![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Vol. 291, Issue 3, 943-952, December 1999
-Subunits1
Laboratoire de Neurobiologie Intégrative et Adaptative, Centre National de la Recherche Scientifique-Université de Provence, Marseille, France (C.M.); Laboratoire de Neurobiologie, Centre National de la Recherche Scientifique, Marseille, France (R.B., G.J., M.G., M.C.); Molecular Medicine and Renal Units, Beth Israel Deaconess Medical Center, Departments of Medicine and Cell Biology, Harvard Medical School, Boston, Massachusetts (M.M.C., S.L.A.); and Laboratoire d'Ingénierie des Protéines, Centre National de la Recherche Scientifique-Université de la Méditerranée, Marseille, France (M.-F.M.-E.)
 |
Abstract |
|---|
 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
|
|---|
The distribution of the binding sites for kaliotoxin (KTX), a blocker
of voltage-dependent K+ channels, was studied with
quantitative autoradiography in adult rat brain and during postnatal
brain maturation. Iodinated KTX bound specifically to tissue sections
with a high affinity (Kd = 82 pM) and a
maximal binding capacity of 13.4 fmol/mg protein. The distribution of
KTX binding sites within the central nervous system was heterogeneous.
The highest densities were found in the neocortex, hypothalamus,
dentate gyrus, bed nucleus of the stria terminalis, and parabrachial
nuclei. The lowest level was observed in the white matter. From
postnatal day 5 onward, KTX binding sites were detectable only in the
hindbrain. The density of KTX binding sites in whole brain drastically
increased after postnatal day 15 to achieve adult levels at postnatal
day 60 in the whole brain. Bath application of KTX to Xenopus
laevis oocytes blocked recombinant Kv1.3 and Kv1.1 channels
potently and Kv1.2 channels less potently, with respective
Kd values of 0.1, 1.5, and 25 nM. KTX
affinities for each of these channels expressed in mammalian cells were
about 10-fold lower. A comparison of the distribution of KTX binding
sites with that of Kv1 channel polypeptides, together with the
pharmacology of KTX block, suggests that the principal targets for KTX
in rat brain are K+ channels containing Kv1.1 and Kv1.3
-subunits.
| |
Introduction |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Excitable
cells express voltage-gated potassium (Kv) channels. These membrane
proteins regulate the resting membrane potential, control the firing
pattern, and modulate the neurotransmitter release. This diversity of
function is reflected in the heterogeneity of Kv channel genes and
their sensitivities to various blockers. Based on their amino acid
sequences, the subunits of Kv channels have been classified into
nine families, Kv1 to Kv9. Each family has several members, and the
Shaker (Kv1) family includes nine cloned genes, Kv1.1 to Kv1.9
(Stühmer et al., 1989
; Pongs, 1992; Chandy and Gutman, 1995). Kv
channels expressed in vivo appear to be homotetramers or
heterotetramers of -subunits, sometimes but not always associated
with regulatory 
-subunits (Sheng et al.; 1993, Rhodes et al., 1997;
Shamotienko et al., 1997). Such combinatorial associations increase
greatly the Kv channel diversity.
As a consequence, our knowledge on the function of a given Kv channel
in situ is presently restricted by several experimental limitations;
these include the similarities in electrophysiological properties among
some subunits and difficulties in determination of in situ Kv
channel subunit composition (Robertson, 1997). One approach to this
problem is the autoradiographic detection of tissue binding sites of a
radiolabeled toxin of well-defined Kv -subunit specificity. For
example, the distribution in mammalian brain of the binding sites of
radiolabeled -dendrotoxin (-DTX) correlated with the localization
of Kv1.1, Kv1.2, and Kv1.6 subunits (Shamotienko et al., 1997) and that
of radiolabeled margatoxin (MgTX) with Kv1.2 and Kv1.3 subunits
(Grissmer et al., 1994; Scott et al., 1994; Koch et al., 1997;
Shamotienko et al., 1997).
