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The Journal of Neuroscience, August 15, 2002, 22(16):7154-7164
The Neural Cell Adhesion Molecule Regulates Cell-Surface Delivery
of G-Protein-Activated Inwardly Rectifying Potassium Channels Via Lipid
Rafts
Markus
Delling1,
Erhard
Wischmeyer2,
Alexander
Dityatev1,
Vladimir
Sytnyk1,
Rüdiger W.
Veh3,
Andreas
Karschin2, and
Melitta
Schachner1
1 Zentrum für Molekulare Neurobiologie,
Universität Hamburg, 20246 Hamburg, Germany,
2 Institut für Physiologie, Universität
Würzburg, 97070 Würzburg, Germany, and
3 Institut für Anatomie, Universitätsklinikum
Charité, 10117 Berlin, Germany
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ABSTRACT |
Mice deficient in the neural cell adhesion molecule (NCAM) exhibit
increased anxiety and anxiolytic sensitivity to serotonin 5-HT1A receptor agonists. Here, we investigate the
relationship between NCAM and 5-HT1A receptor signaling
pathways modulating G-protein-activated inwardly rectifying
K+ (Kir3) channels. When studying this relationship
in cultured hippocampal neurons, we observed that in cells from
NCAM-deficient mice, inwardly rectifying K+ (Kir3)
currents were increased compared with wild-type controls. Analysis of
this modulatory mechanism in Xenopus oocytes and Chinese hamster ovary (CHO) cells revealed that the recombinantly expressed major transmembrane isoforms NCAM140 and NCAM180 specifically reduced
inward currents generated by neuronal Kir3.1/3.2 and Kir3.1/3.3 but not
by cardiac Kir3.1/3.4 channels. Using fluorescence measurements and
surface biotinylation assays, we show that this effect was caused by a
reduced surface localization of Kir3 channels. Furthermore, expression
of flag-tagged Kir3 channels in cultured neurons of NCAM-deficient mice
resulted in a higher transport of these channels into neurites and a
higher cell-surface localization compared with wild-type neurons.
Neuronal Kir3 channels and NCAM isoforms are associated with
cholesterol-rich microdomains (lipid rafts) in CHO cells and in
isolated brain membranes. Mutational and pharmacological disruption of
the lipid raft association of NCAM140 normalizes surface delivery of
channels. We conclude that the transmembrane isoforms of NCAM reduce
the transport of Kir3 channels to the cell surface via lipid rafts.
Thus, regulation of Kir3 channel activity by NCAM may represent a novel
mechanism controlling long-term excitability of neurons.
Key words:
Kir3; GIRK; cell-surface localization; lipid rafts; palmitoylation; NCAM-deficient mice
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INTRODUCTION |
The neural cell adhesion molecule
(NCAM) is involved in neuronal migration, neurite outgrowth and
fasciculation, synapse formation, synaptic plasticity, and emotional
behavior (Schachner, 1997 ; Ronn et al., 1998; Murase and Schuman,
1999). NCAM-deficient mice show increased anxiety and aggression and an
altered response of 5-HT1A receptors to their
agonists 8-hydroxy-dipropylaminotetralin (8-OH-DPAT) and buspirone
(Stork et al., 1997, 1999), suggesting a functional interdependence of
the 5-HT1A receptor and NCAM. 5-HT1A receptor distribution and affinity or
tissue concentrations of 5-HT or its metabolite 5-hydroxyindoacetic
acid were not different between wild-type
(NCAM+/+) and NCAM-deficient
(NCAM /) mice, indicating that NCAM
does not affect 5-HT turnover or 5-HT1A receptor function.
Therefore, we extended our studies on a possible influence of NCAM on
the 5-HT1A receptor, which belongs to the seven
membrane-spanning receptor family activating Gi/o
proteins. In the hippocampus, the main effectors of the
5-HT1A receptor are G-protein-coupled inwardly
rectifying K+ (Kir3) channels localized
presynaptically and postsynaptically. These channels hyperpolarize the
membrane potential and are thus involved in inhibitory activities
(Lüscher et al., 1997). The Kir3 family consists of the Kir3.1,
Kir3.2, Kir3.3, and Kir3.4 subunits, and the majority of functional
Kir3 channels are believed to exist as heterotetramers containing the
Kir3.1 subunit, although some studies report on functional Kir3.2
homomers and Kir3.2/3.3 combinations (Wischmeyer et al., 1997; Inanobe
et al., 1999; Jelacic et al., 2000).
To investigate a possible influence of NCAM on Kir3 channel activity,
we compared inwardly rectifying K+
currents in hippocampal neurons from
NCAM+/+ and
NCAM/ mice. After the observation that
Kir3 channel activity was reduced in the presence of NCAM, we
investigated this modulatory mechanism by recombinantly expressing
combinations of the major glycosyl-phosphatidylinositol-linked NCAM120 and transmembrane NCAM140 and NCAM180 isoforms, Kir3 channels, and 5-HT1A receptor. Here, we show that the
transmembrane isoforms of NCAM cause a reduction of Kir3 currents. This
effect correlated with reduced cell-surface localization of Kir3
channels. We also show that neuronal Kir3 channels are associated with
lipid rafts, and that mutational and pharmacological disruption of the
lipid raft association of NCAM140 reverts the reduced surface delivery of Kir3 channels. We conclude that the transmembrane isoforms of NCAM
reduce the cell-surface transport of Kir3 channels via a novel lipid
raft-associated mechanism.
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MATERIALS AND METHODS |
DNA constructs. Rat NCAM140 and rat NCAM180/pcDNA3
were a gift from P. Maness (University of North Carolina, Chapel Hill, NC), and rat NCAM120 was a gift from E. Bock (University of
Copenhagen, Copenhagen, Denmark). The plasmid coding for enhanced green
fluorescent protein (GFP) was purchased from Clontech (Palo Alto, CA).
Rat Kir2.1, rat Kir3.1, human Kir3.2, rat Kir3.3 and human Kir3.4, and
the three NCAM isoforms were subcloned either into the pcDNA3 vector
(Invitrogen, Leek, The Netherlands) for expression in Chinese hamster
ovary (CHO) cells or into the psGEM vector, which provides the 5' and
3' untranslated regions of the Xenopus  -globin gene, for
expression in Xenopus oocytes. The NCAM140 construct was generated as described previously (Little et al., 1998) with the exception that mutations were performed using an
EcoRI/XhoI subfragment of NCAM140. The
intracellular domain of NCAM140 (NCAM140id; base pairs 2185-2547) was
amplified by PCR and cloned into the pDNA3 vector. The assembly of
concatemeric Kir3.1/3.2 and Kir3.1/3.4 has been described previously
(Wischmeyer et al., 1997). Flag-tagged Kir3.1 was a gift from D. Clapham (Children's Hospital, Boston, MA) and was fused in-frame to
the Kir3.2 and Kir3.4 subunits. RNA was transcribed in vitro
using the Message Machine kit (Ambion, Austin, TX).
Kir3.1/3.2 and Kir3.1/3.4 chimeras and Kir3.2 mutants. For
construction of the C-terminal and N-terminal chimeras of Kir3.2 and
Kir3.4, standard PCR technique was used. Silent restriction sites were
introduced for SalI and XhoI corresponding to
amino acid positions 83 and 193 in Kir3.4 immediately before the first proposed membrane-spanning segment (TM1) and just downstream of the
second membrane-spanning segment (TM2). Subfragments of the N- and
C-terminal regions of Kir3.2 and Kir3.4 were subcloned into pBluescript
II KS. Chimeras were produced by fusing the N or C termini of
Kir3.2 or Kir3.4 with their SalI site in-frame on the
XhoI site of the corresponding core channel. The sequence of
all PCR-amplified products was verified by DNA sequence analysis. The
following Kir3.2 mutants were constructed: (1) exchange of amino acids
1-35 in the N terminus, (2) exchange of amino acids 1-54 in the N
terminus, (3) exchange of amino acids at positions 58, 61, and 72, and
(4) exchange of amino acids at positions 58, 61, 72, 79, and 98 by the
corresponding amino acids of Kir3.4. Amino acids 1-35 and 1-54 were
exchanged between Kir3.2 and Kir3.4 using the PCR technique of
splicing by overlap extension (Retzer et al., 1996). Mutation of
the indicated amino acids in the N terminus of Kir3.2 was performed
using the Quikchange Mutagenesis kit (Stratagene, Amsterdam, The Netherlands).
