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The Journal of Neuroscience, 2001, 21:RC175:1-6
RAPID COMMUNICATION
Somatic Colocalization of Rat SK1 and D class (Cav
1.2) L-type Calcium Channels in Rat CA1 Hippocampal Pyramidal
Neurons
Sarah E. H.
Bowden,
Stephanie
Fletcher,
David J.
Loane, and
Neil V.
Marrion
Department of Pharmacology and Medical Research Council Center for
Synaptic Plasticity, University of Bristol, Bristol, BS8 1TD, United
Kingdom
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ABSTRACT |
In hippocampal neurons, the firing of a train of action potentials
is terminated by generation of the slow afterhyperpolarization (AHP).
Recordings from hippocampal slices have shown that the slow AHP likely
results from the activation of small-conductance calcium-activated
potassium (SK) channels by calcium (Ca2+) entry
through L-type Ca2+ channels. However, the relative
localization of these two channel subtypes is not known. The cloning
and characterization of three subtypes of SK channel has suggested that
SK1 may underlie generation of the slow AHP. Using a novel antibody
directed against rat SK1 (rSK1), it has been determined that the
rSK1 channel is primarily in the soma of hippocampal CA1 neurons. In
conjunction with antibodies directed against C (Cav1.2) and
D (Cav1.3) class L-type Ca2+ channel
 1 subunits, it was observed that rSK1 channels were selectively
colocalized with D class L-type channels. This colocalization supports
the functional coupling of L-type and SK channels previously observed
in cell-attached patches from hippocampal neurons. However, it appears
contrary to the slow rise and decay of the slow AHP. Induction of
delayed facilitation of L-type Ca2+ channels in
cell-attached patches from hippocampal neurons evoked delayed opening
of coupled SK channels. Generation of ensemble currents produced
waveforms identical to the ionic current underlying the slow AHP
(IsAHP). Therefore, these data
indicate that the slow AHP is somatic in origin, resulting from delayed
facilitation of D class L-type Ca2+ channels
colocalized with rSK1 channels.
Key words:
calcium-activated potassium channel; slow
afterhyperpolarization; SK channel; calcium channel; delayed
facilitation; hippocampus
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INTRODUCTION |
In
hippocampal pyramidal neurons, a burst of action potentials is
terminated by generation of a slow afterhyperpolarization (AHP)
(Madison and Nicoll, 1984 ). It has been proposed that the slow AHP is
generated by activation of SK channels (Pedarzani et al., 2001).
However, the observation that afterhyperpolarizations show different
sensitivities to the bee venom toxin apamin suggested the existence of
more than one subtype of SK channel (Pennefather et al., 1985;
Lancaster and Adams, 1986). This was confirmed by the cloning of three
SK channel subtypes (SK1-3; Köhler et al., 1996). These cloned
channels exhibited similar biophysical properties, but were
distinguished by their sensitivity to block by apamin (Köhler et
al., 1996; Hirschberg et al., 1998). Homomeric SK2 and SK3 channels
were blocked by apamin with a high affinity, whereas SK1 was
insensitive (Köhler et al., 1996; Ishii et al., 1997).
Subsequently, it has been reported that hSK1 channels are sensitive to
block by apamin. However, the block is poorly understood because in a
proportion of cells expressing hSK1, a component of current was not
sensitive, and the block displayed a biphasic inhibition (Shah and
Haylett, 2000a; Grunnet et al., 2001). The mRNAs for SK1, 2, and
3 subunits are all present in rat hippocampus (Köhler et al.,
1996). Activation of SK2/SK3 channels is proposed to underlie the
apamin-sensitive medium AHP in the hippocampus (Stocker et al., 1999),
suggesting that SK1 channel activation underlies generation of the
apamin-insensitive slow AHP in these neurons (Köhler et al.,
1996; Vergara et al., 1998).