Kaliotoxin (KTX) is a 38-amino-acid residue toxin purified from the
venom of the Androctonus mauritanicus mauritanicus scorpion (Crest et al., 1992). KTX belongs to a toxin family that includes three
agitoxins (Garcia et al., 1994), Buthus martensi toxin
(Romi-Lebrun et al., 1997), and KTX2 (Laraba-Djebari et al., 1994).
Agitoxin 2 blocks Kv1.1, Kv1.2, Kv1.3, and Kv1.6 channels, and B. martensi toxin blocks Kv1.3 channels. We have previously
characterized KTX as a blocker of Ca2+-activated
K+ channels of intermediate conductance
(IKCa) in Helix neurons (Crest et al.,
1992). Because Kv channels in these neurons were found to be
insensitive to KTX, we proposed KTX as an IKCa
channel blocker. In addition, because charybotoxin (CTX) had been shown to block both Helix IKCa channels
(Hermann and Erxleben, 1987) and mammalian skeletal muscle high
conductance Ca2+-activated K+ (BK)
channels (Miller, 1995), we initially hypothesized that KTX might block
BK-type channels.
Later studies demonstrated, however, that KTX competes with DTX and
mast cell-degranulating (MCD) peptide in rat brain synaptosomes (Romi
et al., 1993; Laraba-Djebari et al., 1994). Grissmer et al. (1994) then
showed that KTX blocks Kv1.3 and Kv1.1 channels, stably expressed in
mammalian cell lines. The KTX binding site on the exofacial surface of
Kv1.3 was later mapped by thermodynamic coupling analysis with mutant
toxins and channels (Aiyar et al., 1996). Although Grissmer et al.
(1994) found Kv1.2 channels expressed in mammalian cells insensitive to
KTX, Hopkins et al. (1996) reported that KTX blocks Kv1.2 channels
expressed in Xenopus laevis oocytes at nanomolar concentrations.
The present study was undertaken to reevaluate the KTX sensitivity of selected mammalian Kv1 channels and then to identify KTX binding sites in adult rat brain. The results demonstrate that KTX potently blocks Kv1.1 and Kv1.3 channels but more weakly blocks Kv1.2. The distribution of KTX receptors in rat brain is heterogeneous, with high densities in neocortex, hypothalamus, and some hindbrain nuclei. KTX binding sites are detectable only after postnatal day 5 (P5) and increase to adult levels by P60.
| |
Materials and Methods |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Heterologous Expression of Kv Channels
Expression in Oocytes.
Mouse Kv1.1, Kv1.3, and Kv3.1 cDNAs
were gifts from K. G. Chandy (University of California, Irvine,
CA). Kv1.1 was subcloned into pXT7, a modified Bluescript vector
bracketing the insert with 5'- and 3'-noncoding sequences derived from
X. laevis -globin. cRNA was transcribed with T7 RNA
polymerase (Megascript Kit; Ambion, Austin, TX) from
PstI-linearized plasmid template. Kv1.3 was subcloned into pSP64T, another X. laevis -globin noncoding
region vector, and cRNA was transcribed with SP6 RNA polymerase from
EcoRI-linearized plasmid template. Kv3.1, subcloned into
pBluescript vector with a poly(A)+ tail, was transcribed
with T3 RNA polymerase from SacI-linearized plasmid
template. Rat Kv1.2 cDNA (a gift from E. Peralta, Harvard University,
Boston, MA) was transcribed with T3 RNA polymerase from
XbaI- or NotI-linearized plasmid
template. cRNAs were stored at 
20°C in water at 1 µg/ml. cRNAs
were injected into X. laevis oocytes in 50-nl volumes at
concentrations of 4 to 10 ng/ml, and currents were recorded 1 to 4 days
after injection.
Expression in Mammalian Cells. Human embryonic kidney cells stably transfected with human Kv1.1 and Kv1.2 cDNAs in pcDNA3 were kindly provided by O. Pongs (Zentrum für Molekulare Neurobiologie, Universität Hamburg, Hamburg, Germany). Cells were maintained in Dulbecco's modified Eagle's medium/Ham's F-12 (BioMedia, Boussens, France) supplemented with 10% FCS, 50 µg/ml streptomycin, 50 U/ml penicillin, and 4 mM L-glutamine (GIBCO BRL Life Technologies, Cergy-Pontoise, France).
