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The Journal of Neuroscience, December 15, 1998, 18(24):10398-10408
Heteromultimeric Delayed-Rectifier K+ Channels in
Schwann Cells: Developmental Expression and Role in Cell
Proliferation
Alexander
Sobko1,
Asher
Peretz1,
Orian
Shirihai2,
Sarah
Etkin1,
Vera
Cherepanova1,
Daniel
Dagan2, and
Bernard
Attali1
1 Neurobiology Department, Weizmann Institute of
Science, Rehovot 76100, Israel, and 2 Bruce Rappaport
Faculty of Medicine, Bernard Katz Minerva Center for Cell Biophysics,
Technion, Haifa 31096, Israel
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ABSTRACT |
Schwann cells (SCs) are responsible for myelination of nerve fibers
in the peripheral nervous system. Voltage-dependent
K+ currents, including inactivating A-type
(KA), delayed-rectifier (KD), and inward-rectifier
(KIR) K+ channels,
constitute the main conductances found in SCs. Physiological studies
have shown that KD channels may play an
important role in SC proliferation and that they are downregulated in
the soma as proliferation ceases and myelination proceeds. Recent
studies have begun to address the molecular identity of
K+ channels in SCs. Here, we show that a large
repertoire of K+ channel  subunits of the
Shaker (Kv1.1, Kv1.2, Kv1.4, and Kv1.5), Shab (Kv2.1), and Shaw (Kv3.1b and Kv3.2)
families is expressed in mouse SCs and sciatic nerve. We characterized
heteromultimeric channel complexes that consist of either Kv1.5 and
Kv1.2 or Kv1.5 and Kv1.4. In postnatal day 4 (P4) sciatic nerve,
most of the Kv1.2 channel subunits are involved in heteromultimeric
association with Kv1.5. Despite the presence of Kv1.1 and Kv1.2 subunits, the K+ currents were unaffected by
dendrotoxin I (DTX), suggesting that DTX-sensitive channel complexes do
not account substantially for SC KD
currents. SC proliferation was found to be potently blocked by
quinidine or 4-aminopyridine but not by DTX. Consistent with previous
physiological studies, our data show that there is a marked
downregulation of all KD channel subunits from P1-P4 to P40 in the sciatic nerve. Our results suggest
that KD currents are accounted for by a
complex combinatorial activity of distinct K+
channel complexes and confirm that KD
channels are involved in SC proliferation.
Key words:
K+ channels; Schwann cells; myelination; proliferation; development; ion channels; heteromultimeric
association
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INTRODUCTION |
Schwann cells (SCs) are responsible
for myelinating axons of the peripheral nervous system, a phenomenon
that is crucial for the maintenance of saltatory conduction of nerve
impulses. In the Schwann cell lineage, multipotent neural crest cells
give rise to SC precursors, which in turn give rise to SCs (for review, see Mirsky and Jessen, 1996 ; Zorick and Lemke, 1996). After a period of
migration and vigorous proliferation, the cells interact with nerve
bundles, and those associating with large axons stop dividing and
myelinate, whereas those associated with smaller axons subsequently
mature as nonmyelin-forming SCs. If the nerve is injured via mechanical
trauma or demyelinating diseases, SCs can reenter the mitotic cycle to
restore the integrity of the myelin sheath and contribute to functional
recovery of the nerve fibers. SCs and neurons exert powerful influences
on each other, during both early development and differentiation, as
well as in the course of peripheral nerve regeneration. Myelinating SCs initiate the process of myelination in response to contact-mediated axonal signals (Mirsky and Jessen, 1996; Zorick and Lemke, 1996). SCs
also provide important support for axons, strongly influencing the
organization of ionic channels along axonal membranes (Rasband et al.,
1998).
Voltage-dependent K+ channels (Kv channels)
constitute the major ionic conductance detected in SCs (Barres et al.,
1990; Chiu, 1991; Ritchie, 1992; Sontheimer, 1994). These include
inactivating A-type (KA),
delayed-rectifier (KD), and
inward-rectifier (KIR) K+ channels (Chiu et al., 1984; Shrager et al.,
1985; Konishi, 1989; Wilson and Chiu, 1990a,b; Amedee et al., 1991;
Verkhartsky et al., 1991a,b; Baker and Ritchie, 1993).
However, the functions of these K+ channel subsets
are still not clear. It has been suggested that SC
KIR channels play a role in buffering
activity-dependent K+ accumulation during early
myelinogenesis and in adult nerves (Konishi, 1990; Wilson and Chiu,
1990a,b). KD channels have been implicated in SC
proliferation during development and after Wallerian degeneration of
sciatic nerves (Chiu and Wilson, 1989; Konishi, 1989; Wilson and Chiu,
1990a). Interestingly, physiological studies have revealed that
KD and KIR channels are
downregulated in the soma as myelination proceeds and proliferation
ceases (Konishi, 1990; Wilson and Chiu, 1990a). However, developmental
studies at the protein level are not documented.