In cultured neurons, the slow AHP is evoked by
Ca2+ entry through L-and N-type
Ca2+ channels, with a contribution from
Ca2+-induced
Ca2+ release reported from organotypic
cultured neurons (Tanabe et al., 1998; Shah and Haylett, 2000b). In
hippocampal slices it appears that Ca2+
entry through L-type channels is the primary source of
Ca2+ for activation of the slow AHP
(Rascol et al., 1991; Moyer et al., 1992). However, the relative
localization of SK and L-type channels in hippocampal neurons is not
known. Using indirect methods it has been proposed that SK channels are
in the soma (Lancaster and Zucker, 1994), the basal dendrites (Bekkers,
2000), or the apical dendrites (Sah and Bekkers, 1996). Patch-clamp
recordings have shown that SK channels are present in the soma of
hippocampal neurons (Marrion and Tavalin, 1998; Hirschberg et al.,
1999). Similarly, immunohistochemical studies have shown that L-type Ca2+ channels are primarily somatic (Hell
et al., 1993). A novel antibody has been raised against the rat SK1
(rSK1) channel. The antibody recognized a protein of appropriate
molecular weight only in tissues previously demonstrated to contain SK1
subunit mRNA. Using immunocytochemistry, rSK1 channels were shown to be
primarily somatic in acutely dissociated hippocampal CA1 neurons. The
subcellular location of C and D class L-type channels was resolved to
examine whether a specific subtype is involved in generation of the
slow AHP. In this study, rSK1 channels were found to be selectively
colocalized with D class L-type calcium channels. This colocalization
appears inconsistent with the slow rise and decay of the slow AHP.
Using cell-attached patch recordings, induction of delayed facilitation
of L-type channels evoked delayed openings of coupled SK channels.
Generated ensemble currents were identical to the ionic current
underlying the slow AHP. The observed somatic colocalization of rSK1
and D class L-type channels suggests that the slow AHP is somatic in
origin. This is supported by the observation that induction of delayed
facilitation of L-type channels evokes a slow AHP waveform from coupled
SK channels.
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MATERIALS AND METHODS |
Antibody production. An antibody was raised in guinea
pig against a 26 amino acid peptide (QAQQEELEARLAALESRLDVLGASLQ)
corresponding to a unique region in the C terminus of the rat SK1
channel. Antiserum was collected, titer was determined by ELISA, and
antibodies were purified by affinity chromatography (Research Genetics).
Tissue preparation. Tissues were removed from 10- to
13-d-old rat pups (Sprague Dawley) and homogenized (Polytron blender, full power, 10 sec) in ice-cold Tris buffer (25 mM Tris, pH 7.4). The homogenate was washed twice
by centrifugation (25,000 × g, 10 min, 4°C), and the
pellet was resuspended in Tris buffer. Solubilization buffer (25 mM Tris, 2 mM EDTA, 0.1 mM PMSF, and 0.5% Triton X-100, pH 7.4) was
added, and the preparation was agitated for 60 min at 4°C, after
which insoluble material was removed by centrifugation at 100,000 × g for 60 min at 4°C.
For Western blot analysis of calcium and potassium channels in rat
brain (Fig. 1A,C),
whole brain was removed from 10- to 13-d-old rat pups and stored at
 80°C. Brain tissue was slowly thawed and homogenized in ice-cold
HSE buffer (10 mM HEPES, 350 mM sucrose, and 5 mM EDTA,
pH 7.4) containing protease inhibitors [pepstatin A (1 µg/ml),
leupeptin (1 µg/ml), aprotinin (1 µg/ml), Pefabloc SC (0.2 mM), benzamidine (0.1 mg/ml), and calpain
inhibitors I and II (8 µg/ml)]. The homogenate was centrifuged at
2000 × g for 5 min, and the supernatant was removed
and centrifuged at 100,000 × g for 1 hr. The resulting
pellet was resuspended in ice-cold HSE buffer containing protease
inhibitors. For all tissue preparations, protein content was determined
using the Bradford assay (Bio-Rad, Hemel Hempstead, UK).