Jurkat JH 6.2 T cells constitutively expressing Kv1.3 (kindly provided by E. Beraud-Juven, Laboratoire d'Immunologie, Faculté de Médecine, Marseille, France) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% FCS and 100 µg/ml streptomycin, 100 U/ml penicillin (GIBCO BRL Life Technologies). Cells were plated onto Nunc (Roskilde, Denmark) dishes 3 days before use in electrophysiological experiments.Electrophysiological Recordings
Voltage-Clamp Experiments in Oocytes.
Two-electrode
voltage-clamp recordings were performed using a Gene Clamp 500 amplifier (Axon Instruments, Burlingame, CA). Oocytes were bathed in
calcium-free saline containing 88 mM NaCl, 1 mM KCl, 0.8 mM
MgSO4, 2.4 mM Na2CO3, and 10 mM
HEPES, pH 7.4. Intracellular electrodes were filled with 3 M KCl. The
holding potential was set at 80 mV. The perfusion system, designed to deliver a fast change in toxin concentration, was as previously described (Cotton et al., 1997). KTX was synthesized by J. van Rietschoten (CNRS UMR 6560, Marseille), and the steady-state effects of
KTX were determined after 15 min of treatment.
Patch-Clamp Experiments in Mammalian Cells.
Whole-cell
recordings were performed on cells bathed in 137 mM NaCl, 5.3 mM KCl,
0.8 mM MgSO4, 0.4 mM Na2HPO4, 1.8 mM CaCl2, and 10 mM HEPES, pH 7.4. Patch pipettes were
pulled on a P87 Sutter puller (Novato, CA) from borosilicate glass
capillaries (Clark Instruments, Pangbourne, UK) and filled with 130 mM
KCl, 2 mM MgCl2, 10 mM EGTA, and 10 mM HEPES, pH 7.4. Holding potential was set at 80 mV. KTX was delivered to the bath via
pneumatic picopump system (WPI, Aston, UK) by the application of
pressure to a broken patch pipette.
Whole-Cell Current Analysis
Currents from voltage- and patch-clamp experiments were sampled at 2 kHz. Software for stimulation, acquisition, and analysis was custom-written by H. Chagneux (CNRS UPR 9024). Dose-response curves were determined by the successive addition of KTX at increasing concentrations. Each point was the mean ± S.E. of three to nine experiments. Experimental points were fitted to the theoretical hyperbolic curve: y = 1/[1 + ([T]/IC50)n], where y is the fraction of unblocked current, IC50 is the concentration of toxin inducing 50% block, and n is the Hill coefficient corresponding to the number of molecules required to block one channel. When n = 1, the interaction KTX/channel is bimolecular, and IC50 values corresponds to the dissociation constant (Kd).
Animals and Tissue Preparation
Adult male Sprague-Dawley rats weighed 150 to 200 g. Postnatal rats were raised in litters of five pups of either sex, and the day of birth was designated P0. The minimum number of rats used in each experiment at each postnatal stage (P0, P3, P5, P7, P9, P15, P21, P60) was three infants from three different Sprague-Dawley females. They were given food and water ad libitum at constant room temperature under a 12-h light/dark cycle.
Anesthetized rats were sacrificed by decapitation, and their brains
were immediately removed and frozen in isopentane at 40°C. Coronal
or sagittal cryostat sections (15 µm) were collected, thaw-mounted
onto cold chrom-alum/gelatin-coated glass slides, and stored at
60°C until used.
Binding and Autoradiographic Procedures
The binding of KTX to Kv channel proteins was performed on
tissue sections using a highly radioactive KTX synthesized and radiolabeled using 125I as previously described
(Romi et al., 1993). The brain sections were incubated with 1 to 200 pM
125I-KTX (for binding studies) or with 20 pM
125I-KTX (for autoradiographic procedures), at
4°C in a 20 mM Tris-Cl buffer, pH 8, containing 50 mM NaCl and 1%
BSA. The nonspecific binding component was measured by the addition of
a large excess of native KTX (0.2 mM) 30 min before the addition of
125I-KTX. After a 60-min incubation, the sections
were rinsed four times (with each wash lasting 10 s) in a 20 mM
Tris-Cl buffer, pH 8, containing 150 mM NaCl and 1% BSA and then
rinsed once in the same buffer containing 0.1% BSA.
