodies specific for Kv1 and Kv2
to examine their expression, subcellular distribution, and
co-association in adult rat brain. We observed that these subunits
are widely expressed, and that immunoreactivity for Kv1 and Kv2
is concentrated in multiple subcellular domains including neuronal
somata and dendrites, the paranodal segments of myelinated axons, and
in the terminal fields of several cortical and subcortical projection
systems. We also observed that Kv2 is the predominant -subunit
isoform in rat brain; although Kv1 mRNA is widely expressed, the
Kv1 polypeptide is not a major component of the total rat brain
-subunit pool, and almost all of the Kv1 that is present is in
K+ channel complexes that also contain Kv2.
Together, these observations suggest that subunits are integral
components of K+ channel complexes, and that the
inclusion of Kv1 in Kv2-containing complexes may serve to fine
tune the electrophysiological properties of channels in specific brain
regions.
MATERIALS AND METHODS
Materials. All reagents were molecular biology grade
from Sigma (St. Louis, MO) or Boehringer Mannheim (Indianapolis, IN),
except where otherwise noted.
Preparation of 35S-labeled cRNA probes. DNA
templates for riboprobe synthesis were prepared by the PCR using
plasmid clones containing the full-length rat Kv1 or Kv2 cDNAs
(Nakahira et al., 1996) or a partial Kv2 cDNA (K. Nakahira, S. Nawoschik, J. Trimmer, unpublished observations) as PCR templates. Two
independent riboprobes targeted to unique, nonoverlapping regions of
Kv1 or Kv2 were used in the present study. The probe sequences
were checked versus the Genbank database to ensure that they only
recognize the appropriate targets among all deposited sequences. To
generate riboprobes for the Kv1 subunit, one pair of oligonucleotide
primers was designed to amplify a 264 bp region spanning nucleotides
46 to 218 of the rat Kv1 cDNA and, in addition, to add the
promoter sequences for T3 RNA polymerase. These primers contained
the following sequences: 5
-CGGATCCG- CTGTGCTGTGGGGTTCTGAGAGGAC-3
(forward); 5-AATTAACC- CTCACTAAAGGGATATTTCATGCCAGTCTGCT-3
(reverse). The forward primer for this Kv1 probe contained eight
nucleotides of vector sequence at the 5 end from the BamHI
site used for cloning. A second pair of oligonucleotide primers was
designed to amplify a 271 bp sequence spanning nucleotides 321 to
51 of the rat Kv1 cDNA and, in addition, to add the promoter
sequences for T3 RNA polymerase. These primers had the following
sequences: 5-CCTGACCACATGGATCTGGC-3 (forward);
5-AATTAACCCTCACTAAAGGGCGGCAGAGGGTGAGACGTT-3
(reverse). To generate riboprobes for the Kv2 subunit, one pair of
primers was designed to amplify a 228 bp fragment from the 3
untranslated region (nucleotides 1130-1357) of the rat Kv2 cDNA
and, in addition, to add the promoter sequences for T3 RNA polymerase.
These primers had the following sequences:
5-CCCAGCTCGGACAGTTCCTGGTTCC-3 (forward);
5-AATTAACCCTCACTAAAGGGGCATCCAGCGAGG AAG- CGGC-3 (reverse). A
second riboprobe for Kv2 was generated using oligonucleotide primers
designed to amplify a 300 bp fragment spanning nucleotides 809-1108 of
the rat Kv2 cDNA and, in addition, to add the promoter sequences for
T3 RNA polymerase. These primers contained the following sequences:
5-ACCAGTGGTTGAAGGACAAG-3 (forward);
5-AATTAACCCTCACTAAAGGGTGACTTAGGATCTATAGTCC-3 (re-verse). All
PCR products were gel purified on 1.5% low-melt agarose gels, and
bands containing the Kv1 or Kv2 products were excised, phenol and
phenol-chloroform extracted, and ethanol precipitated. The pellet then
was dried and resuspended in 1× TE buffer containing (in
mM): 10 Tris/HCl, 1 EDTA, pH 7.4. Fifty nanograms
of DNA template were used for transcription reactions using
35S-CTP (New England Nuclear, Boston, MA) and the
Riboprobe Gemini System (Promega, Madison, WI). Probes were examined by
Northern analysis of rat brain RNA and by Southern analysis of plasmids
containing the Kv1 and Kv2 cDNAs. Each probe reacted with a
single band of appropriate size on Northern blots and with only the
appropriate cDNA on the plasmid Southern blots, indicating that these
riboprobes specifically recognized Kv1 and Kv2 transcripts (data
not shown).
In situ hybridization. Eight adult male Sprague-Dawley rats
were used for analysis of Kv1 and Kv2 mRNA expression by in
situ hybridization histochemistry. Animals were killed by
asphyxiation with CO2, and the brains were
removed, immediately frozen in a bed of pulverized dry ice, and stored
at 70°C. Sections were cut at 10 µm on a Hacker-Brights cryostat
and thaw mounted onto chilled (20°C) slides coated with Vectabond
reagent (Vector Labs, Burlingame, CA). All solutions were prepared in
dH2O treated with 0.1% (v/v)
diethylpyrocarbonate and autoclaved. Sections were fixed by immersion
in 4% paraformaldehyde in PBS, pH 7.4, then immersed sequentially in
2× SSC (1× SSC is 0.150 M sodium chloride,
0.015 M sodium citrate),
dH2O, and 0.1 M
triethanolamine, pH 8.0. The sections then were acetylated by immersion
in 0.1 M triethanolamine containing 0.25% (v/v)
acetic anhydride; washed in 0.2× SSC; dehydrated in 50, 70, and 90%
ethanol; and rapidly dried. One ml of prehybridization solution
containing 0.9 M NaCl, 1 mM
EDTA, 5× Denhardt's solution, 0.25 mg/ml single-stranded herring
sperm DNA (Gibco, Gaithersburg, MD), and 50% deionized formamide (EM
Sciences, Gibbstown, NJ) in 10 mM Tris, pH 7.6, was pipetted onto each slide, and the slides incubated for 3 hr at
50°C in a humidified box. The sections then were dehydrated by
immersion in 50, 70, and 90% ethanol and air dried.