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Figure 1.
Characterization of the antibody to rat SK1.
A, Western blot analysis of protein extracts from
different rat tissues illustrating the distribution of proteins
detected by the antibody to rSK1. A protein of ~57 kDa was recognized
in brain and heart but not in lung, liver, spleen, or kidney.
B, Western blots showing selective detection of the
GST-rSK1 fusion constructs by the antibody directed against rSK1.
Extracts of E. coli only (a),
bacteria transformed with a plasmid encoding GST
(b), and bacteria transformed with a plasmid
encoding a fusion protein of GST and the C terminus of rSK1
(c) were probed with antibodies to GST and rSK1.
The rSK1 antibody recognized the GST-rSK1 fusion protein but not
protein extracts from bacteria expressing GST alone. C,
Western blot probed with antisera to the Ca2+
channel 1C subunit (a), the
Ca2+ channel 1D subunit
(b), and the rSK1 subunit
(c). Immunoblotting showed that the rSK1 antibody
did not detect protein bands at the predicted molecular weights for the
Ca2+ channel subunits. Similarly, neither of the
Ca2+ channel antibodies detected a protein
corresponding to the molecular weight for the rSK1 subunit.
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Glutathione S-transferase fusion proteins.
The cDNA encoding the C-terminal region of the rSK1 channel (amino
acids 429-533) was amplified using PCR and subcloned into the
glutathione S-transferase (GST) fusion vector pGEX-6P1
(Amersham Pharmacia Biotech, Little Chalfont, UK). The resulting
plasmid was used to transform Escherichia coli strain BL21
(Stratagene, Amsterdam, The Netherlands). Expression of fusion
proteins was induced by adding
isopropylthio- -D-galactoside (1 mM). Cells were sedimented by centrifugation and
resuspended in PBS before separation of bacterial proteins by
SDS-PAGE.
SDS-PAGE and Western blotting. Samples were solubilized in
SDS sample buffer, incubated at 80°C for 20 min, and applied to acrylamide gels. After SDS-PAGE, protein was transferred to
polyvinylidene difluoride membrane using a transfer cell (Bio-Rad). The
membrane was washed in blocking buffer and incubated with solutions of primary and IgG peroxidase-conjugated secondary. Immunoreactive proteins were visualized using an ECL detection system (ECL Plus, Amersham, UK).
Cell preparation. Acutely dissociated hippocampal CA1
neurons were obtained from the brains of 9- to 12-d-old Sprague Dawley rats as described previously (Cloues et al., 1997). Dissected hippocampi were sectioned into 300- to 400-µm-thick slices. After enzyme treatment, slices were stored in solution at room temperature under an oxygen atmosphere. Slices were removed, and the CA1 region was
dissected when required. Pyramidal neurons were released by gentle
trituration through fire-polished Pasteur pipettes and plated onto
multiwell glass slides (ICN Biomedicals, Basingstoke, UK) for
immunocytochemical studies or Primaria-coated dishes (Falcon) for electrophysiology.
Antibody labeling. Cells were fixed and permeabilized
with PBS containing 3% PFA and 0.1% Triton X-100 for 10 min at room temperature and then rinsed with PBS containing 1% BSA for 30 min.
After incubation with the appropriate primary antibody (1:200) for 12 hr at 37°C, excess antibody was removed by washing with PBS-BSA. The
appropriate fluorochrome-conjugated secondary antibody (1:100; Jackson
ImmunoResearch, West Grove, PA) was then applied for 2 hr at 25°C.
Finally, cells were rinsed in PBS-BSA for 20 min at room temperature,
then mounted using Vectashield mount (Vector Laboratories, Orton
Southgate, Peterborough, UK). Images were captured on a Leica
(Nussloch, Germany) confocal laser-scanning microscope.