For biochemical studies, a portion of the labeled brain sections was
removed. Radioactivity was measured with a Packard spectrometer (Crystal II multidetector system). Protein was determined according to
Bradford (1976). Equilibrium binding constants were determined by
Scatchard plot analysis, and linear regression was performed to obtain
equilibrium dissociation constant (Kd) and
maximal receptor concentration (Bmax).
Kd and Bmax
values (mean ± S.E.) were obtained from three independent experiments.
For autoradiographic investigations, the slices were dried with a
stream of cold air and exposed to Kodak BioMax MR film. After a 7-day
exposure, the films were processed in Kodak Industrex developer at room
temperature for 2 min, fixed, and then washed. Azur II-stained sections
were used for reference. The autoradiograms were analyzed and
quantified using NIH Image software. Plastic standards (Amersham Corp.,
Paisley, UK) were used to calibrate 125I
concentrations. Receptor densities were expressed in fmol/mg protein. A
mean receptor density value for each nucleus was calculated from six to
eight bilateral measurements in three animals. The specific binding
value was determined as the difference between total and nonspecific
binding components for a given area. Rat brain regions were identified
and named according to the rat brain atlas of Paxinos and Watson
(1986).
| |
Results |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Kv1.1, Kv1.2, and Kv1.3 Channels Are Blocked by KTX.
Figure
1 shows the reversible inhibitory effect
of various concentrations of KTX (1-50 nM) on currents in X.
laevis oocytes expressing either Kv1.1, Kv1.2, and Kv1.3. Block
by KTX was voltage independent. Even at 1 µM, KTX had no effect on
Kv3.1 channel (not illustrated). The relative blocking potency was
determined by measurement of current remaining after stepwise increases
in KTX concentration (Fig. 2).
Experimental points (mean ± S.E.) of three to nine experiments
were fitted to hyperbolic curves. For Kv1.3 channel, we found an
IC50 value of 0.1 ± 0.04 nM and a Hill coefficient
value of 1.08 ± 0.1. The corresponding values for Kv1.1 and Kv1.2
channels were 1.1 ± 0.2 and 25 ± 1.5 nM for IC50 and 1.03 ± 0.4 and 0.9 ± 0.1 for the Hill
coefficient. Because the Hill coefficient was very close to 1, these
results confirm that KTX blocks Kv channels via a bimolecular
mechanism. Therefore, the IC50 values were considered
dissociation constants (Kd).
|
|
). To estimate the Kd value of KTX for
Kv1.1, Kv1.2, and Kv1.3 channels expressed in mammalian cells, we used
human embryonic kidney 293 cells stably expressing human Kv1.1 and
Kv1.2 genes and Jurkat cells constitutively expressing the Kv1.3
channel. Figure 3A shows the percentage
of Kv1.3, Kv1.1, and Kv1.2 currents blocked at various voltages by 2, 100, and 400 nM KTX, respectively. To estimate precisely any shift in
affinity compared with the oocyte data, we performed dose-response
experiments with various KTX concentrations (Fig. 3B). The
Kd values measured were 2, 14, and 270 nM
for Kv1.3, Kv1.1, and Kv1.2, respectively, and the Hill coefficient for
each was very close to 1. These results indicate that KTX affinity for
Kv1.1, Kv1.2, and Kv1.3 channels expressed in mammalian cells was 10- to 20-fold lower than that in X. laevis oocytes.
|
).
Binding Assays of KTX in Adult Rat Brain Sections.