Labeled riboprobes were denatured in a small volume (100 µl) of
hybridization solution containing 0.9 M NaCl, 1 mM EDTA, 1× Denhardt's solution, 0.1 mg/ml
yeast tRNA, 0.1 mg/ml single-stranded salmon sperm DNA, dextran sulfate
(10%), 0.08% BSA, 10 mM DTT (Boehringer
Mannheim, Indianapolis, IN), and 50% deionized formamide in 10 mM Tris, pH 7.6, at 95°C (1 min); placed on ice
(5 min); and added at a final concentration of 50,000 cpm/µl to 10 ml
of prewarmed (to 55°C) hybridization solution. The hybridization
solution then was pipetted onto the sections and allowed to hybridize
overnight at 55°C in a humidified chamber. The sections were
subsequently washed once for 45 min at 37°C in 2× SSC containing 10 mM DTT, once for 30 min at 37°C in 1× SSC
containing 50% formamide, and once for 30 min at 37°C in 2× SSC.
Single-stranded and nonspecifically hybridized riboprobe was digested
by immersion in 10 mM Tris, pH 8.0, containing
bovine pancreas RNase A (Boehringer Mannheim) (40 µg/ml), 0.5 M NaCl, and 1 mM EDTA. The
sections then were washed in 2× SSC for 1 hr at 60°C, followed by
0.1× SSC containing 0.5% (w/v) sodium thiosulfate for 2 hr at 60°C.
The sections then were dehydrated in 50, 70, and 90% ethanol
containing 0.3 M ammonium acetate, and dried.
The slides then were loaded into X-ray cassettes and exposed to
Hyperfilm -Max (Amersham, Arlington Heights, IL) for 3-7 d. Once a
satisfactory exposure was obtained, the slides were coated with
nuclear-track emulsion (NTB-2) (Eastman Kodak, Rochester, NY) and
exposed for 7-21 d at 4°C. The emulsion autoradiograms were
developed and fixed according to the manufacturer's instructions, and
the underlying tissue sections were stained with hematoxylin.
To assess nonspecific labeling in the in situ hybridization
procedure, a control probe was generated from a template provided in
the Riboprobe Gemini System kit (Promega catalog #P2651). This vector
was linearized using ScaI, and transcribed using T3 RNA
polymerase. The resulting transcription reaction generates two
products, a 250 bp and a 1525 bp riboprobe, containing only vector
sequence. This control probe mixture was labeled as described above and
added to the hybridization solution at a final concentration of 50,000 cpm/µl. No specific hybridization was observed in control sections;
i.e., these sections gave a very weak uniform hybridization signal that
did not follow neuroanatomical landmarks (data not shown).
Production of synthetic peptides and antibodies. Synthetic
peptides corresponding to amino acids 7-28 from the N terminus of the
rat Kv1 polypeptide (CTEHNLKSRNGEDRLLSKQSST) (Rettig et al., 1994)
and amino acids 1-17 of the rat Kv2 polypeptide
(MYPESTTGSPARLSLRQC) (Rettig et al., 1994) were synthesized (Quality
Controlled Biochemicals, Hopkinton, MA) and conjugated to keyhole
limpet hemocyanin (KLH) (1 mg peptide/mg carrier protein) using
sulfo-m-maleimidobenzoyl-N-hydroxysuccinimide
ester (Pierce, Rockford, IL). These peptide/KLH conjugates were
injected into rabbits for the production of polyclonal antisera (Pocono
Rabbit Farm, Canadensis, PA), and into mice for the production of
monoclonal antibodies (mAbs). Production of mAbs and purification of
monoclonal immunoglobulins were performed essentially as described
previously (Trimmer et al., 1985), and will be described in detail
elsewhere (Z. Bekele-Arcuri, M. Matos, J. Trimmer, unpublished
observations). For affinity purification of the polyclonal antibodies,
the Kv1 and Kv2 peptides were conjugated to SulfoLink coupling
gel (Pierce) via the terminal cysteine residues. Polyclonal
antibodies were affinity purified from immune serum by standard
procedures (Harlow and Lane, 1988).
Immunofluorescence. Immunofluorescence staining of
transiently transfected cells expressing Kv1 and Kv2 was
performed essentially as described previously (Shi et al., 1994).