Kir3.1/3.2- and Kir3.1/3.4-GFP chimeras. Fusion proteins of
the Kir channels with GFP chimeras (Kir3-GFP chimeras) were constructed by removing the stop codon and introducing an XhoI site at
the 3' end of the coding sequences of Kir3.2 and Kir3.4 using a
standard PCR technique. SalI and XhoI sites were
introduced by PCR into the 5' and 3' ends, respectively, of the GFP
cDNA. The SalI site of GFP cDNA was fused in-frame to the 3'
end XhoI site of the Kir3.2 and Kir3.4 subunits, and the
resulting construct was subcloned into pcDNA3.
Culturing and transfection of CHO cells. CHO cells were
maintained in Glasgow modified Eagle's medium (GMEM) containing 10% fetal calf serum (FCS). CHO cells were seeded in six well plates and
transfected at 90% confluence using the Lipofectamine Plus kit
(Invitrogen, Gaithersburg, MD) according to the manufacturer's instructions. Cells were triple transfected with 0.4 µg of GFP cDNA,
0.6 µg of DNA of the indicated K+
channel, and 1 µg of DNA of the specified NCAM isoform. Cell-surface localization of Kir3 and NCAM was studied 48 hr after transfection. For
electrophysiological recordings, cells were detached with Versene
solution (Invitrogen) 24 hr after transfection and seeded onto
poly-L-lysine-coated glass coverslips. Recordings
were performed 48 hr after transfection. For biochemical analysis and
surface biotinylation assays, cells were detached with Versene solution 24 hr after transfection and reseeded in six well plates at a density
of 40%. Where indicated, 10 µM lovastatin
(Calbiochem, Bad Soden, Germany) together with 250 µM mevalonate (Sigma, Deisenhofen, Germany) (48 hr before analysis) were added to the medium. Lovastatin was used in
the presence of mevalonate to allow synthesis of nonsterol products
from mevalonate (Brown and Goldstein, 1980). Lovastatin was prepared in
its open acid form before use as described previously (Fenton et al.,
1992), and mevalonate was applied in its lactone form.
Calcium phosphate transfection of hippocampal neurons. Cells
were transfected 24 hr after seeding using the calcium phosphate method
(Ethell and Yamaguchi, 1999). Before transfection, cells were incubated
for 1 hr at 3% CO2 and 35°C in serum-free
culture medium. The DNA/calcium phosphate precipitate was prepared
using a Mammalian Transfection Kit (Stratagene) according to the
manufacturer's instructions. Fifty microliters of the DNA/calcium
phosphate precipitate was mixed with 1 ml of serum-free medium and
added to cultures for 3 hr at 3% CO2 and 35°C.
The incubation was stopped by 5% glycerol in serum-free medium. Cells
were then washed three times with medium and returned to 5%
CO2 and 37°C for 11 d.
Surface biotinylation and determination of Kir3 channel
internalization in transfected CHO cells. Internalization kinetics were measured after surface biotinylation as described previously (Schmidt et al., 1997). In brief, 48 hr after transfection, cells were
washed twice with ice-cold PBS containing 2 mM
MgCl2 and 0.2 mM
CaCl2 (PBSCM). Surface proteins were biotinylated
by incubating cells with 0.5 mg/ml
sulfo-N-hydroxysuccinimide-disulfide-biotin (Pierce,
Rockford, IL) in PBSCM for 10 min at 4°C. Biotinylation was
terminated by incubation with 20 mM glycine in
PBSCM at 4°C for 10 min followed by extensive washes with PBSCM.
Biotinylated cells then either were returned to 37°C in GMEM, 10%
FCS for 15 min, 30 min, or 1 hr or were lysed directly in
radioimmunoprecipitation assay (RIPA) buffer containing 10 mM Tris, pH 7.5, 150 mM
NaCl, 1 mM EDTA, 1% Triton X-100, and protease
inhibitor mix (COMPLETE; Roche Diagnostics, Mannheim, Germany) and
centrifuged for 15 min at 4°C. To determine the amount of protein
that was internalized at 37°C after the indicated time periods,
remaining surface-bound biotin was stripped off the surface proteins.
Cells were washed twice with XXX (NT) buffer [20
mM
N-tris(hydroxymethyl)methyl-3-aminopropane-sulfonic acid, pH 8.6, 150 mM NaCl, 0.5 mM CaCl2, 2 mM MgCl2, and 0.2% BSA]
at 4°C and incubated twice for 10 min with NT buffer containing 10 mM sodium-2-mercaptoethanesulfonate at 4°C. The
reaction was terminated by extensive washing with NT buffer. Finally,
cells were lysed in RIPA buffer and centrifuged at 14,000 × g at 4°C for 15 min. The supernatants were
collected, and protein concentrations were determined using the
bicinchoninic acid kit (Pierce). The amounts of surface-localized
proteins and internalized proteins were determined by precipitating
biotinylated proteins with streptavidin-coupled agarose beads (Pierce)
at 4°C overnight. Agarose beads were pelleted by centrifugation and
washed twice with RIPA buffer. Precipitated proteins were solubilized
by the addition of 2× SDS sample buffer to the agarose beads. Proteins
were separated by SDS-PAGE and quantified by immunoblot analysis using
polyclonal Kir3.1 and NCAM (Simon et al., 1991) antibodies.
Isolation of detergent-resistant membrane fractions. Lipid
rafts were isolated as described previously (Melkonian et al., 1999).
In brief, monolayers of cotransfected CHO cells grown in 150 cm2 plates were detached using 4 ml of
Versene solution and pelleted by centrifugation for 5 min at 500 × g and 4°C. Pellets were homogenized in TNE buffer (in
mM: 25 Tris-HCl, pH 7.5, 150 NaCl, 5 EDTA) and COMPLETE protease inhibitor at 4°C using a Dounce homogenizer. Lysates were cleared by centrifugation at 500 × g and
4°C for 5 min to remove unlysed cells and debris. A small fraction of the supernatant was used to verify equal Kir3.1/3.2 and NCAM expression by immunoblot analysis. Membranes were collected by centrifugation at
100,000 × g for 1 hr at 4°C and resuspended in 500 µl of TNE buffer, pH 11, containing 1% Triton X-100. Membranes were
incubated for 30 min on ice, adjusted to 40% sucrose, and placed in an
SW 55Ti ultracentrifuge tube. The 40% fraction was overlaid with 1.5 ml of TNE 36% sucrose and 2 ml of 10% sucrose. After centrifugation (16 hr, 100,000 × g, 4°C), six 750 µl fractions
were collected from the top, diluted with 3 ml of TNE buffer, pH 11, and centrifuged for 1.5 hr at 100,000 × g at 4°C.
This procedure permits isolation of only the Triton X-100-insoluble
proteins in the pelleted fractions, whereas Triton X-100-soluble
proteins remain in the supernatant. Pellets were resuspended in 50 µl
of 2× SDS sample buffer and subjected to SDS-PAGE. For preparation of
lipid rafts from brain homogenates, 10 forebrains from 2-d-old C57BL/6J
mice were homogenized in TNE buffer. Low-density fractions were
prepared exactly as described for CHO cells.
Endoglycosidase H digestion. Cell lysates of transfected CHO
cells were diluted with 2× citrate buffer (150 mM sodium-citrate, pH 5.5) and incubated
overnight at 37°C with 0.1 U of endoglycosidase H (EndoH; Roche
Diagnostics). Controls were treated identically without the addition of
EndoH. Lysates were subjected to immunoblot analysis with Kir3.1 antibodies.