The binding
data are presented in Fig. 4. Tissue
sections were incubated in Tris-Cl buffer with increasing concentration
of 125I-KTX until equilibrium was achieved. The labeled
toxin bound in a concentration-dependent manner. The specific
125I-KTX binding was found to be saturable and of high
affinity, in the range of studied concentrations. Scatchard plot
analysis was consistent with the presence of a single class of
noninteracting binding sites, with a Kd
value for the 125I-KTX-receptor complex of 82 ± 17 pM
and a Bmax value of 13.4 ± 1.3 fmol/mg
protein.
|
Distribution of KTX Binding Sites in Adult Rat.
To minimize
nonspecific binding, the concentration of 125I-KTX used in
autoradiographic experiments was 20 pM. The nonspecific component of
binding was homogeneously distributed in brain regions, and no
difference was detectable between gray and white matter (Fig.
5J).
|
|
Postnatal Ontogenesis of KTX Binding Sites in Rat Brain. Table 2 presents the mean density of KTX binding sites during the maturation of the rat central nervous system. At birth and up to P3, whole brain was almost devoid of high-affinity KTX receptors. From P5 onward, a distinct increase in KTX binding site density was observed in the midbrain, particularly in the central gray, tegmental nuclei, and hindbrain. From P5 to P7, the binding increased in mid and hindbrain, whereas it was still low in forebrain. From P9 to P13, most binding sites in forebrain increased slowly and then more dramatically by P15 (Fig. 6). At P15, significant contrast remained between elevated labeling of midbrain and lower levels of labeling in forebrain and hindbrain. In many structures, a slight decrease in KTX receptor densities was observed out to P21. Nevertheless, the heterogeneous distribution characteristically observed in adult animals was already present at P21 (Fig. 6).
|
|
| |
Discussion |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
The pharmacological identification of Kv channel subtypes expressed in rat brain requires the use of blockers with unambiguously defined activity profiles. Toward this goal, we determined that 1) KTX blocks the Kv1.3 and Kv1.1 channels with a high affinity and blocks the Kv1.2 channel with a low affinity, 2) KTX binds to specific receptor sites in the rat brain with a maximal capacity of 13.4 fmol/mg protein, 3) the distribution of KTX binding sites in rat brain is heterogeneous, and 4) KTX receptors appeared during brain ontogenesis only at P5.
KTX Blocks Kv1 Channels in Mammals.
KTX blocks Kv1.1, Kv1.2,
and Kv1.3 channels expressed in X. laevis oocytes with
subnanomolar to nanomolar affinity. When these channels are expressed
in mammalian cell lines, KTX affinity was shifted to the right by one
order of magnitude. This result suggests that the binding potency of
KTX for Kv channels expressed in situ in rat brain is weak for the
channels containing Kv1.2 -subunits and higher for channels
containing Kv1.3 and KV1.1 -subunits. According to the cell system
used to express Kv channels, similar differences in KTX affinity have
been reported (Grissmer et al., 1994; Hopkins, 1996). However, the
origin of such differences remains undetermined and may result from
different levels of channel glycosylation or phosphorylation, inducing,
in turn, variability in voltage sensitivity, activation threshold, and
gating kinetics (Robertson, 1997). For various Kv1 channels,
differences in affinity for ligands like DTX, MCD peptide, CTX, and
4-aminopyridine have also been observed in different heterologous
expression systems (Stühmer et al., 1989; Grissmer et al., 1994).
In addition, KTX blocks very weakly skeletal muscle BK channel
(Kd = 13 µM; not illustrated).
KTX Binding Sites in Rat Brain. Our results reveal a relatively heterogeneous distribution of KTX binding sites in adult rat brain. The highest levels of KTX receptors were found in the neocortex, bed nuclei of the stria terminalis, most of the hypothalamus, dentate gyrus, central gray, and parabrachial nuclei. The white matter contained the lowest level of KTX binding. The autoradiographic data are in good agreement with biochemical studies with synaptosome preparations. Indeed, for a 20 nM concentration of 125I-KTX, the average value for KTX binding site densities determined by autoradiographic procedures was similar to the biochemical values of KTX binding sites measured in brain homogenates (present data) and in P2 synaptosomal membranes (17-22 fmol/mg protein; Martin-Eauclaire, personal communication).