Briefly, green monkey fibroblast COS-1 cells were cultured on glass
coverslips that had been previously coated with 25 µg/ml
poly-L-lysine. For staining, cells were washed
three times in ice-cold PBS, then fixed and permeabilized by treatment
for 20 min at 4°C with a freshly prepared ice-cold fixative
containing 3% paraformaldehyde/0.1% Triton X-100 in PBS. Cells then
were washed three times in TBS containing 0.1% Triton X-100 (TBS-T),
and nonspecific protein binding sites were blocked by incubation in
TBS-T containing 4.5% w/v nonfat dry milk powder (Blotto-T) (Johnson
et al., 1984). Cells then were incubated in primary antibodies diluted
in Blotto-T for 1 hr at room temperature, washed three times in
Blotto-T for 30 min total, and incubated in the appropriate secondary
antibodies for 30 min. After three washes in TBS-T for 15 min total,
cells were mounted in PBS medium containing 90% glycerol and 1 mg/ml
p-phenylenediamine and viewed on a Zeiss Axiophot microscope
using epifluorescence illumination.
Brain membrane preparations. A crude synaptosomal membrane
fraction was prepared from freshly dissected adult rat brains,
essentially as described previously (Trimmer, 1991, 1993). Briefly,
brains were homogenized in 0.3 M sucrose, 10 mM sodium phosphate, pH 7.4, and 10 mM sodium fluoride, containing a protease
inhibitor cocktail (1 mM phenylmethyl sulfonyl
fluoride, 1 µg/ml leupeptin, 2 µg/ml aprotinin, and 1 µg/ml
pepstatin). The resultant homogenate was centrifuged at 3,000 × g for 10 min to remove nuclei and cellular debris. The
supernatant then was centrifuged at 45,000 × g for 60 min to pellet the membranes. Aliquots of the membrane preparations were
suspended in the homogenization buffer, and protein was determined
using the BCA method (Pierce).
SDS/polyacrylamide gels and immunoblotting. For immunoblots,
50 µg of membrane protein was added to SDS sample buffer, boiled, and
fractionated on 7.5 or 12% SDS/polyacrylamide gels (Maizel, 1971).
Disulfide bonds were reduced by the addition of 20 mM 2-mercaptoethanol to the sample buffer. Lauryl
sulfate (Sigma) was the SDS source used for all SDS-PAGE (Shi et al.,
1994). After electrophoretic transfer to nitrocellulose paper, the
resulting blots were blocked in TBS containing 4% low fat milk
(Blotto) (Johnson et al., 1984), incubated in affinity-purified
antibody diluted 1:50-1:100 in Blotto for 1 hr or undiluted mAb tissue
culture supernatants, and washed three times in Blotto for 30 min
total. Blots then were incubated in HRP-conjugated secondary antibody
(1:2000 dilution in Blotto) (Cappel, West Chester, PA) for 1 hr and
then washed in TBS three times for 30 min total. The blots then were
incubated in substrate for enhanced chemiluminescence (ECL) (Amersham,
Arlington Heights, IL) for 1 min and autoradiographed on preflashed (to
OD545 = 0.15) Kodak X-OMAT LS film.
Immunoprecipitation. Immunoprecipitation reactions were
performed using detergent lysates of rat brain membranes. All
procedures were performed at 4°C. Membranes (1 mg membrane
protein/tube) were solubilized to 1 ml final volume/tube in lysis
buffer (1% Triton X-100, 0.15 M NaCl, 1 mM EDTA, 10 mM sodium
azide, 10 mM Tris/HCl, pH 8.0, containing a
protease inhibitor cocktail (see above). Affinity-purified antibodies
were added and the samples incubated for 2 hr on a rotator, followed by
addition of 20 µl of a 50% suspension of protein A sepharose and
additional incubation for 45 min. After incubation, protein A sepharose
was centrifuged at 10,000 × g for 20 sec, and the
resulting pellets were washed by resuspension and centrifugation six
times with lysis buffer. The final pellets were resuspended in 50 µl
reducing sample buffer, and 20 µl electrophoresed on 12% SDS-PAGE
and subjected to immunoblotting as described above.
Immunohistochemistry. Twelve adult male Sprague-Dawley rats
were deeply anesthetized with sodium pentobarbital (60 mg/kg, i.p.) and
perfused through the ascending aorta with 150 ml of 0.9% saline,
followed by 500 ml of fixative containing freshly prepared 4%
paraformaldehyde in 0.1 M
NaPO4 buffer, pH 7.4 (PB). The brains were
removed, cryoprotected for 18-48 hr in 20% sucrose in PB, frozen in a
bed of pulverized dry ice, and then cut into 35-µm-thick sections on
a sliding microtome. Consecutive 1-in-10 series of sections were
collected in 0.05 M PB and processed for light
microscopic immunohistochemistry as described previously (Rhodes et
al., 1995). Affinity-purified rabbit polyclonal anti-Kv1 and rabbit
anti-Kv2 antibodies were used at dilutions of 1:1500 and 1:4000,
respectively. Mouse mAbs (purified IgG fractions and tissue culture
supernatants) raised against Kv1 (clone K9/40) and Kv2 (clone
K17/70) were used at dilutions of 1:100-1:1500. To verify the
specificity of the immunohistochemical reactions, some sections were
processed either without addition of the primary antibody or using
antibodies incubated previously (1 hr) in vehicle containing an excess
of the synthetic peptide/BSA antigen (5-25 µg/ml). No specific
staining was observed in these control sections (data not shown).
Analysis of sections processed for in situ hybridization and
immunohistochemistry was performed using a Zeiss Axiophot
photomicroscope. Low-magnification photographs of immunohistochemically
stained sections and autoradiograms generated on film were taken using
a Nikon Multiphot macrophotography system.