Electrophysiological recordings. Cultures of hippocampal
neurons were prepared by a combination of enzymatic and mechanical dissociation (Dityatev et al., 2000) from 1- to 3-d-old, age-matched C57BL/6J or NCAM/ mice (Cremer et al.,
1994) that had been inbred for at least nine generations onto the
C57BL/6J background. Cells were maintained in vitro for
12-20 d. K+ currents were recorded from
pyramidal-like neurons in the whole-cell mode at room temperature. The
extracellular solution used for recordings from hippocampal neurons
contained (in mM): 140 NaCl, 4 KCl, 10 HEPES, 2 CaCl2, 1.5 MgCl2, 30 glucose, and 12 sucrose, pH 7.25. CHO cells were perfused with the
solution at elevated concentrations of KCl (25 mM) and reduced concentrations of NaCl (119 mM). CHO cells with bright GFP signal were
patched without knowledge of the identity of the plasmids used for
transfection. Recordings of inward currents in neurons were performed
in the presence of tetrodotoxin (1 µM;
Calbiochem) and 0.1 mM
CdCl2. Patch pipettes were filled with (in
mM): 125 K-gluconate, 20 KCl, 10 HEPES, 0.2 EGTA,
2 Mg-ATP, 0.2 Na-GTP, and 10 glucose. The pH was adjusted with KOH to
7.2, and the osmolarity was 310-315 mOsm. G-protein-coupled inwardly
rectifying K+ channels were activated in
neurons either with 30 µM 8-OH-DPAT or 50 µM (RS)-baclofen (both from Tocris,
Bristol, UK). Double whole-cell voltage-clamp recordings were performed
using an EPC-9 amplifier and PULSE software (Heka Electronics,
Lamprecht, Germany). During experiments, serial resistance as well as
cell resistance and capacitance were measured routinely, and leak
currents were digitally subtracted. There was no significant difference
between neurons of different genotypes regarding these parameters.
For electrophysiological recordings from Xenopus oocytes,
cDNAs of all NCAM and Kir3 isoforms were subcloned into the
polyadenylating transcription plasmid psGEM, and capped run-off
poly(A)+ cRNA transcripts (~3 ng each)
were injected into defolliculated oocytes (Wischmeyer et al., 1997).
Oocytes were incubated at 20°C in ND96 solution containing (in
mM): 96 NaCl, 2 KCl, 1 MgCl2, 1 CaCl2, and 5 HEPES, pH 7.4, supplemented with 100 µg/ml gentamicin and 2.5 mM sodium pyruvate. Seventy-two hours after
injection, two-electrode voltage-clamp measurements were performed with
a TURBO TEC-10 C amplifier (NPI Electronics, Tamm, Germany) in ND96 or
high K+ solution containing (in
mM): 96 KCl, 2 NaCl, 1 MgCl2, 1 CaCl2, and 5 HEPES, pH 7.4. Measurements were standardized in each experiment using
cotransfected Kir3 channel/5-HT1A receptor
oocytes as a positive control. Data were acquired and controlled by
PULSE/PULSEFIT software (Heka Electronics).
Indirect immunocytochemistry. Before fixation, transfected
neurons were stained with flag antibody (Sigma) and detected with Cy5-coupled secondary antibodies (Dianova, Hamburg, Germany). This
procedure induces clustering of cell-surface-associated Kir3.1flag/3.2 channels. Cells were fixed with 4% paraformaldehyde in PBS for 15 min
at room temperature and permeabilized with 0.25% Triton X-100 in PBS.
Cells were then blocked with 3% BSA in PBS. Antibodies to Kir3.1 were
applied in PBS containing 3% BSA for 1 hr at room temperature.
Cultures were then washed with PBS and incubated with Cy3-coupled
secondary antibodies (Dianova) for 1 hr at room temperature. Cultures
were washed and postfixed with 2% paraformaldehyde in PBS. Finally,
coverslips were embedded in Aqua Poly/Mount medium (Polysciences, Inc.,
Warrington, PA), and images were taken on a Zeiss (Jena, Germany)
LSM510 confocal laser-scanning microscope.
Quantification of immunofluorescence. Cell-surface
fluorescence in oocytes was measured with a Zeiss LSM410 argon-crypton laser-scanning microscope equipped with a 16 × oil-immersion
objective lens. For quantification of fluorescence intensity, confocal
images were taken under constant parameters from 10 average scans at different locations (n = 5 cells). Cell-surface
fluorescence of transfected NCAM+/+ and
NCAM/ neurons was quantified using
Scion Image software (Scion Corp., Frederick, MD).
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RESULTS |
Kir3 inward currents are increased in hippocampal neurons
of NCAM/ mice
To explore the interaction of NCAM and
5-HT1A receptor signaling, we investigated Kir3
channels in hippocampal neurons cultured from
NCAM+/+ and
NCAM/ mice as a likely target for
5-HT1A receptor signaling (Lüscher et al.,
1997). This analysis was also instigated by our previous findings that
NCAM influences the activity of outward K+
currents in cultured oligodendrocyte precursor cells (Sontheimer et
al., 1990). Expression of Kir3 channels in pyramidal cell-like neurons
could be demonstrated both immunocytochemically (data not shown) and by
patch-clamp analysis. Whole-cell, voltage-clamp experiments revealed
several features typical of Kir3 currents: slow current activation
(Fig. 1A), current
potentiation by the 5-HT1A receptor agonist
8-OH-DPAT (Fig. 1B) and the
GABAB receptor agonist baclofen (Fig.
1C), complete current block by
Ba2+ (Fig. 1B,C), and
current dependence on extracellular [K+]
(data not shown). When compared in neurons from
NCAM+/+ and
NCAM/ mice, it was found that the
density of Kir3-like currents significantly increased to 215% in the
absence of NCAM (Fig. 1E) (1.7 ± 1.7 pA/pS,
n = 29, in NCAM+/+ neurons
vs 3.6 ± 2.6 pA/pS, n = 20, in
NCAM/ neurons at a holding potential
of  130 mV). In contrast, transient and sustained outward
K+ currents remained unchanged (Fig.
1D,E).
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Figure 1.
Increased inward currents in
NCAM-deficient cultured hippocampal neurons. A, Slowly
activating inward currents were evoked in wild-type
(NCAM+/+) and NCAM-deficient
(NCAM/) hippocampal neurons by voltage steps
from 60 to 90, 110, and 130 mV. Inward currents activated in
neurons by a voltage ramp from 130 to 40 mV were augmented by 30 µM 8-OH-DPAT (B) and by 50 µM (RS)-baclofen
(C). These currents were blocked by the addition
of 1 mM Ba2+ into the
extracellular solution (B, C). D,
Transient and sustained K+ outward currents were
evoked in neurons by voltage steps from 60 to 30, 10, and +10 mV.
E, Cumulative data show a significant
difference in inward currents recorded in neurons from
NCAM/ and NCAM+/+ mice
(*p = 0.001; Student's t
test). The horizontal dotted line indicates current density
in neurons from NCAM+/+ mice to which other current
densities were normalized. Bars represent mean values of current
density recorded in NCAM/ relative to
NCAM+/+ neurons; error bars represent SD. Absolute
values corresponding to the bars are in pA/pS (number of cells):
3.6 ± 2.6 (n = 20), 1.7 ± 1.7 (n = 29), 63.1 ± 20.1 (n = 10), 55.5 ± 17 (n = 9), 30.5 ± 16.7 (n = 10), and 28.4 ± 10.9 (n = 9). Calibration: A, 100 pA, 50 msec; B, 100 pA, 200 msec; C, 50 pA, 200 msec; D, 1 nA, 50 msec.