The histological resolution of the autoradiograms does not allow the identification of KTX binding sites as exclusively neuronal. Indeed, these receptors can also be present in glial, endothelial, and arterial smooth muscle cells. With the use of electrophysiological recording, various glial Kv currents have been characterized in tissue slices from the central nervous system (Akopian et al., 1997; Bordey and
Sontheimer, 1997). Nevertheless, autoradiographic analysis indicated a
very low density of KTX receptors in adult white matter, suggesting
very weak KTX binding in oligodendrocytes. However, a contribution to
KTX binding from astrocytes cannot be excluded.
Several facts favor the attribution of the measured KTX binding sites
to neurons: 1) no published immunological or in situ hybridization
study has mentioned the contribution of glial cells to the labeling of
Kv channels in brain (Kues and Wunder, 1992; Sheng et al., 1994; Wang
et al., 1994; Veh et al., 1995); 2) the Kv1.1 channel is only weakly
detected in mature astrocytes, and the Kv1.3 channel was not found in
glial cells (Smart et al., 1997; Hallows and Tempel, 1998); 3) the
expression of Kv channels is reduced in spinal cord astrocytes
cocultured with dorsal root neurons (Thio et al., 1993); and 4) Kv
currents decrease 3-fold between P5 and P20 in astrocytes of
hippocampal slices (Bordey and Sontheimer, 1997), whereas KTX binding
site density increases concomitantly in the same region during brain maturation.
Maximal KTX binding capacity (13.4 fmol/mg protein of brain tissue) is
very low compared with values determined for DTX (500-1100 fmol/mg
protein; Bidard et al., 1989), MgTX (800-900 fmol/mg protein; Knaus et
al., 1995; Koch et al., 1997), and MCD peptide (160 fmol/mg protein;
Bidard et al., 1989). Studies of mutual inhibition of 125I-KTX binding by DTX and of
125I-DTX binding by KTX reveals that KTX
recognizes only a small fraction of the DTX binding sites (Harvey et
al., 1995).
Electrophysiological data show that DTX blocks Kv1.2, Kv1.1, and Kv1.6,
and, with a lower affinity, and Kv1.3 channels. MgTX blocks Kv1.2 and
Kv1.3 channels, and MCD peptide blocks Kv1.1, Kv1.6, and, less
potently, Kv1.2 (Stühmer et al., 1989; Grissmer et al.,
1994). These data suggest that the low maximal binding capacity of KTX
may result from its very low affinity for the Kv1.2 channel.
In agreement with this hypothesis, the distribution of KTX binding
sites in rat brain was found to be heterogeneous, whereas the density
of DTX and MCD peptide binding sites was evenly distributed in gray
matter except in hippocampus and cerebellum (Bidard et al., 1987, 1989;
Mourre et al., 1988; Awan and Dolly, 1991).
In the hippocampus, the highest levels of DTX binding sites were found
in the CA3 field and the dentate gyrus (Bidard et al., 1989), and the
highest density of MCD peptide binding sites was found in the stratum
lucidum of the CA3 field. The stratum lacunosum moleculare of the CA1
field was poor in density of MCD peptide binding. The KTX binding sites
were particularly located in the outer molecular layer of the dentate
gyrus and in the stratum lucidum.
In the cerebellum, the KTX receptor levels were similar in the granular
and molecular layers with a higher density in the Purkinje layer,
whereas the granular and molecular layers, respectively, contained high
and moderate levels of DTX and MCD peptide binding sites.
Comparison of Distribution of KTX Binding Sites and Distribution of
Kv1.1 and Kv1.3 Subunits.
In most of the structures analyzed,
and particularly in olfactory bulb, hippocampal CA3 subfield, and
cerebellum, a comparison of the distribution of Kv1.1 and Kv1.3
-subunits with the localization of 125I-KTX binding
sites suggests that KTX recognizes channels containing these subunits.