All standard reagents were obtained from Sigma with the exception
of CaCl2 (Fluka, Neu-Ulm, Germany) and HEPES
(Calbiochem, La Jolla, CA).
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RESULTS |
Characterization of an antibody to the rat SK1
channel (anti-rSK1)
An antibody was raised to a sequence unique to rSK1. This region
was selected because it displayed little sequence identity with
corresponding regions in rSK2 and rSK3 (31 and 39%, respectively). Western blot analysis of protein extracts from different rat tissues showed that the novel antibody recognized a protein in brain and heart
of ~57 kDa, the predicted molecular weight for the rSK1 subunit. No
such protein band was recognized in lung, liver, spleen, or kidney
(n = 7) (Fig. 1A,C). Specific binding
was confirmed by elimination of labeling by preincubating the antibody
with the peptide antigen (1:100; data not shown). This tissue
distribution is comparable with that of mRNA for the rSK1 subunit
(Köhler et al., 1996). Labeling was not observed at the predicted
molecular weight for rSK3 (82 kDa). In addition, labeling of an 82 kDa
protein was not observed in liver, a tissue rich in rSK3 (Fig.
1A) (Barfod et al., 2001). The predicted molecular
weight for rSK2 (64 kDa) is close to that estimated for rSK1. However,
the novel antibody did not recognize protein in Western blot analysis
of extracts from either human embryonic kidney 293 or tSA201
cells transiently expressing rSK2 (data not shown). Therefore, the size
and distribution of the protein detected are consistent with the
antibody recognizing the rSK1 protein, with no cross-reactivity with
either rSK2 or rSK3 subunits.
The expression of human SK1 and rat SK2 and 3 clones has been reported
in both Xenopus oocytes and mammalian cell lines
(Köhler et al., 1996; Shah and Haylett, 2000a). We and others
have attempted to express a full-length clone of rat SK1 in a mammalian
cell line, but have not been successful (L. Kaczmarek, personal
communication). As an alternative approach to confirm the
specificity of the antibody, a GST-rSK1 fusion protein was produced.
Western blot analysis of extracts from bacteria expressing either GST
alone or the GST-rSK1 fusion protein showed that an antibody directed
against GST recognized both a 29 kDa protein corresponding to GST and a
protein of 40 kDa corresponding to the GST-rSK1 fusion protein
(n = 7) (Fig. 1B). In contrast,
anti-rSK1 only recognized protein in extracts from bacteria expressing
the GST-rSK1 fusion protein. This demonstrates that the antibody
specifically recognized the rSK1 subunit epitope (n = 6) (Fig. 1B).
Subcellular distribution of class C and D L-type calcium channels
in hippocampal CA1 neurons
Antibodies specific for the C and D class 1 subunits were used
to identify and localize the L-type calcium channel subtypes in
hippocampal CA1 neurons. C class L-type calcium channels were distributed at a very low density on the central region of the cell
bodies. In contrast, dense immunoreactivity was observed at the base of
the apical and basal dendrites and extended only into their proximal
regions. The staining was further characterized by punctate clusters of
intense labeling along the length of the apical dendrite (Fig.
2a). Visualization of the
1D subunit revealed a diffuse distribution of
D class channels over the surface of the cell body. This staining
extended only into the proximal portions of the apical and basal
dendrites and diminished along the length of the apical dendrite (Fig.
2b). Denser immunoreactivity was occasionally seen at the
base of the basal and apical dendrites in some neurons. The observed
labeling corresponded well with the expression of
1C and 1D subunits
reported in hippocampal slices (Hell et al., 1993), confirming that
channel distribution had been retained in the acutely dissociated cell
preparation.
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Figure 2.
Subcellular distribution of L-type calcium and SK1
channels in hippocampal neurons. Confocal images show the labeling
detected with specific antibodies to 1C
(a), 1D (b),
and rSK1 subunits (c, d) using FITC-conjugated secondary
antibodies; magnification 63×. Scale bar, 10 µm. In all cases, the
specificity of labeling was confirmed by elimination of
immunoreactivity after preincubation of the primary antibody with the
respective antigenic peptide (1:1).