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NCAM140 and NCAM180 reduce neuronal Kir3 currents in
Xenopus oocytes and CHO cells
To determine the functional relationship between NCAM and Kir3
channels, we coinjected Xenopus oocytes with equimolar cRNA concentrations of NCAM isoforms and concatenated pairs of Kir3 subunits, which mimic the Kir3 subunit composition in native cells (Wischmeyer et al., 1997). 5-HT1A receptor cRNA
was coinjected to assay the activity of receptor-activated Kir3
currents with ramp and voltage-step protocols. With 96 mM K+ and 10 µM 5-HT in the bath solution, a Kir3.1/3.2
combination (present in neurons) gave rise to robust inwardly
rectifying K+ currents, which averaged
23.1 ± 7.1 µA (n = 100) at a holding potential of 100 mV. Independent of the presence of NCAM isoforms, both basal and ligand-activated macroscopic Kir3.1/3.2 currents exhibited biophysical properties, e.g., slow current activation (Fig.
2A),
K+ permeability, dependence of conductance
on extracellular K+, or block by
Ba2+ and Cs+,
typical of native G-protein-activated Kir3 channels (data not shown).
After coexpression with NCAM180 or NCAM140, total current amplitudes of
Kir3.1/3.2 were strikingly reduced to 36 ± 14%
(n = 10) and 25 ± 12% (n = 10),
respectively (Fig. 2A,B). These values are of a
magnitude similar to the reduction of Kir3-like currents in
NCAM+/+ mice (42% reduction) compared
with NCAM/ mice. However, coexpression
of NCAM120, which is devoid of an intracellular domain, or a receptor
tyrosine kinase (e.g., the trkB receptor) used as additional control
had no significant effect on Kir3.1/3.2 currents. When probed in CHO
cells, cotransfected NCAM140 and NCAM180 isoforms suppressed
Kir3.1/3.2 channel activity in a quantitatively similar manner (Fig.
2C).
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Figure 2.
Suppression of neuronal Kir3
channels by NCAM180 and NCAM140 in Xenopus oocytes and
CHO cells. Whole-cell currents of Xenopus oocytes
injected with cRNAs encoding Kir3.1/3.2 channel subunits,
5-HT1A receptor, and one of the indicated NCAM isoforms are
shown. A, Currents are responses to 2 sec voltage ramps
between 150 and +60 mV and 500 msec voltage jumps to 80, 100, and
120 mV, respectively, in the presence of 10 µM 5-HT and
96 mM K+. Activation time constants at 0 mV were 20.3 msec in the absence and 19.7 msec in the presence of
NCAM140. B, Bar graph showing the relative modulation of
Kir3.1/3.2 currents by NCAM120 (1.1 ± 0.43; amplitude relative to
control), NCAM140 (0.36 ± 0.14), NCAM180 (0.25 ± 0.12), and
NCAM140id (expression vector encoding the intracellular domain of
NCAM140) (0.9 ± 0.18) in oocytes. C, Bar graph
showing the relative modulation of Kir3.1/3.2 currents by NCAM120
(1.24 ± 0.67, n = 10), NCAM140 (0.29 ± 0.33, n = 13), and NCAM180 (0.17 ± 0.09, n = 11) in transfected CHO cells. D,
Macroscopic current responses to 2 sec voltage ramps between 150 and
+60 mV of oocytes injected with NCAM180, 5-HT1A
receptor, and different Kir3 channels. E, Bar graph
showing the relative inhibition by NCAM180 of Kir3.1/3.2 channels
(0.25 ± 0.12 amplitude relative to control in the absence of
NCAM), Kir3.1/3.3 (0.18 ± 0.12), Kir3.1/3.4 (0.92 ± 0.38),
and Kir2.1 (0.86 ± 0.26). Error bars represent SD.
Horizontal dotted lines in A and D
indicate baseline. Vertical dotted lines in A and
D indicate 0 mV holding potential. Horizontal dotted
lines in B, C, and E indicate
current amplitudes to which other current densities were normalized.
*Statistical significance (Student's t test;
p < 0.01).
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To investigate the specificity of this reduction in channel activity by
NCAM isoforms, other Kir3 channel combinations were also tested. We
found that the Kir3.1/3.3 channel expressed in neurons was also
severely suppressed by NCAM180 to 18 ± 12% (n = 10). In contrast, both a Kir3.1/3.4 channel, constituting the cardiac
KACh channel, as well as other
neuronal, constitutively active Kir channels (e.g., Kir2.1), were not
significantly affected (Fig. 2D,E).
NCAM140 reduces surface localization of GFP-tagged Kir3.2 channels
in Xenopus oocytes
To visualize channel targeting to the cell surface of
Xenopus oocytes, Kir3.2 and Kir3.4 channel subunits were
tagged with enhanced GFP at their C termini. cRNAs were injected with
or without NCAM120, NCAM140, and NCAM180, and the membrane GFP
fluorescence of intact oocytes was inspected by confocal microscopy 48 hr after cRNA injection. Although uninjected oocytes showed no
background fluorescence, injection of Kir3.1/3.2-GFP and
Kir3.1/Kir3.4-GFP constructs resulted in strong fluorescence signals at
the cell surface, which were quantified by line-scan luminometry using a photomultiplier (Fig. 3). Coexpression
of NCAM140 caused a prominent decrease by ~58% of Kir3.1/3.2-GFP
fluorescence in the membrane (Figs. 3A,B), whereas the
Kir3.1/3.4-GFP signal remained unaffected by coexpressed NCAM140 (Fig.
3C,D). Similar results were obtained by coinjection of
Kir3.1/Kir3.2-GFP with NCAM180 (data not shown). Coinjection of
NCAM120, however, did not alter Kir3.1/3.2-GFP fluorescence levels.
These results are in accordance with the measurements of ensemble and
unitary Kir3 currents (see below) and indicate that NCAM140 and NCAM180
control membrane surface localization of neuronal Kir3 channels.
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Figure 3.
Cell-surface localization of Kir3 channels is
reduced by NCAM. Representative confocal images of oocytes injected
with cRNAs of GFP-tagged Kir3.1/3.2 (A, B) and
Kir3.1/3.4 (C, D), respectively, in the absence
(A, C) and presence (B, D) of NCAM140 are
shown. Graphs show average fluorescence measurements of 10 line
scannings perpendicular to the cell surface representative of five
oocytes measured. Data are shown as mean ± SD of fluorescence
signal.
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The N terminus of Kir3.2 is the major structural determinant for
NCAM sensitivity
From the differential sensitivity of Kir3 channels to NCAM
inhibition, we sought to identify the structural determinants for the
reduced Kir3 channel membrane localization. Because it was unlikely
that Kir3.1 subunits, present in all tested combinations, conferred the
sensitivity for NCAM, we analyzed hybrid channels in which the N or C
termini of the Kir3.2 and Kir3.4 subunits were exchanged. When the C
termini were exchanged, the hybrid channels maintained the NCAM
sensitivity of the core channel (Fig. 4A,B), whereas exchange
of the N termini reverted the sensitivity of the core channel toward
NCAM180 (Fig. 4C,D). Thus, Kir3.1/3.4 channels were
suppressed by NCAM180 when the N terminus of Kir3.4 was substituted by
the N terminus of Kir3.2. Although current suppression of hybrid
channels was not as pronounced as in Kir3.1/3.2 wild-type channels, we
conclude that the N terminus of the Kir3.1/3.2 channel harbors the
major structural determinants for NCAM inhibition. To obtain additional
insights into functionally important domains and amino acids, we
exchanged subdomains and single amino acid residues that differ between
the N termini of Kir3.2 and Kir3.4 (see Materials and Methods). The
inhibitory effect of NCAM140 was not abolished in any of these mutants
(data not shown), suggesting that multiple allosteric effects are
involved in the NCAM-mediated Kir3 inhibition.
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Figure 4.