Recent studies have begun to address the molecular identity of
K+ channels in SCs. The Shaker-like
delayed-rectifier Kv1.1, Kv1.2, and Kv1.5 and the inward-rectifier IRK1
and IRK3 subunits were shown to be expressed in SCs of the rat
sciatic nerve (Chiu et al., 1994; Mi et al., 1995, 1996; Rasband et
al., 1998). We recently found that Kv2.1 and Kv3.1b delayed-rectifier
subunits are abundantly expressed in mouse SCs and sciatic nerve
(Sobko et al., 1998). Thus, the KD currents are
probably accounted for by the activity of different
K+ channel complexes. However, it is not known
whether these channel complexes assemble as homomultimers or
heteromultimers in vivo.
The goals of this study were (1) to identify at the mRNA and protein
levels the K+ channel subunits underlying the
KD currents in mouse SCs and to determine
whether they could assemble as heteromultimeric complexes, (2) to
analyze the developmental profile of the various subunits in the
sciatic nerve, and (3) to compare the ability of K+
channel blockers to depress the SC K+ currents with
their capacity to inhibit SC proliferation. Our results indicate that a
complex repertoire of Kv channel subunits is expressed in cultured
SCs and sciatic nerve. We found heteromultimeric association between
Kv1.5 and either Kv1.2 or Kv1.4. A marked downregulation of all
delayed-rectifier K+ channel subunits was
detected in the sciatic nerve from postnatal day 1 (P1)-P4 to P40. Our
findings suggest further that KD channels are
important for SC proliferation.
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MATERIALS AND METHODS |
Materials. Quinine, quinidine, 4-aminopyridine
(4-AP), tetraethylammonium (TEA), and protein A/G Sepharose were
purchased from Sigma (St. Louis, MO). Charybdotoxin (CTX),
dendrotoxin I (DTX), and Agitoxin-2 (AgTx) were kindly provided
by Alomone Labs (Jerusalem, Israel). The source of antibodies was as
follows: monoclonal and polyclonal antibodies to Kv1.1, Kv1.2, Kv1.4,
Kv1.5, and Kv2.1 were from Upstate Biotechnology (Lake Placid, NY);
anti-Kv1.1, anti-Kv1.2, Kv1.4, Kv1.5, anti-Kv3.1b, and anti-Kv3.2
polyclonal antibodies were purchased from Alomone Labs (Jerusalem,
Israel); and rabbit anti-S-100 was from Sigma. Goat anti-mouse, goat
anti-rabbit fluorescein (FITC)- or rhodamine (TRITC)-conjugated and
horseradish peroxidase (HRP)-conjugated secondary antibodies
were from Jackson ImmunoResearch (West Grove, PA).
[3H]Thymidine (29 Ci/mmol) was purchased from
Amersham (Arlington Heights, IL).
Schwann cell cultures, proliferation, and
immunocytochemistry. Primary SC cultures were prepared from P4
mouse sciatic nerves according to Brockes et al. (1979). Cells were
plated on poly-D-lysine-coated Petri dishes or glass
coverslips and grown in DMEM-F-12 supplemented with 2 mM
glutamine, 10% fetal calf serum (FCS), and antibiotics. Under these
conditions, which allowed proliferation and inhibited the expression of
the myelin phenotype, we obtained 90-95% pure Schwann cell cultures,
as controlled by S-100 immunofluorescence. [3H]Thymidine incorporation (0.2 µCi/well) was
assayed in SCs cultured as indicated, in serum-containing medium, in
serum-deprived medium (DMEM-F-12), or in serum-free defined medium
supplemented with either 5 µg/ml insulin, 5 µg/ml transferrin and 5 ng/ml sodium selenite (SATO1), or with SATO1 containing 5 ng/ml PDGF
and 5 ng/ml basic FGF (bFGF) (SATO2). Cells were treated with
various concentrations of channel blockers as indicated, and the assay was performed as described previously (Attali et al., 1997).
Immunocytochemistry was performed (Attali et al., 1997; Sobko et al.,
1998), and cells were viewed using a Zeiss (Oberkochen, Germany)
Axioplan microscope equipped with phase-contrast and epifluorescent optics.
Preparation of cell lysates and membrane solubilization.