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Localization of rSK1 channels in hippocampal CA1 neurons
The antibody raised against a unique sequence in the rSK1 channel
(anti-rSK1) was used to determine the subcellular distribution of the
SK1 channel. Immunofluorescent staining of the rSK1 channel subunit
detected a diffuse somatic distribution. Similar to the labeling of the
1D subunit, immunoreactivity for rSK1 channels was also seen at the base of the basal and apical dendrites in some
neurons. Weaker staining, which diminished along the length of the
dendrites, was also observed (Fig. 2c,d).
Colocalization of rSK1 with D class L-type calcium channels in rat
hippocampal CA1 neurons
Comparison of the subcellular distributions of the rSK1 and
1D calcium channel subunits suggested that the
two channel types may be colocalized (Fig. 2b-d). However,
it was necessary to exclude the possibility that antibody
cross-immunoreactivity could underlie the similarity in the staining
patterns observed. Western blot analysis of rat brain proteins probed
with antibodies to calcium channel subunits 1C
and 1D revealed two forms for each of the L-type calcium channel subunits with apparent molecular weights of
~220 and ~190 kDa (Hell et al., 1993). The antibodies to calcium channel subunits 1C and
1D did not detect any reactivity in the
molecular weight region corresponding to that predicted for the rSK1
channel (n = 5) (Fig. 1Ca,b). Similarly,
probing with anti-rSK1 detected a single protein band of ~57 kDa,
with no reactivity observed at the predicted molecular weights for the
calcium channel subunits (n = 19) (Fig.
1Cc). In each case, the specific binding was eliminated by
preincubation of the antibody with its corresponding peptide antigen at
a ratio of 1:1 for 1C and
1D and 100:1 for rSK1. In addition, it was
demonstrated that the immunoreactivity of the
1D antibody was not affected by preincubation
with the rSK1 peptide (1:1), and vice versa (100:1; data not shown).
With immunocytochemistry, preincubation of the rSK1 antibody with the 1D peptide (1:1) did not alter the pattern of
staining compared with that observed with rSK1 antibody alone (data not
shown). Taken together, these findings excluded the possibility of
cross-reactivity between the antibodies to the
1D and rSK1 subunits verifying the patterns
observed in the single labeling immunocytochemistry studies.
Using the fact that the primary antibodies for the two channel subtypes
were raised in different species (rabbit for
anti-1D and guinea pig for anti-rSK1),
multiple labeling studies were performed to determine if the rSK1 and D
class channels were colocalized. Figure 3
shows the patterns of labeling obtained from the simultaneous acquisition of the immunoreactivity for each of the channel types within an individual neuron. Significant colocalization of the rSK1 and
D class channels was detected, particularly in the cell soma and
proximal region of the apical dendrite (Fig. 3C). It was
noted that the expression patterns were not identical, with areas of
distinct labeling for each channel type also being observed. In most
cases, a slight excess of somatic staining for the
1D subunit could be discerned. This is
consistent with previous reports from single channel studies that
suggested the possibility of "spare" L-type calcium channels in the
soma of hippocampal neurons (Marrion and Tavalin, 1998).
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Figure 3.
Colocalization of rSK1 with D class L-type calcium
channels in fixed hippocampal neurons. Two examples of the pattern of
immunoreactivity observed for 1D (secondary
antibody: donkey anti-rabbit Cy5, red;
A) and rSK1 (secondary antibody: donkey anti-guinea pig
FITC, green; B) subunits in acutely
dissociated hippocampal neurons. In each case, areas of colocalization
appear as yellow (C). Scale bar, 10 µm.