-subunit polypeptides are strongly expressed in the internal plexiform layer, granular layer, and mitral soma layer, and all are
moderately present in the glomerular layer. Moreover, the outer
plexiform layer, which is weakly labeled by KTX, shows a strong Kv1.2
immunoreactivity (Veh et al., 1995). In situ hybridization experiments
also indicate that high levels of Kv1.3 mRNA are found only in the
internal granule layer (Kues and Wunder, 1992).
In the hippocampus, the dentate gyrus contains high densities of Kv1.1
and Kv1.3 mRNAs, and the CA3 field contains a high density of Kv1.1
mRNA. These structures contain high or intermediate densities of KTX
binding sites. In both regions, the distribution of immunoreactivity to
Kv1.1 and Kv1.3 -subunits overlaps the distribution of KTX binding
sites. However, surprisingly, in CA1 subfield, the density of KTX
receptors is low when Kv1.1 immunoreactivity is intense (Wang et al.,
1994; Veh et al., 1995; Rhodes et al., 1997).
In cerebellum, as in neocortex, thalamic nuclei, and in many areas of
the hindbrain and medulla, Kv1.1 mRNA and Kv1.1 subunit polypeptide
are detected in high or intermediate density (Kues and Wunder, 1992;
Wang et al., 1994; Veh et al., 1995). These structures also contain
high or intermediate density of KTX binding sites.
These data suggest that in every structure containing intermediate or
high densities of KTX binding sites, Kv1.1 or Kv1.3 mRNA (or protein)
have been detected. In contrast, in CA1 subfield where the Kv1.1
-subunit was intensively detected, the KTX binding sites density was
weak. It has been demonstrated that the binding of KTX to Kv1 channel
requires at least two KTX-sensitive -subunits (Aiyar et al., 1994).
Therefore, one Kv1.1 (or Kv1.3) -subunit detected with
immunohistochemistry may be included in an insensitive KTX channel.
Because in bovine brain Kv1.1 and Kv1.3 have not been described as
partners in heterotetrameric Kv channels (Scott et al., 1994;
Shamotienko et al., 1997), we hypothesize that KTX receptors are
channels containing at least two Kv1.1 or Kv1.3 -subunits.
Postnatal Development of KTX Binding Sites in Rat Brain.
Very
few studies has been performed on the expression and distribution of
Kv1 -subunits during the maturation of rat brain. Hallows and Tempel
(1998) reported that Kv1.1 expression in mouse brain is low at birth
and increases dramatically at the end of the first postnatal week.
These data are consistent with the measured development of KTX binding sites.
showed that
Kv1.1 mRNA in this region was present at nearly constant level from P1
to the adult stage. In contrast, Kv1.3 mRNA expression was maximal at
P8 and, surprisingly, down-regulated in the CA1 and CA3 fields by P15
to undetectable levels, while remaining constant in the dentate gyrus.
We observed that the density of KTX receptors is much higher in dentate
gyrus than in CA1 and CA3 fields.
Previous developmental studies with Kv toxins have been performed only
with MCD peptide (Mourre et al., 1988). The main difference with KTX is
that the MCD peptide receptors are initially present in the forebrain,
whereas the KTX binding sites appear first in the hindbrain.
In conclusion, the pharmacological specificity of KTX determined with
recombinant Kv channels and the autoradiographically determined
distribution of KTX receptors in rat brain should be useful tools for
future investigations of channels containing Kv1.3 and Kv1.1
-subunits. KTX can be used to purify these channels, to discern
their roles in shaping neuronal excitability, and to define their
involvement in cerebral functions.
| |
Acknowledgments |
|---|
We thank Michèle André and Hélène Chagneux for technical assistance, Raymond Fayolle for manufacturing the bilayer amplifier, and Régine Romi for help in KTX synthesis.
| |
Footnotes |
|---|
Accepted for publication July 27, 1999.
Received for publication April 20, 1999.
1 This work was supported by the Centre National de la Recherche Scientifique and by the Association Française contre les Myopathies (Grant 5030). M.N. and S.L.A. were supported by NIH Grants HL15157 (Boston Sickle Cell Center) and DK34854 (Harvard Digestive Diseases Center). S.L.A. is an Established Investigator of the American Heart Association.