RESULTS
Localization of Kv1 and Kv2 mRNA
General features
Analysis of autoradiograms prepared on film indicated that the
Kv1 and Kv2 mRNAs are widely and heterogeneously expressed in
adult rat brain (Table 1). The pair of
riboprobes for Kv1 generated patterns of hybridization signal that
were indistinguishable from one another, as did the pair of riboprobes
for Kv2. Taken together with the result of Northern and Southern
blotting using these probes, this result strongly suggests that the
riboprobes used in the present study specifically reveal the presence
of Kv1 or Kv2 mRNA.
A very high density of Kv1 expression was observed in the striatum,
nucleus accumbens, olfactory tubercle, CA1 subfield of the hippocampus,
and entorhinal and posterior cingulate cortices, and in several
midbrain and brainstem motor nuclei (Fig. 1).
Intermediate levels of Kv1 expression were observed in the piriform
cortex; neocortex; medial septal-diagonal band complex; anterior,
mediodorsal, and ventral tier thalamic nuclei; and the cerebellar
cortex and deep nuclei. Intermediate levels of Kv1 expression also
were observed in the laterodorsal, ventral posteromedial (VPM), ventral
posterolateral (VPL), and dorsal lateral geniculate, and medial
geniculate nuclei of the thalamus. Low levels of Kv1 expression were
observed in the globus pallidus and hypothalamus. The highest levels of
Kv2 expression were observed in the piriform cortex, hippocampal
formation, and in layer II of the entorhinal cortex, with somewhat
lower levels in the neocortex, medial septal-diagonal band complex,
and the anterior, VPM, and VPL nuclei of the thalamus. Low and levels
of Kv2 expression were observed in the remaining thalamic nuclei and
in the striatum, globus pallidus, and hypothalamus.
Fig. 1.
Expression of Kv1 and Kv2 mRNA in adult rat
brain. Horizontal and coronal sections of rat brain were processed by
in situ hybridization histochemistry to localize Kv1
(A, B, E-G) and Kv2
(C, D, H-J) mRNA.
Areas containing a high density of hybridization signal appear dark in
these bright-field images. The autoradiograms in
A-D were exposed for 3 d, whereas those in
E-J were exposed for 7 d. At the shorter
exposure time, subtle differences in expression levels are more easily
discernible. For example, there is a comparatively greater density of
Kv1 mRNA in the CA1 subfield of the hippocampus as compared with the
adjacent CA3 subfield (B). CA1, Hippocampal
subfield; CB, cerebellum; CPu, caudate putamen;
EC, entorhinal cortex; RN, red nucleus;
ms, medial septal nuclei; AN, anterior thalamic
nucleus; MD, mediodorsal thalamic nucleus.
[View Larger Version of this Image (143K GIF file)]
Neocortex and hippocampus
Examination of high-resolution emulsion autoradiograms of sections
processed to visualize hybridization to Kv1 and Kv2 mRNAs
indicated that these two transcripts are expressed within cellular
profiles with the size and morphology of neurons as opposed to glial
cells (Fig. 2). Kv1 mRNA was expressed in large
pyramidal cells in the deep half of layer III and in layers V and VI.
Very high levels of Kv1 mRNA were observed in small to medium
interneurons in cortical layers II, III, V, and VI. Interestingly, high
levels of Kv1 mRNA also were observed in small cells juxtaposed to
the subcortical white matter (Fig. 1). In virtually all regions of the
neocortex, Kv2 mRNA was highly expressed in pyramidal cells in
layers II, III, V, and VI. In proisocortical areas such as the
entorhinal and cingulate cortices, Kv1 and Kv2 mRNA also were
highly expressed in large multipolar neurons in layer II (Fig. 2).
Fig. 2.
Cellular localization of Kv1 and Kv2 mRNA
expression. Emulsion autoradiograms were prepared to localize Kv1
and Kv2 mRNA within individual cells. Cells containing a high
density of mRNA contain a correspondingly high density of silver
grains, which appear as bright spots in these dark-field
images. In posterior cingulate cortex (area 23), Kv1 mRNA
(A) is highly expressed in many small cells in layers II and
III and larger cells in layer V (arrows), whereas Kv2
(B) is expressed predominantly in small cells in layer II
and larger cells in layer V (arrows). In the entorhinal
cortex, Kv1 mRNA (C) is expressed in large and small
cells in layers II, III, V, and VI, whereas Kv2 mRNA (D)
is expressed at high density in layer II cells and with a lower density
in cells in the remaining layers of this structure. In the caudate
nucleus, there is a very high level of Kv1 expression (E)
and a lower level of Kv2 expression (F) in
virtually all cells (arrows). The bundles of myelinated
axons that course through this structure do not contain hybridization
signal (arrowheads). In the basal forebrain, Kv1
(G) and Kv2 (H) mRNAs are expressed in
large cells (arrows) with the distribution and frequency
characteristic of the large cholinergic neurons present in this region.
The short arrows in G and H mark the
midline of the brain.
[View Larger Version of this Image (213K GIF file)]
In the hippocampus, Kv1 mRNA was expressed in dentate granule cells
and in pyramidal cells of all subfields. However, pyramidal cells in
the CA1 subfield expressed higher levels of Kv1 mRNA than the
adjacent CA2 subfield and subiculum (Fig. 1). In addition, Kv1 was
expressed in large interneurons located in stratum oriens and radiatum
of all subfields. The distribution of Kv2 mRNA was strikingly
similar to that observed for Kv1, with the exception that there
appeared to be a uniformly high density of Kv2 expression across all
subfields, and Kv2 mRNA did not appear to be expressed at an
appreciably greater density in hippocampal interneurons.