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DISCUSSION |
A novel antibody directed against a unique sequence in the C
terminus of the rat SK1 channel showed the channels to be diffusely distributed, primarily in the soma and proximal regions of the dendrites of acutely dissociated hippocampal CA1 neurons (Fig. 2c,d). In contrast, C class L-type channels were found at
the base of the apical and basal dendrites, around the periphery of the
soma, and in punctate clusters along the length of the apical dendrite
(Fig. 2a). D class L-type channels were more diffusely distributed in the soma with a diminishing presence along the length of
the apical dendrite (Hell et al., 1993). Overlay of immunofluorescent
staining of individual neurons in multiple labeling studies revealed a
somatic colocalization of SK1 and D class channels.
It has been proposed that SK channel activation underlies generation of
the slow AHP in hippocampal neurons (Pedarzani et al., 2001). The
observed somatic colocalization of SK1 and L-type D class channels
suggests that the slow AHP is generated in the soma of these neurons.
Support for a somatic location for SK1 channels comes from
single-channel studies, which reported the presence of SK channels in
somatic patches (Marrion and Tavalin, 1998; Hirschberg et al., 1999).
In addition, their somatic colocalization with L-type calcium channels
identified with the low resolution of light microscopy is consistent
with the functional coupling of L and SK channels observed in somatic
cell-attached patches from these neurons (Marrion and Tavalin, 1998).
The calcium sensitivity of SK channel gating also suggests that the two
channel types must be in close proximity. Both native and cloned SK
channel subtypes exhibit an open probability
(Po) of 0.5 at a calcium concentration
of ~0.5-0.7 µM (Köhler et al., 1996;
Hirschberg et al., 1998). The Po of
the SK channel at the peak of the slow AHP has been estimated to be
0.5-0.7 (Sah and Isaacson, 1995; Valiante et al., 1997), predicting a
requirement for an intracellular calcium concentration of ~1
µM (Hirschberg et al., 1999). However, bulk
increases of intracellular calcium of only 30 nM
have been measured during the slow AHP (Knöpfel et al., 1990).
For SK channels to experience 1 µM calcium,
microdomain models predict that they must be within 150 nm of a calcium
channel (Marrion and Tavalin, 1998). Therefore, the observed
colocalization of L and SK channels is sufficient to account for the
slow AHP.
Although close proximity of the two channel types would permit
sufficient calcium to be present at the SK channels, their distribution
within the soma is apparently inconsistent with the slow time course of
the slow AHP in the hippocampus. It has previously been proposed that
diffusion of calcium from its point of entry to the SK channels
underlies the slow kinetics of the slow AHP in hippocampal cells
(Lancaster and Adams, 1986). Accordingly, SK channels have always been
proposed to be at some distance from calcium channels, although their
exact location has been disputed. How then can a model of
colocalization of the channels account for the kinetics of activation
of the slow AHP? It has been proposed that the slow AHP time course may
be attributable to potassium channels reacting slowly to increases in
intracellular calcium (Sah and Clements, 1999). However, cloned and
native SK channels have been demonstrated to activate rapidly in
response to a rise in calcium (Xia et al., 1998). Furthermore,
calcium-dependent channels that may underlie the slow AHP in
hippocampal neurons have been shown to respond rapidly to
photolytically released calcium (Lancaster and Zucker, 1994). Therefore
it is apparent that SK channel kinetics alone are not sufficient to
explain the time course of the slow AHP (Hirschberg et al., 1999).
An alternative explanation is that the calcium channel kinetics can
account for the time course of the slow AHP in these neurons. It has
been reported that a train of action potentials that would evoke the
slow AHP induces an enhanced activity of L-type calcium channels at
membrane potentials negative to 50 mV. This behavior is termed
delayed facilitation and has been proposed to provide a prolonged
source of calcium entry at negative membrane potentials (Cloues et al.,
1997). Because both the time course and modulation of delayed
facilitation closely resembles those of the slow AHP, delayed
facilitation of L-type calcium channels has been proposed to dictate
the time course of the slow AHP in hippocampal neurons (Cloues et al.,
1997).