Send reprint requests to: Dr. C. Mourre, Laboratoire de Neurobiologie Intégrative et Adaptative, UMR 6562, CNRS-Université de Provence, Escadrille Normandie-Niemen, Case 351, 13397, Marseille, Cedex 20, France. E-mail: mourre{at}newsup.univ-mrs.fr
| |
Abbreviations |
|---|
Kv, voltage-gated potassium; CTX, charybdotoxin; DTX, dendrotoxin; KTX, kaliotoxin; MCD, mast cell-degranulating; IKCa, intermediate conductance Ca2+-activated K+; MgTX, margatoxin; BK, high conductance Ca2+-activated K+.
| |
References |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
-bungarotoxin, - and 
-dendrotoxins.
Neuroscience
40:
29-39[Medline].
receptor sites.
Biochem Biophys Res Commun
143:
383-389[Medline].-subunits with Kv1 -subunits in mammalian brain K+ channel complexes.
J Neurosci
17:
8246-8258This article has been cited by other articles:
|
|
 |
|
  O. Tschritter, F. Machicao, N. Stefan, S. Schafer, C. Weigert, H. Staiger, C. Spieth, H.-U. Haring, and A. Fritsche A New Variant in the Human Kv1.3 Gene Is Associated with Low Insulin Sensitivity and Impaired Glucose Tolerance J. Clin. Endocrinol. Metab., February 1, 2006; 91(2): 654 - 658. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Szabo, J. Bock, A. Jekle, M. Soddemann, C. Adams, F. Lang, M. Zoratti, and E. Gulbins A Novel Potassium Channel in Lymphocyte Mitochondria J. Biol. Chem., April 1, 2005; 280(13): 12790 - 12798. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Yu, W. Fu, H. Liu, X. Luo, K. X. Chen, J. Ding, J. Shen, and H. Jiang Computational Simulations of Interactions of Scorpion Toxins with the Voltage-Gated Potassium Ion Channel Biophys. J., June 1, 2004; 86(6): 3542 - 3555. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Xu, P. Wang, Y. Li, G. Li, L. K. Kaczmarek, Y. Wu, P. A. Koni, R. A. Flavell, and G. V. Desir The voltage-gated potassium channel Kv1.3 regulates peripheral insulin sensitivity PNAS, March 2, 2004; 101(9): 3112 - 3117. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Xu, P. A. Koni, P. Wang, G. Li, L. Kaczmarek, Y. Wu, Y. Li, R. A. Flavell, and G. V. Desir The voltage-gated potassium channel Kv1.3 regulates energy homeostasis and body weight Hum. Mol. Genet., March 1, 2003; 12(5): 551 - 559. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. A. Castle, D. O. London, C. Creech, Z. Fajloun, J. W. Stocker, and J.-M. Sabatier Maurotoxin: A Potent Inhibitor of Intermediate Conductance Ca2+-Activated Potassium Channels Mol. Pharmacol., February 1, 2003; 63(2): 409 - 418. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Devaux, M. Gola, G. Jacquet, and M. Crest Effects of K+ Channel Blockers on Developing Rat Myelinated CNS Axons: Identification of Four Types of K+ Channels J Neurophysiol, March 1, 2002; 87(3): 1376 - 1385. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Beeton, J. Barbaria, P. Giraud, J. Devaux, A.-M. Benoliel, M. Gola, J. M. Sabatier, D. Bernard, M. Crest, and E. Beraud Selective Blocking of Voltage-Gated K+ Channels Improves Experimental Autoimmune Encephalomyelitis and Inhibits T Cell Activation J. Immunol., January 15, 2001; 166(2): 936 - 944. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Wrisch and S. Grissmer Structural Differences of Bacterial and Mammalian K+ Channels J. Biol. Chem., December 8, 2000; 275(50): 39345 - 39353. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||
) was assessed from the
difference between total (
) and nonspecific binding (
). Inset,
Scatchard plot of the data. A Kd value
(mean ± S.E.) of 82 ± 17 pM and a
Bmax value of 13.4 ± 1.3 fmol/mg
protein were obtained from three similar experiments.