N termini of Kir3 subunits determine sensitivity
to NCAM. A, C, Macroscopic current responses to 2 sec
voltage ramps between 150 and +60 mV of oocytes injected with
NCAM180, 5-HT1A receptor, and different combinations of
hybrid Kir3.2/Kir3.4 subunits connected to Kir3.1 subunits. N and C
termini of injected Kir3 subunits were exchanged as shown in the
drawings with Kir3.2 components in gray and Kir3.4 in
black. B, Bar graph showing the relative
inhibition by NCAM180 of Kir3.1/3.2 and Kir3.1/3.4 with the C terminus
exchanged, Kir3.1/3.2 + C terminus 3.4 (0.6 ± 0.26), and
Kir3.1/3.4 + C terminus 3.2 (0.82 ± 0.39). D,
Relative inhibition by NCAM180 of Kir3.1/3.2 and Kir3.1/3.4 with the N
terminus exchanged, Kir3.1/3.2 + N terminus 3.4 (0.84 ± 0.2), and
Kir3.1/3.4 + N terminus 3.2 (0.56 ± 0.27; n = 10 each). Error bars represent SD. Horizontal dotted lines
in A and C indicate baseline. Vertical
dotted line in A and D indicates 0 mV
holding potential. Horizontal dotted lines in B
and D indicate current amplitudes to which the other
currents were normalized. *Statistical significance (Student's
t test; p < 0.01).
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Because cell-surface localization of Kir3 channels is specifically
reduced by the NCAM140 and NCAM180 isoforms that harbor an
intracellular domain, we tested whether the intracellular domain of
NCAM140 alone was sufficient to cause this effect. When a construct that encodes NCAM140id and that is membrane associated because of the presence of palmitoylated intracellular cysteines (Little et
al., 1998) was coinjected with Kir3.1/3.2, it did not suppress Kir3
channel cell-surface localization or Kir3 inward currents (90 ± 18% of control) (Fig. 2B).
Stimulation of NCAM140- or NCAM180-associated signal transduction
pathways do not alter surface localization of Kir3.1/3.2
We subsequently asked which mechanisms could account for the
reduced surface expression of neuronal Kir3 channels in the presence of
NCAM140 or NCAM180. First, we noted that acute or prolonged stimulation
of NCAM by NCAM-specific antibodies known to induce NCAM-dependent signaling (Beggs et al., 1997) was without effect on the
activity of Kir3.1/3.2 channels in Xenopus oocytes. Under our recording conditions, no significant differences in Kir3.1/3.2 current amplitudes were measured for NCAM140 or NCAM180 after application of polyclonal NCAM antibodies (200 µg/ml) for 30 min and
48 hr, respectively. Second, suppression was unaltered when assayed for
known mediators of NCAM signaling [i.e., incubation of
oocytes for 48 hr with 2'-amino-3'-methoxyflavone, a
selective inhibitor of microtubule-associated protein kinase kinases
(Kolkova et al., 2000), or
1-tert-butyl-3-[6-(3,5-dimethoxy-phenyl)-2-(4-diethylamino-butylamino)-pyrido [2,3-d]pyrimidin-7-yl]-urea
(PD173074), a specific inhibitor (Mohammadi et al., 1998) of the
FGF receptor known to be functionally linked to NCAM]. Third,
single-channel measurements in the cell-attached configuration showed
that both unitary conductance and open probability of Kir3.1/3.2
channels remained unchanged in the presence of NCAM180 (Fig.
5A). These observations
support the view that Kir3 current reduction by NCAM is attributable to
a reduction in the number of functional channels at the cell surface,
as revealed by immunofluorescence measurements. Finally, we were unable
to demonstrate a direct physical interaction of Kir3.1/3.2 and NCAM140
by cocapping, coimmunoprecipitation, or yeast two-hybrid assays that
tested the interaction of the NCAM140 intracellular domain with the N
and C termini of Kir3.2 (data not shown).
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Figure 5.
Exclusion of NCAM140 from lipid rafts or
depletion of cholesterol neutralizes the inhibitory effect of NCAM140
on Kir3 channel activity. A, Single channel
cell-attached recordings at +60, 60, and 100 mV are shown from
oocytes coinjected with Kir3.1/3.2 and NCAM180 (left
panel). No significant difference in channel activity is
observed in the presence or absence of NCAM180 (middle
panel). The bar graph quantifies the product of open
probability (Po) and unitary
conductance ( ) for Kir3.1/3.2 channels in the presence and absence
of NCAM. B, Schematic diagram of NCAM140 palmitoylation
sites. Mutated cysteine residues are indicated in gray.
Circular areas indicate immunoglobulin-like domains, and
the fibronectin type III-like domains are shown as gray
boxes. The surface membrane is indicated by the pair of
vertical lines. C, Immunoblot analysis of
sucrose gradient fractions of Kir3.1/3.2 and NCAM140
(left) or NCAM140
(right)-cotransfected CHO cells. Kir3.1/3.2 (top
panels) is present both in the low-density raft fractions
(lanes 3 and 4) and in the
high-density fraction (lane 6), possibly
containing cytoskeleton-associated proteins. Analysis of the
low-density sucrose gradient fractions shows that the amount of
Kir3.1/3.2 in lipid rafts is identical in the presence of NCAM140 or
NCAM140. The blot was stripped and reprobed with polyclonal NCAM
antibodies verifying that, in contrast to NCAM140, NCAM140 is hardly
detectable in the lipid raft fractions (bottom panel,
lanes 2-4). NCAM140 and the Kir3 channel are
both present in the major lipid raft fraction (lane 3).
D, In forebrain homogenates, the Kir3.1 subunit is
present in the low-density fractions (lanes 3 and
4) representing the lipid raft fraction and in
the high-density, Triton X-100-insoluble fraction (lane
6, top panel). The blot was reprobed with
an antibody for the raft-associated nonreceptor tyrosine kinase fyn
(bottom panel) to confirm isolation of lipid
rafts. E, Bar graph showing the relative inhibition of
Kir3.1/3.2 currents in CHO cells mediated by NCAM140 (0.14 ± 0.04), NCAM140 (0.79 ± 0.19), and NCAM120 (1 ± 0.23).
Lovastatin treatment rescued NCAM140-mediated inhibition (0.47 ± 0.07), leaving NCAM120-mediated inhibition unchanged (0.86 ± 0.14). Mean amplitudes relative to Kir3.1/3.2-NCAM120-cotransfected
cells are shown. *Statistical significance from control.
#Statistical significance between Kir3.1/3.2 + NCAM140 with
and without lovastatin (Student's t test;
p < 0.01). Error bars represent SEM.
Horizontal dotted lines in A indicate the open
and closed state of the channel. The horizontal dotted line
in E indicates the current amplitude to which the other
currents were normalized. The vertical dashed line in
E separates the results without lovastatin from the results
with lovastatin. IB, Immunoblot for the molecules
indicated.
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Impairment of NCAM140 raft association abrogates the
inhibitory effect of NCAM140 on Kir3.1/3.2 cell-surface
localization
We have reported recently that all NCAM isoforms are associated
with lipid rafts, and that lipid raft association of NCAM140 is
essential for its function as a neuritogenic receptor (Niethammer et
al., 2002). Therefore, we asked whether lipid raft association of
NCAM140 is essential to modulate Kir3 channel surface localization. These experiments were instigated by our observation that Kir3.1/3.2 is
partly associated with lipid rafts in transfected CHO cells and brain
homogenates using the well established criteria for identification of
lipid raft-associated proteins, namely insolubility in cold nonionic
detergents and flotation in defined sucrose density gradients (Hooper,
1997). As shown in Figure 5C,D, Kir3.1 immunoreactivity is
present in the upper fractions (lanes 3 and
4) of a sucrose density gradient of Triton
X-100-insoluble protein fractions of transfected CHO cell lysates and
mouse forebrain homogenates, indicating a partial association with the
lipid raft fraction in CHO cells and mouse brains. Kir3.1 subunits were
also present in the Triton X-100-insoluble bottom fraction of the
gradient (Fig. 5C,D, lane 6), which is
likely to contain cytoskeleton-associated Kir3.1 subunits (Montixi et
al., 1998; Kennedy et al., 1999).