Striatum and basal forebrain
The greatest density of Kv1 expression was observed in the
caudate putamen, nucleus accumbens, and olfactory tubercle. Virtually
all neurons in these structures expressed extremely high levels of
Kv1 mRNA (Figs. 1, 2E). Kv1 mRNA also was highly
expressed in the medial septal nuclei and the vertical and horizontal
limbs of the diagonal band of Broca in a distribution that closely
resembled the distribution of cholinergic neurons in these structures
(Fig. 2G) (Mesulam et al., 1983). In contrast to Kv1,
there was a low level of Kv2 expression in the caudate putamen,
nucleus accumbens, and olfactory tubercle (Figs. 1, 2F).
However, there was a high level of Kv2 expression in the medial
septal and diagonal band nuclei in a pattern that overlapped
with, but was somewhat more extensive than, that observed for Kv1
(Figs. 1, 2H).
Thalamus and hypothalamus
A moderate density of Kv1 expression was observed throughout
the thalamus, with a somewhat greater density in the anterior,
mediodorsal, and entopeduncular nuclei (Fig. 1), and a moderate density
in the laterodorsal, VPM, and VPL nuclei. A low level of Kv1
expression was observed in the hypothalamus, but with a somewhat more
intense hybridization signal in the ventromedial hypothalamic nucleus.
A low to moderate level of Kv2 expression was observed throughout
the thalamus, with a somewhat greater density in the anterior,
mediodorsal, paracentral, and ventroposterior nuclei. Low levels of
Kv2 expression also were observed in the hypothalamus, however, as
for Kv1, there was a somewhat greater density of Kv2 expression
in the ventromedial nucleus.
Midbrain
Several midbrain motor nuclei, including the red nucleus and
all cranial nerve nuclei, contained a very high density of Kv1 and
Kv2 expression (Fig. 1). In addition, cells in the superficial and
deep layers of the superior and inferior colliculi, the periaqueductal
gray, and the pars compacta and pars reticulata of the substantia nigra
expressed intermediate levels of Kv1 mRNA. Similarly, large neurons
in the deep layers of the superior and inferior colliculi expressed
moderate levels of Kv2 mRNA, and cells scattered throughout the
substantia nigra pars compacta and pars reticulata expressed moderate
to low levels of Kv2 mRNA (Fig. 1).
Cerebellum and brainstem
In the cerebellar cortex, Purkinje cells and granule cells
displayed high and moderate levels of Kv1 expression, respectively,
and small neurons scattered throughout the molecular layer expressed
much lower levels of Kv1 mRNA. In contrast, Purkinje cells and
granule cells displayed intermediate levels of Kv2 expression, and
small neurons with a size and distribution similar to basket cells
expressed moderate levels of Kv2 mRNA. Large neurons in all deep
cerebellar nuclei expressed moderate levels of Kv1 and Kv2 mRNA
(Fig. 1).
In the pons and medulla, all cranial nerve sensory and motor nuclei
displayed moderate to high levels of Kv1 and Kv2 expression. In
addition, mRNA for both subunits was highly expressed in large
pontine and medullary reticular neurons and in neurons of the superior
and inferior olivary complexes.
Generation and characterization of Kv1- and Kv2-specific
polyclonal and mAbs
A polyclonal antiserum was produced by the immunization of rabbits
with a synthetic Kv1 peptide immunogen, corresponding to N-terminal
amino acids 7-28 of the deduced rat brain Kv1 sequence (Rettig et
al., 1994). This sequence is not present in the deduced sequence of the
rat brain Kv2 subunit, although some overlap is seen to a
recently identified rat brain Kv3 subunit (11/22 positions
identical with changes spread throughout the sequences) (Heinemann et
al., 1995; Pongs, 1995). Thus, it is likely that antibodies generated
to this peptide are specific for Kv1. Rabbit polyclonal antibodies
raised against this peptide exhibited a high titer against the peptide
on ELISA assays and displayed a monospecific reaction on immunoblot
assays versus rat brain membranes to a 44 kDa polypeptide (Fig.
3). That this peptide was Kv1 was supported by
analysis of Kv1 and Kv2 expressed in COS-1 cells, which showed
that the expressed recombinant Kv1 polypeptide shared
immunoreactivity and electrophoretic mobility with the putative rat
brain Kv1 polypeptide (Fig. 3). No corresponding immunoreactivity
was seen to the recombinant 38 kDa Kv2 polypeptide expressed in
COS-1 cells or present in the crude rat brain membranes on the same
immunoblot (Fig. 3). Immunofluorescence staining of transfected COS-1
cells revealed a similar pattern of immunoreactivity in that the
anti-Kv1 antibody exhibited a strong immunofluorescence staining of
Kv1- , but not Kv2- , transfected cells (Fig.
4).
Fig. 3.
Immunoblot analyses of the Kv1 and Kv2
-subunit polypeptides in rat brain membranes and in transfected
COS-1 cells. Crude rat brain membranes (RBM) (50 µg) and
the detergent extracts of COS-1 cells transfected with Kv1/RBG4
(Kv1), Kv2/RBG4 (Kv2), or RBG4 alone
(vector) were fractionated on a 12.5% SDS gel and
transferred to nitrocellulose, and the resultant immunoblot probed with
either rabbit anti-Kv1 polyclonal antibody at 1:50 (left)
or mouse anti-Kv2 mAb K17/70 tissue culture supernatant neat
(right). Signals were visualized using ECL (left,
30 min; right, 1 min). Numbers on left
refer to mobility of prestained molecular weight standards.