If SK channels respond to a colocalized calcium source, it would be
expected that an ensemble waveform of SK channel activity after
induction of delayed facilitation should mimic the macroscopic slow
AHP. Figure 4 shows SK channel activity
recorded in a cell-attached patch with an electrode containing 2 mM CaCl2 and KCl. Induction of
delayed facilitation of colocalized L-type channels by a train of
action potential waveforms activated SK channels within the patch.
Openings occurred after some delay and were observed to decrease in
frequency during the 5 sec voltage step to 0 mV (Fig. 4A). Generation of an ensemble current produced a
waveform that displayed a slow rise (peaking within ~0.5-1 sec) and
decayed with an exponential time course ( ~ 1.6 sec) that was
reminiscent of the current underlying the slow AHP (Fig.
4B).
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Figure 4.
IsAHP-like waveform of
SK channel activity induced by delayed facilitation of colocalized
L-type calcium. A, Selected sweeps show outward (upward)
channel openings evoked by a train of action potential waveforms
(holding potential, 60 mV; post-train voltage, 0 mV) recorded in the
cell-attached configuration from acutely dissociated hippocampal
neurons. Electrodes were filled with a solution containing (in
mM): NMDG (150); aspartic acid (150); HEPES (10); 4-AP (1);
3-4-DAP (1); CaCl2 (2); KCl (2), supplemented with and
dendrotoxins (200 nM) and TTX (10 µM). In
these conditions, individual L-type channels could not be resolved.
Their presence was confirmed by subsequent block of SK channel activity
by application of 3 µM nimodipine (data not shown).
Evoked channels had a single-channel amplitude consistent with a
conductance of 10 pS, as previously reported for hippocampal SK
channels measured under these conditions (Marrion and Tavalin, 1998).
Open-state kinetics were consistent with those observed for SK channels
in patches excised from hippocampal CA1 neurons (Hirschberg et al.,
1999). B, Generation of an ensemble current (average of
15 sweeps) gave a waveform resembling that of the
IsAHP. A slow rise of outward current was
observed, peaking ~600 msec after the termination of the train. The
decay of the waveform was fit with an exponential time course ( ~ 1.6 sec). The dashed line represents zero
current.
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In summary, our immunocytochemical studies have directly demonstrated
the somatic distribution of rSK1 channels in isolated rat hippocampal
CA1 neurons and their colocalization with the D subclass of L-type
calcium channels. In addition, we have provided evidence for how such a
channel distribution may underlie the slow AHP observed in these
neurons. These findings support the view that local rather than global
rises in calcium are involved in the coupling of calcium-dependent
processes. They also provide direct evidence of how selective
functional coupling may be achieved through specific colocalization of
different channel subtypes.
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FOOTNOTES |
Received June 15, 2001; revised July 25, 2001; accepted July 30, 2001.
This work was supported by grants from the Human Frontiers Science
Program and the Medical Research Council (United Kingdom). We are
grateful to Dr. D. Shepherd for the critical reading of this
manuscript. In addition, we gratefully acknowledge the provision of
research facilities within Wellcome Trust-funded laboratories and the
Medical Research Council-funded School of Medical Sciences Cell Imaging Facility.
Correspondence should be addressed to Dr. Neil V. Marrion, Department
of Pharmacology, School of Medical Sciences, University of Bristol,
University Walk, Bristol, BS8 1TD, UK. E-mail:
N.V.Marrion{at}bristol.ac.uk.
This article is published in
The Journal of Neuroscience, Rapid Communications Section,
which publishes brief, peer-reviewed papers online, not in print. Rapid
Communications are posted online approximately one month earlier than
they would appear if printed. They are listed in the Table of Contents
of the next open issue of JNeurosci. Cite this article as:
JNeurosci, 2001, 21:RC175 (1-6). The
publication date is the date of posting online at
www.jneurosci.org.
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ABSTRACT
INTRODUCTION