We then asked whether removal of NCAM140 from lipid rafts would affect
Kir3 channel cell-surface localization. We have shown that raft
association of an NCAM140 mutant (denoted NCAM140) is reduced
compared with NCAM140 when the palmitoylation sites, and thus a
raft-targeting signal, are removed (Fig. 5B) (Niethammer et
al., 2002). NCAM140 but not NCAM140 colocalizes with Kir3.1/3.2 in
the lipid raft fraction of transfected CHO cells (Fig. 5C, left panel, lanes 3 and 4 vs Fig.
5C, right panel, lanes 3 and 4). Lipid raft localization of the Kir3 channel
is not altered when NCAM140 is removed from the lipid rafts by ablation
of palmitoylation sites (Fig. 5C, top panels). It
needs to be emphasized in this context that NCAM140 and NCAM140 are
present in equal amounts in total cell lysates and at the cell surface
(Niethammer et al., 2002). The inhibitory effect of NCAM140 on Kir3
channel surface localization is abolished when NCAM140 is deprived of
palmitoylation sites and therefore not present in lipid rafts (Fig.
5E). Lipid rafts may thus function as a platform from which
NCAM controls the surface localization of Kir3.1/3.2 channels.
To further document the involvement of lipid rafts in the
NCAM-mediated surface localization of Kir3 channels, we disrupted lipid
rafts in CHO cells by depletion of cholesterol using lovastatin, a
blocker of the 3-hydroxy-3-methylglutaric acid CoA reductase and
thus of cholesterol biosynthesis. This treatment not only disperses
lipid rafts in the surface membrane but also affects assembly of lipid
rafts in intracellular organelles, such as the trans-Golgi
network. As determined electrophysiologically, disruption of lipid
rafts by lovastatin partially rescued Kir3 channel surface localization
in Kir3.1/3.2-NCAM140-cotransfected cells (53 ± 7% reduction by
NCAM140 in the presence of lovastatin vs 86 + 4% reduction by NCAM140
in the absence of lovastatin) (Fig. 5E). Treatment with
lovastatin per se did not impair the surface localization or channel
properties of the Kir3.1/3.2, because currents were unaffected by
lovastatin in Kir3.1/3.2-NCAM120-cotransfected cells (Fig.
5E). One reason why NCAM140-mediated Kir3 channel inhibition was only partly rescued by lovastatin is most likely because of the
incomplete block of cholesterol biosynthesis by this treatment (Keller
and Simons, 1998).
Reduction of Kir3 channel cell-surface localization by NCAM140
altered delivery to or internalization from the surface membrane?
To further investigate the mechanisms of Kir3 channel cell-surface
localization, we assayed the internalization of Kir3.1/3.2 and
Kir3.1/3.4 biochemically by surface biotinylation in CHO cells after
transfection with Kir3.1 that was tagged by a flag-epitope inserted
into the first extracellular loop of the subunit (Kennedy et al.,
1999). Single channel properties of Kir3.1flag/3.2 and Kir3.1flag/3.4
and current inhibition by NCAM140 were unaffected by the flag epitope
(data not shown). Quantification of Kir3.1/3.2 and Kir3.1/3.4
cell-surface localization showed that NCAM140 but not NCAM140
reduced the amount of Kir3.1/3.2 protein (but not Kir3.1/3.4, data not
shown) in the surface membrane by ~50% (Fig. 6A, lanes 8 vs 7 and 9, B). Overall, protein
expression of Kir3.1flag/3.2 was not significantly affected by NCAM140
(Fig. 6A, lanes 10-12, C).
Lovastatin treatment also did not alter the overall protein expression
of Kir3.1flag/3.2 (Fig. 6A, lanes 4-6 vs
10-12).
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Figure 6.
NCAM140 reduces cell-surface
localization of Kir3.1/3.2 in CHO cells but does not alter
internalization rates of Kir3.1/3.2. A, Immunoblot
analysis of Kir3.1/3.2 cell-surface expression in CHO cells. CHO cells
were cotransfected with Kir3.1/3.2 and either NCAM120 (lanes 3, 6, 9, and 12), NCAM140 (lanes 2, 5, 8, and 11), or NCAM140 (lanes 1, 4, 7, and 10). Cell lysates (lanes
4-6 and 10-12) and proteins bound to
streptavidin-agarose (lanes 1-3 and
7-9) were separated by SDS-PAGE, and the amount of
Kir3.1/3.2 or NCAM was quantified by immunoblot analysis using
polyclonal Kir3.1 antibodies. NCAM140 reduces surface localization of
Kir3.1/3.2 compared with NCAM120 and NCAM140 (lane 8
vs lanes 7 and 9), whereas the overall
intensity of Kir3.1/3.2-immunoreactive bands in the cell lysate is not
altered by NCAM140 (lane 11 vs lanes 10
and 12). Incubation with lovastatin does not influence
Kir3.1/3.2 expression (lanes 4-6 vs
10-12) but enhances surface localization of Kir3.1/3.2
(lane 2 vs lanes 1 and
3). B, Normalized surface
localization of Kir3.1/3.2 in the presence of NCAM140 (0.48 ± 0.03) and NCAM140 (0.84 ± 0.09) in comparison with NCAM120.
Kir3.1-immunoreactive bands of streptavidin-agarose-precipitated
proteins were quantified by densitometric analysis and expressed
relative to NCAM120 (100%). *Values significantly different from
that of NCAM120 (Student's t test;
p < 0.01). Error bars represent SEM.
C, Normalized protein expression of Kir3.1/3.2 in the
presence of NCAM140 (0.86 ± 0.05) and NCAM140 (1.03 ± 0.04) in comparison with NCAM120. Kir3.1-immunoreactive bands in total
cell lysates were quantified by densitometric analysis and expressed
relative to NCAM120. Bars represent means ± SEM.
D, Internalization kinetics of Kir3.1/3.2 in the
presence of NCAM120 (lanes 1 and
4), NCAM140 (lanes 2 and
5), and NCAM140 (lanes 3 and
6). Lanes 1-3, The initial amount
of Kir3.1/3.2 channels present at the cell surface at 0 min.
Lanes 4-6, The amount of Kir3.1/3.2 and the individual
NCAM isoforms internalized after 60 min at 37°C. E,
Internalization rates were calculated by dividing the relative
intensities of Kir3.1-immunoreactive bands (normalized to
cotransfection with NCAM120) after cells had been exposed to 60 min at
37°C (lanes 4-6 in D) by the band
intensities at 0 min (lanes 1-3 in D).
Kir3.1/3.2 channels are internalized with the same kinetics when
cotransfected with NCAM140 (0.97 + 0.04) or NCAM140 (1 + 0.05)
compared with cotransfection with NCAM120. Bar graphs show
internalization relative to NCAM120. Bars show the mean ± SEM.
F, EndoH digestion of CHO cell lysates transfected with
Kir3.1/3.2 and NCAM140 (lanes 1 and
2), Kir3.1/3.2 NCAM140 (lanes 3 and
4), and the cell adhesion molecule L1
(lanes 5 and 6), in which the
homophilic-binding site was deleted (Zhao et al., 1998). Cell lysates
were either treated with EndoH (lanes 1, 3, and
5) or treated without EndoH (lanes 2, 4,
and 6). EndoH digestion does not result in a
shift of the immunoreactive bands in their apparent molecular weights,
indicating that Kir3.1/3.2 does not acquire an increased EndoH
sensitivity in the presence of NCAM140. As a positive control, CHO
cells were transfected with a protein known to be retained in the ER,
namely the mutated L1 molecule (A. Rünker, personal
communication). The L1 molecule is shifted to a lower apparent
molecular weight by EndoH digestion (lane 5 vs
6). The horizontal dotted lines in
B, C, and E indicate the current
amplitude to which the other currents were normalized.
IB, Immunoblot for the molecules
indicated.