[View Larger Version of this Image (47K GIF file)]
Fig. 4.
Analysis of antibody specificity.
Immunofluorescence staining of Kv1 and Kv2 subunits expressed
in COS-1 cells. COS-1 cells were transfected with Kv1/RBG4
(A-D) or Kv2/RBG4
(E-H) cDNAs. Transfected cells then were
fixed, permeabilized, and incubated with rabbit anti-Kv1 polyclonal
antibody at 1:100 (A, E), rabbit anti-Kv2
polyclonal antibody at 1:200 (B, F), mouse
anti-Kv1 mAb K9/40 at 1:2 (C, G), or
mouse anti-Kv2 mAb K17/70 at 1:2 (D,
H). Cells then were incubated with Texas Red-conjugated
anti-rabbit (A, B, E,
F) or anti-mouse (C, D,
G, H) secondary antibody.
[View Larger Version of this Image (72K GIF file)]
mAbs generated against the same Kv1-specific peptide exhibited
similar properties. All of the mAbs exhibited strong reactions in ELISA
assays against the Kv1 peptide immunogen and in immunofluorescence
staining of COS-1 cells expressing recombinant Kv1 polypeptide (Fig.
4). Staining was not observed in nontransfected or Kv2-transfected
cells (Fig. 4). Immunoblot analysis of crude rat brain membranes
revealed that none of the mAbs isolated recognized the SDS-denatured
rat brain Kv1 polypeptide, although specific immunoreactivity was
seen to the recombinant Kv1 (but not Kv2) polypeptide present in
COS-1 cell extracts, perhaps because of the higher expression levels of
Kv1 in these extracts compared with that observed in rat brain
membranes (data not shown). All of these mAbs, and the rabbit
polyclonal antibody, selectively immunoprecipitate the recombinant
Kv1 polypeptide present in 35S-methionine
labeled COS-1 cell extracts (data not shown). Additional details on the
generation and characterization of these mAbs will be described
elsewhere.
A polyclonal antiserum was produced by the immunization of rabbits with
a synthetic Kv2 peptide immunogen, corresponding to N-terminal amino
acids 1-17 of the deduced rat brain Kv2 sequence (Rettig et al.,
1994). This sequence is not present in the deduced sequence of the rat
brain Kv1 or Kv3 subunit (Heinemann et al., 1995; Pongs,
1995). Thus, it is likely that antibodies generated to this peptide
would be specific for Kv2. Rabbit polyclonal antibodies raised
against this peptide exhibited a high titer against the Kv2 peptide
on ELISA assays and displayed a monospecific reaction on immunoblot
assays versus rat brain membranes to a 38 kDa polypeptide (Fig. 3).
Additional immunoreactivity was exhibited to a minor, 41 kDa
polypeptide (Fig. 3) that is also recognized by the pan--subunit
antibody (Rhodes et al., 1995). The molecular identity of this 41 kDa
polypeptide is not known; however, the fact that it exhibits
immunoreactivity to both the N-terminally directed anti-Kv2 antibody
and the C-terminally directed pan- antibody indicates that it is
either a post-translational variant of Kv2 or an alternative splice
variant of the Kv2 gene. That the 38 kDa polypeptide was Kv2 was
supported by analysis of Kv1 and Kv2 expressed in COS-1 cells,
which showed that the expressed recombinant Kv2 polypeptide shared
immunoreactivity and electrophoretic mobility
(Mr = 38 kDa) with the putative rat brain
Kv2 polypeptide (the 41 kDa variant was not detected). No
corresponding immunoreactivity was seen to the recombinant 44 kDa
Kv1 polypeptide expressed in COS-1 cells or present in the crude rat
brain membranes on the same immunoblot (Fig. 3). Immunofluorescence
staining of transfected COS-1 cells revealed a similar pattern of
immunoreactivity in that the anti-Kv2 antibody exhibited a strong
immunofluorescence staining of Kv2- but not Kv1- transfected
cells (Fig. 4).
Anti-Kv2 mAbs were generated against a
glutathione-S-transferase fusion protein containing the
entire Kv2 polypeptide. All of the N-terminally directed mAbs
exhibited monospecific immunofluorescence staining of COS-1 cells
expressing recombinant Kv2, but not Kv1, polypeptide (Fig. 4). No
staining to untransfected cells was seen (Fig. 4). Immunoblot analysis
of crude rat brain membranes revealed that only the K17/70 mAb
recognizes the SDS-denatured rat brain Kv2 polypeptide and
recombinant Kv2 (but not Kv1) polypeptide present in COS-1 cell
extracts (Fig. 3). Additional details on the generation and
characterization of these mAbs will be described elsewhere.