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Cell-surface biotinylation of the Kir3 channels allows the measurement
of their internalization rates. Kir3 channel-transfected CHO cells
showed that fewer Kir3 channels were internalized after 1 hr at 37°C
in the presence of NCAM140 (Fig. 6D, lane
5) compared with NCAM120 and NCAM140 (Fig.
6D, lanes 4 and 6).
Because initially fewer Kir3 channels were surface localized in the
presence of NCAM140 (Fig. 6D, lane 2 vs
lanes 1 and 3), the ratio between the initial
amount of Kir3.1/3.2 present at the cell surface (lanes 1-3) and the amount internalized after 1 hr (lanes
4-6) was identical among the individual NCAM isoforms
(Fig. 6E). These results were also obtained for
shorter (15 and 30 min) internalization periods (data not shown). Our
data thus show that NCAM140 does not enhance the internalization rates
of Kir3 channels, suggesting that NCAM140 and NCAM180 reduce the
delivery of Kir3.1/3.2 to the surface membrane.
To determine whether Kir3 channels remain in the endoplasmic reticulum
(ER) in the presence of NCAM140, we measured the endoglycosidase H
sensitivity of Kir3.1/3.2 in cells transfected with NCAM140 or
NCAM140. Sensitivity of the protein carbohydrate moieties toward
EndoH is an indicator for the retention of proteins in the ER. We
observed that Kir3 channels were not more sensitive to EndoH in
NCAM140- compared with NCAM140-transfected cells, indicating that
the channels were not retained in the ER by NCAM140 (Fig.
6F). Thus, control of the number of Kir3 channels at
the cell surface by NCAM140 is likely to occur in compartments
downstream of the ER en route to the surface membrane, presumably at
the level of the Golgi network.
Kir3 channel transport in NCAM-deficient neurons
We also investigated whether increased Kir3 currents in neurons of
NCAM-deficient (NCAM/) mice (Fig.
1E) would correlate with altered surface targeting of
Kir3.1/3.2. Cultured hippocampal neurons of wild-type
(NCAM+/+) and
NCAM/ mice were cotransfected with
Kir3.1flag/Kir3.2 channel and GFP. Transfected neurons were identified
by the GFP signal, and live neurons were stained by an antibody
recognizing the extracellular flag epitope of the Kir3 channel,
allowing quantification of Kir3 channels at the cell surface. In live
NCAM+/+ neurons, weak flag immunostaining
was detectable at the neuronal cell surface (Fig.
7A,c), showing that only small
amounts of Kir3.1/3.2 are targeted to the cell surface. Immunostaining
of permeabilized neurons, also monitoring intracellular localization of
Kir3 channels, showed Kir3 channel accumulation primarily in the
cytoplasm of the somata in vesicular structures around the nucleus.
Only weak Kir3 staining was detected in the neurites of the
permeabilized cells (Fig. 7A,e).
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Figure 7.
Kir3.1flag/3.2 channels are enriched at the cell
surface and in the dendrites of NCAM/ neurons.
A, Confocal images of NCAM/
(a, c, e) and NCAM+/+ (b, d,
f) neurons cotransfected with Kir3.1flag/3.2 channels
and GFP. Only cells expressing comparable amounts of GFP were selected
for quantification (a, b). Neurons were stained with
flag antibody before fixation (c, d). After
permeabilization, cells were stained with Kir3.1 antibody to visualize
intracellular Kir3.1flag/3.2 channels (e, f). In
NCAM/ neurons, more flag-immunoreactive clusters
are detectable on the cell surface than in NCAM+/+
neurons (d vs c), demonstrating that more
Kir3.1flag/3.2 channels are present at the cell surface in
NCAM/ neurons. In NCAM+/+
neurons, intracellular Kir3.1 immunoreactivity is prominent in the cell
soma around the nucleus, whereas it is only weakly present in neurites
(e). In contrast, NCAM/
neurons show a more diffuse intracellular Kir3.1 immunoreactivity in
neurites and cell soma (f).
B, Bar graph showing in arbitrary units (AU) the mean
fluorescence signal ± SEM of cell-surface flag immunostaining of
Kir3.1flag/3.2-transfected NCAM/ (7.45 ± 0.96 AU) and NCAM+/+ (3.7 ± 0.53 AU) neurons
(n = 30 each), demonstrating that Kir3.1flag/3.2
surface localization is reduced by ~50% in
NCAM+/+ versus NCAM/ neurons.
Scale bar, 20 µm. *Statistically significant difference between
genotypes (Student's t test; p < 0.01).
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In contrast, live NCAM/ neurons
transfected with Kir3.1flag/3.2 showed prominent flag immunostaining at
the cell surface, indicating considerable targeting of the Kir3 channel
to the cell surface. Quantification of flag surface fluorescence showed
that Kir3.1flag/3.2 surface localization was increased by 50% in
NCAM/ compared with
NCAM+/+ neurons (3.7 ± 0.53 vs
7.45 ± 0.96; n = 30) (Fig. 7B).
Moreover, immunostaining for the Kir3 channel in permeabilized
NCAM/ neurons showed that the Kir3
channel was not retained in the somata around the nucleus as in
NCAM+/+ neurons but was transported into
the neurites to be inserted into the surface membrane.
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DISCUSSION |
In a previous study on NCAM-deficient mice,
5-HT1A receptor signaling was identified to be
hypersensitized to the receptor agonists 8-OH-DPAT and buspirone (Stork
et al., 1999). The functions of the 5-HT1A
receptor itself, however, were unlikely to be altered, because receptor
distribution and affinity or 5-HT metabolism in different brain regions
of NCAM-deficient and wild-type mice proved to be independent of the
genotype. Furthermore, coupling of the 5-HT1A
receptor to adenyl cyclase in transfected CHO cells was independent of
the presence of any of the NCAM isoforms (M. Delling, unpublished
observations), indicating that NCAM neither influenced the
5-HT1A receptor itself nor coupling of the
receptor to G-proteins. Based on these results, we investigated the
possibility that NCAM influences Kir3 channel activity as a downstream
effector of the 5-HT1A receptor.
NCAM140 and NCAM180 reduce the cell-surface localization of
Kir3 channels
In cultured hippocampal neurons of
NCAM+/+ and
NCAM/ mice and in heterologous
expression systems, Kir3 channel currents (Kir3.1/3.2 or Kir3.1/3.3)
are reduced by ~70% by the NCAM140 and NCAM180 isoforms. The
reduction of Kir3 currents by NCAM140 and NCAM180 correlated with a
decrease in the cell-surface localization of the Kir3 channel, whereas
overall expression of the channel was not reduced. In addition, in
NCAM+/+ hippocampal neurons, Kir3.1/3.2
surface localization was reduced by ~50% compared with
NCAM/ neurons, and Kir3 channels were
accumulated in intracellular organelles around the nucleus, suggesting
that the Kir3 channel is retained by NCAM in Golgi apparatus-like structures.
Our results are in agreement with previous studies in which Kir3.1
subunits were not only detectable on dendritic spine membranes but also
in the intracellular compartments of cell somata in the CA1 region of
the hippocampus. These intracellular compartments were identified as
Golgi network structures and as other, not fully characterized small
vesicles (Drake et al., 1997). In primary thyrotroph cells, Kir3.1
subunits were found to be associated with intracellular dense core
vesicles. After stimulation with thyrotropin-releasing hormone, they
fuse with the surface membrane and thus enhance
KG currents (Morishige et al., 1999).
Characterization of Kir3 channel internalization kinetics suggests that
NCAM also regulates transport of Kir3 channels to the cell surface
rather than internalization of the channel. Interestingly, activation of NCAM-dependent signaling cascades by antibody clustering and blockage of NCAM-linked kinase pathways did not influence Kir3 channel
delivery to the cell surface, suggesting that NCAM regulates this
delivery not via the known signaling pathways but via a novel mechanism.