Reciprocal co-immunoprecipitations: subunit association
To determine whether Kv1 and Kv2 were present in the same
rat brain K+ channel complexes, we performed
reciprocal co-immunoprecipitation experiments with the anti-Kv1 and
anti-Kv2 antibodies. Detergent lysates were prepared from rat brain
membranes under conditions shown previously to preserve - or
-subunit interactions (Sheng et al., 1993; Wang et al., 1993; Rhodes
et al., 1995; Nakahira et al., 1996; Shi et al., 1996). These lysates
then were used in immunoprecipitation reactions performed with the
anti-Kv1 and anti-Kv2 polyclonal antibodies and with a pan-
polyclonal antibody that recognizes both Kv1 and Kv2 (Rhodes et
al., 1995). Immunoprecipitations also were performed using a polyclonal
antibody against the Kv2.1 subunit, which should not
co-immunoprecipitate either of these subunits (Rhodes et al.,
1995). Immunoprecipitation reactions then were subjected to immunoblot
analyses to assay for the presence of Kv1 with the anti-Kv1
polyclonal antibody, for Kv2 with the K17/70 mAb, and for the
-subunit-associated subunit Kv1.2 with the Kv1.2C polyclonal
antibody (Rhodes et al., 1995).
As expected, the anti-Kv1 antibody immunoprecipitated the 44 kDa
Kv1 polypeptide from these rat brain extracts (Fig.
5), as did the anti-pan- polyclonal antibody, whereas
the anti-Kv2.1 antibody did not immunoprecipitate detectable amounts of
Kv1. That the same amount of rabbit IgG was precipitated in each of
these reactions is demonstrated by the ~50 kDa band present in each
immunoprecipitation lane that is attributable to immunoreactivity of
the rabbit IgG from the immunoprecipitating antibody, with the
anti-rabbit secondary antibody used to develop the immunoblots.
Surprisingly, levels of Kv1 comparable to those observed in the
anti-Kv1 reaction were co-immunoprecipitated by the anti-Kv2
polyclonal antibody. Because these immunoprecipitation reactions were
performed under conditions in which antigen is limiting, these data
demonstrate that a large proportion of the Kv1 found in rat brain is
present in complexes that also contain the Kv2 subunit (Fig.
5).
Fig. 5.
Presence of Kv1 and Kv2 in rat brain
K+ channel complexes. Samples of adult rat brain
membranes (RBM) (60 µg) and aliquots of products of
immunoprecipitation reactions from detergent extracts of 500 µg RBM
with polyclonal antibodies specific for both Kv1 and Kv2
(pan-, 1:100), Kv1 (anti-Kv1,
1:200), Kv2 (anti-Kv2, 1:500), or the Kv2.1 subunit (anti-Kv2.1, 1:200) were size fractionated by 12.5%
SDS-PAGE. Samples were transferred to nitrocellulose and probed with
rabbit anti-Kv1 polyclonal antibody at 1:50 (left
panel), mouse anti-Kv2 mAb K17/70 neat (middle
panel), or rabbit anti-Kv1.2 polyclonal antibody at 1:100
(right panel). Bound antibody detected by
ECL-autoradiography for 40 min (left panel) or 3 min
(middle and right panels). Arrows
point to mobility of the heavy-chain polypeptides of the rabbit
immunoglobulins used in the immunoprecipitation reactions and of the
respective K+ channel polypeptides;
numbers at left of the panels denote
Mr of prestained molecular weight
standards. Bands at ~50 kDa (left and right
panels) are heavy chains of rabbit IgG used for
immunoprecipitations, which react with anti-rabbit, but not anti-mouse,
secondary antibody.
[View Larger Version of this Image (29K GIF file)]
These same immunoprecipitation reactions then were assayed for the
presence of Kv2; because these immunoblots were performed with the
anti-Kv2 mouse mAb K17/70, the rabbit IgG bands typically seen in
the immunoprecipitation reactions are not visualized. The rabbit
anti-Kv2 polyclonal antibody was able to immunoprecipitate the 38 kDa Kv2 polypeptide present in these samples. Comparable amounts of
Kv2 also were observed in the immunoprecipitation reaction performed
with the anti-pan- polyclonal antibody (Fig. 5). Low levels of
Kv2 also were observed in the reactions performed with the
anti-Kv1 antibody, confirming the observation above and indicating
that individual K+ channel complexes may contain
both of these -subunit polypeptides. As expected, the anti-Kv2.1
antibody also was unable to co-immunoprecipitate detectable amounts of
Kv2. All of the immunoprecipitation reactions performed with the
anti--subunit antibodies contained Kv1.2 (Fig. 5), showing that each
of these antibodies could effectively recognize and isolate intact
K+ channel complexes.
Immunohistochemical localization of Kv1 and Kv2
Analysis of immunohistochemically stained sections indicated that
Kv1 and Kv2 are present in cell bodies, dendrites, the
juxtaparanodal regions of myelinated axons, and terminal fields of
several major projection systems. The pattern of immunohistochemical
staining observed with mAbs and polyclonal antibodies to the same subunit was indistinguishable. The areal and laminar distribution of
labeled cells suggests and comparisons of sections processed by
in situ hybridization and immunohistochemistry suggest that
cells expressing Kv1 and Kv2 mRNA also are immunoreactive for the
corresponding proteins. Regions containing moderate to intense
immunoreactivity for Kv1 and Kv2 were the neocortex, hippocampus,
piriform cortex, striatum, thalamus, cerebellum, cranial nerve nuclei,
and virtually all major white matter pathways. Although it is beyond
the scope of this paper to provide a comprehensive account of
-subunit immunoreactivity in every brain region, the salient
features of the immunohistochemical staining are described below and
summarized in Table 1.
Neocortex and hippocampus
In the neocortex, immunoreactivity for Kv1 was concentrated
within pyramidal cells and was particularly intense in smaller bipolar
and multipolar interneurons in layers II, III, V, and VI (Fig.