Hence, our study extends the reports on the controlled transport of
Kir3 channels to the membrane (Morishige et al., 1999; Ma et al., 2001)
by the idea that the number of Kir3 channels in the membrane can also
be controlled by a recognition molecule.
Lipid rafts are involved in the transport of Kir3.1/3.2 channels to
the membrane
We have shown that both NCAM140 and the Kir3.1/3.2 channel are
partially associated with lipid rafts, as demonstrated previously for a
specific subgroup of K+ channels, the
Kv1.5 and Shaker K+ channels (Martens et
al., 2000a,b). Ablation of intracellular palmitoylation and thus raft
targeting sites of NCAM140 recovered cell-surface localization of
Kir3.1/3.2. In addition, disruption of lipid rafts by lovastatin
treatment partly rescued the inhibition of NCAM140 on Kir3 channel
delivery to the cell surface. Our observations underline the importance
of lipid rafts in cellular protein trafficking, which has already been
demonstrated in previous studies (for review, see Simons and Ikonen,
1997). It was also shown that lovastatin impairs surface
delivery of the influenza virus hemagglutinin in transfected baby
hamster kidney-21 and Madin-Darby canine kidney cells (Keller
and Simons, 1998) and leads to a mis-sorting of the raft-associated
aminopeptidase N in enterocytes (Hansen et al., 2000). However, in the
present study, disruption of lipid rafts did not affect channel
delivery per se, because lovastatin had no effect on current amplitudes
in Kir3.1/3.2 and NCAM120 cotransfected cells. Instead, we observed an
indirect effect of lipid raft disruption on Kir3 channel delivery,
namely that removal of NCAM from lipid rafts enhances the transport of
Kir3 channels to the plasma membrane. We cannot define whether
NCAM-dependent modulation of Kir3 channels takes place in lipid rafts
of the Golgi apparatus intracellularly or at the cell surface in the plasma membrane. Although treatment of CHO cells with
methyl-cyclodextrin, an agent extracting cholesterol from the plasma
membrane and thus dispersing plasma membrane lipid rafts, had no
influence on NCAM140-mediated inhibition of Kir3 channel trafficking
(our unpublished observations), suggesting that plasma membrane
lipid rafts are not involved in the inhibitory effect, we cannot
determine at which stage of intracellular processing NCAM blocks Kir3
channel delivery to the cell surface.
We thus hypothesize that NCAM140 modulates the retention of Kir3
channels via its presence in lipid rafts. The precise NCAM-mediated mechanism of channel retention operant within lipid rafts is presently unknown and deserves additional attention. Because we were unable to
demonstrate a direct physical interaction of Kir3 channels with
NCAM140, we suggest that intracellular retention of the Kir3 channel is
controlled via an NCAM-linked mediator acting on the N terminus of the
Kir3 channel (see Fig. 8 for a
hypothetical model) that has been implicated in the cell-surface
delivery of Kir3 channels (Stevens et al., 1997). Alternatively,
yet unknown raft-associated second messenger systems under the control
of NCAM140 and NCAM 180 might be involved in the retention of the Kir3
channel. However, raft localization of NCAM per se is not sufficient, because NCAM120, although present in lipid rafts
(Niethammer et al., 2002), does not modulate Kir3 delivery, most likely
because of the absence of an intracellular domain. Furthermore, the
intracellular domain of NCAM140 per se is also not sufficient for
inhibition of Kir3 transport, possibly because of incorrect
configuration of this domain. Because raft localization of NCAM140 and
NCAM180, and thus the degree of Kir3 channel inhibition, are determined by palmitoylation (Fig. 8), the regulatory mechanisms controlling palmitoylation deserve major attention (Resh, 1999). An important role
for palmitoylation has been demonstrated by previous studies showing
that a reduction in palmitoylation of growth cone proteins is
sufficient to stop neurite extension (Hess et al., 1993; Patterson and
Skene, 1994). There is growing evidence that different subtypes of
lipid rafts exist in the plasma membrane (Pierini and Maxfield, 2001).
Further analysis of the exact lipid raft subtype containing NCAM and
the Kir3 channels may yield additional insights into the mechanisms by
which NCAM inhibits Kir3 channel delivery to the cell surface.
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Figure 8.
Hypothetical scheme of NCAM-mediated inhibition of
Kir3 channel transport to the cell surface. Kir3 channels are
transported along the secretory pathway to the cell surface
(pathway A) or to yet-unidentified intracellular
compartments (pathway B) (Drake et al., 1997).
Palmitoylation of the intracellular domain of NCAM140 or NCAM180 and
thus targeting to lipid rafts favors the transport of Kir3 channels
into intracellular compartments rather than to the cell surface. Both
NCAM140 and Kir3 channels are present in lipid rafts. Disruption of
lipid rafts by lovastatin or mutation of NCAM140 palmitoylation sites
directs the Kir3 channels into pathway A. Because no direct molecular
association of the transmembrane isoforms of NCAM and the Kir3 channel
could be demonstrated, a raft-associated functional linker
(arrow) might be involved. Both the N terminus of the
Kir3.2 subunit and the intracellular domain of NCAM140 in its complete
transmembrane configuration favor pathway B.
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Physiological relevance of recognition molecule-dependent
regulation of Kir3 channel activity
The combined observations tempt us to speculate on the involvement
of NCAM in the 5-HT1A receptor-mediated
regulation of Kir3 channels. We have shown previously that the number
of NCAM180-positive spine synapses in the hippocampus is increased
after induction of long-term potentiation (Schuster et al.,
1998). This increase has been suggested to enhance synaptic strength
(Schachner, 1997). As suggested by the present study, another
consequence of NCAM180 upregulation would be a downregulation of the
inhibitory Kir3 subgroup of potassium channels in the cell membrane and
thus a reduction in the amplitudes of slow inhibitory postsynaptic
potentials triggered by several neurotransmitters. These
neurotransmitter receptors would be coupled to the G-protein-activated
Kir3 channels and thereby control the degree of postsynaptic
hyperpolarization. As a consequence of reduced Kir3 channel activity
during periods of NCAM protein upregulation correlating with increase
in synaptic efficacy, postsynaptic cell impedance increases, and
neurons become more sensitive to synaptic inputs. These effects would
also be important during neuritogenesis and synapse formation. In
NCAM/ mice, increased activity of
5-HT1A receptor-stimulated Kir3 channels may be
causal for a lower excitability of target neurons for serotonergic fibers in the limbic system, and thus altered levels of aggression and
anxiety (Stork et al., 1999, 2000). Although the behavioral consequences of NCAM-dependent Kir3 channel regulation remain to be
elucidated at the cellular level, our study provides the first insights
into some of the molecular determinants regulating neuronal long-term
excitability by controlling the number of active K+ channels in the surface membrane.
| |
FOOTNOTES |
Received Jan. 28, 2002; revised May 14, 2002; accepted May 20, 2002.
This work was supported by Deutsche Forschungsgemeinschaft Grants
Scha 185/17-1 (M.S.), Ka 1175/1-3 (A.K.), and Ve 187/1-2 (R.W.V.). M.D.
is a scholar of the Studienstiftung des Deutschen Volkes. We thank Dr.
Harold Cremer for his gift of NCAM-deficient mice, Galina Dityateva for
hippocampal cultures, Drs. Patricia Maness and Elisabeth Bock for the
gift of NCAM cDNAs, Dr. D. Clapham for the gift of the flag-tagged
Kir3.1 channel, Dr. Olaf Pongs for his comments on this manuscript, and
Dr. Günter Gercken for support. We thank M. Honemann and A. Sporning for oocyte preparation and P. Niethammer for help with the
sucrose gradients. The FGF inhibitor PD173074 was a gift from
Parke-Davis (Ann Arbor, MI).
Correspondence should be addressed to Melitta Schachner, Zentrum
für Molekulare Neurobiologie, Universität Hamburg, 20246 Hamburg, Germany. E-mail: melitta.schachner{at}zmnh.uni-hamburg.de.
| |
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