6A). In each of these cell populations,
immunoreactivity for Kv1 was concentrated within the cell body and
proximal portions of the dendritic tree. Immunoreactivity for Kv2
also was observed in cortical pyramidal cells, with very intense
labeling of large pyramidal cells in layer V (Fig. 6C).
However, in contrast to immunoreactivity for Kv1, immunoreactivity
for Kv2 was concentrated throughout the entire apical dendrite of
layer V pyramidal cells, including fine branches and apical tufts.
Interestingly, the small interneurons that displayed strong
immunoreactivity for Kv1 were not intensely immunoreactive for
Kv2. In addition to the somatodendritic staining described above, in
many cortical regions, moderate to intense immunoreactivity for Kv1
and Kv2 was observed in axonal profiles coursing throughout the
cortical neuropil and in a pattern consistent with labeling of terminal
fields. This pattern of labeling was particularly intense in the
piriform, posterior cingulate, retrosplenial, and entorhinal cortices.
Myelinated axons in the subcortical white matter as well as other
regions also contained immunoreactivity for Kv1 and Kv2. The
staining in these fibers was discontinuous, with a greater density of
reaction product in the juxtaparanodal segments at nodes of Ranvier
(Shi et al., 1996).
Fig. 6.
Immunohistochemical localization of Kv1 and
Kv2 in the cerebral cortex and striatum. In parietal cortex,
immunoreactivity for Kv1 (K9/40 mAb) (A) is concentrated
in large pyramidal cells in layer V and in small interneurons
(arrows) concentrated primarily in layers II and III.
Immunoreactivity for Kv2 (affinity-purified polyclonal antibody)
(C) is concentrated in the somas and apical dendrites of
layer V pyramidal cells. The small interneurons that contain a high
density of immunoreactivity for Kv1 contain a much lower density of
immunoreactivity for Kv2. In the striatum, there is a very high
density of immunoreactivity for Kv1 (B) and Kv2
(D) in the globus pallidus (GP) and a lower
density in the caudate nucleus (CD). In the globus pallidus,
immunoreactivity for Kv1 and Kv2 is primarily concentrated in
terminal fields and is present in both the supra- and subcommissural
segments. AC, Anterior commissure.
[View Larger Version of this Image (159K GIF file)]
In the hippocampal formation, there was a heterogeneous
distribution of Kv1 and Kv2 immunoreactivity with moderate to
intense staining of granule and pyramidal cell somata (data not shown).
Immunoreactivity for Kv1 and Kv2 also was concentrated within
interneurons in stratum oriens and stratum radiatum of the CA1-CA3
subfields. In the dentate gyrus, granule cells contained a moderate
density of Kv1 and Kv2 immunoreactivity. In these cells, staining
was concentrated in the cell soma and did not extend into dendritic
branches, suggesting that this staining is associated with subunits
synthesized within these cells. In the molecular layer of the dentate
gyrus, there was a distinct band of Kv1 and Kv2 immunoreactivity
in the middle third and somewhat less intense labeling in the outer
third. As described previously (Rhodes et al., 1995), the location of
this band of intense immunoreactivity corresponds closely to the
termination zone of the medial perforant path. In the CA subfields and
subiculum, immunoreactivity for Kv1 and Kv2 was distributed
diffusely throughout the neuropil, with a greater density in stratum
radiatum and stratum oriens than in stratum moleculare, and
immunoreactivity for Kv2, but not Kv1, was concentrated
throughout the apical dendrites and apical dendritic tufts of
hippocampal pyramidal cells. In the CA3 subfield, there was a moderate
density of Kv1 immunoreactivity in the mossy fiber zone.
Striatum and basal forebrain
In the caudate nucleus and nucleus accumbens, there was a moderate
density of immunoreactivity for Kv1 and Kv2 within neurons of all
sizes and morphologies and distributed diffusely throughout the
neuropil. Interestingly, there was a higher density of immunoreactivity
for Kv1 and Kv2 in bundles of axons coursing through the
ventromedial portion of the caudate nucleus than in similar fiber
bundles in the dorsolateral component of this structure. These fibers
appeared to originate from cells within the caudate nucleus and could
be followed in consecutive sections to their apparent termination
throughout the supra- and subcommissural segments of the globus
pallidus (Fig. 6B,D). Additional fibers showing
intense immunoreactivity for Kv1 and Kv2 appeared to continue
beyond the globus pallidus and run through the medial forebrain bundle
and cerebral peduncle to terminate within the substantia nigra pars
reticulata (Fig. 7A,C). Intense
terminal-field labeling for Kv1 and Kv2 also was observed in the
nucleus accumbens and olfactory tubercle, and in the deep half of the
molecular layer of pyriform cortex.
Fig. 7.
Immunohistochemical localization of Kv1 and
Kv2 in the midbrain and cerebellum. In the midbrain,
immunoreactivity for Kv1 (K9/40 mAb) (A) and Kv2
(affinity-purified polyclonal antibody) (C) is concentrated
in terminal fields in the pars reticulata of the substantia nigra
(SNr) and in large neurons in the red nucleus
(RN). In the cerebellar cortex, immunoreactivity
for Kv1 (B) and Kv2 (D) is concentrated in
the cell bodies of Purkinje cells (P) and in terminal fields
throughout the molecular layer. Immunoreactivity for Kv2 also is
concentrated in the dendrites of Purkinje cells and in the axon
terminals of basket cells, which form a characteristic synaptic plexus
(arrows) that terminates on the initial segment of Purkinje
Previous Article | Next Article