Confluent cell cultures (60 or 100 mm dishes) were washed twice with cold PBS and scraped on ice in PBS containing 1 mM
PMSF. After centrifugation, cell pellets or acutely isolated sciatic
nerves were either frozen in liquid nitrogen and kept at  80°C until use or homogenized in the buffer containing 50 mM Tris, pH
7.4, 1 mM EDTA, 1 mM PMSF, 10 µg/ml
aprotinin, and 10 µg/ml leupeptin and centrifuged at 21,000 × g for 30 min at 4°C. The pellet (crude membranal fraction)
was sonicated and resuspended in either SDS-sample buffer (Laemmli,
1970) for direct immunoblot analysis or in the solubilization buffer
(glycerol 10%, 50 mM HEPES, pH 7.4, 10 mM EDTA, NaCl 150 mM, 1.5 mM
MgCl2, 1% Triton X-100, 1 mM PMSF, 10 µg/ml aprotinin, and 10 µg/ml leupeptin) for immunoprecipitation. Protein concentration was determined using Bio-Rad (Hercules, CA)
protein assay solution with bovine serum albumin and human immunoglobulin as a standard.
Immunoprecipitation. Samples were incubated with shaking in
the solubilization buffer for 1 hr at 4°C and spun at 21,000 × g. Using identical amounts of input proteins for the various
treatments, the extracts (supernatants) were precleared with 1:1 slurry
of protein A/G Sepharose (30 µl/ml of extract) and equilibrated in lysis buffer for 1 hr at 4°C with shaking. After a short spin, the
supernatant was transferred into a new tube and incubated with the
antibodies or the respective preimmune serum at 4°C for 4 hr or
overnight with shaking. Immune complexes were pooled-down after the
addition of 30 µl of protein A/G Sepharose for 1 hr at 4°C with
shaking. Where indicated, the supernatant was transferred into a new
tube and was reused in a second round of immunoprecipitation. Immunoprecipitates were washed three times in the solubilization buffer, and beads were finally resuspended in 30 µl of 2× SDS-sample buffer and boiled at 100°C for 5 min to elute the precipitated proteins. The eluent was electrophoresed on SDS-PAGE with
standard molecular weight markers.
Immunoblot analysis. After separation on SDS-PAGE, proteins
were electrotransferred to nitrocellulose blots. Blots were blocked in
PBS containing 10% nonfat milk and 0.05% Tween 20 (Western buffer)
for 1 hr at room temperature with shaking. Then, blots were incubated
with the respective antibody overnight at 4°C or for 4 hr at room
temperature. After three washes with Western buffer, blots were
incubated for 1 hr with HRP-conjugated secondary antibody, followed by
extensive washes in PBS. Labeled proteins were detected by enhanced
chemiluminescence (ECL) (Amersham). Band signals corresponding to
immunoreactive proteins were measured and scanned by image densitometry
using NIH Image 1.54 and Adobe Photoshop 3.0 software. Densitometric
data were normalized to respective protein input assessed by Ponceau
staining of blots. In addition to tests performed previously in
transfected HEK 293 cells (Attali et al., 1997), we checked directly
for antibody specificity in SC extracts by preadsorbing the anti-Kv
channel antibodies with their respective antigens for 1 hr at room
temperature as follows (see Fig. 3B): anti-Kv1.1,
anti-Kv1.2, and anti-Kv1.5 antibodies (Upstate Biotechnology and
Alomone Labs) were preadsorbed with the respective glutathione
S-transferase (GST) channel fusion proteins (3 mg/mg
antibody; Alomone Labs) spanning similar or overlapping C-terminus
domains of the immunizing antigens (residues 416-495, 417-498, and
513-602 of mouse Kv1.1, rat Kv1.2, and mouse Kv1.5,
respectively). Because the antigen (residues 13-37 of rat Kv1.4) of anti-Kv1.4 antibodies from Upstate Biotechnology was not
available to us, we also used anti-Kv1.4 antibodies from Alomone Labs
(residues 589-655 of rat Kv1.4) to test for specificity by preadsorbing antibodies with the corresponding GST channel fusion protein (3 mg/mg antibody). The N-terminus-directed (Upstate
Biotechnology) and C-terminus-directed (Alomone Labs) anti-Kv1.4
antibodies labeled the same molecular weight immunoreactive protein in
SC extracts, further demonstrating the specificity of the anti-Kv1.4
antibodies from both sources. Anti-Kv2.1 (Upstate Biotechnology),
anti-Kv3.1b, and anti-Kv3.2 (Alomone labs) antibodies were preadsorbed
with their immunizing peptide antigens (1 mg peptide/mg antibody)
consisting of residues 837-853, 567-585, and 184-204 of the
respective rat channel sequences. In all cases, the labeling was
specific and was unaffected by unrelated (BSA, 3 mg/mg antibody) or
irrespective antigens.
Molecular cloning and semiquantitative PCR. Molecular
cloning of Kv channel subunits was performed by screening a rat
Schwann cell cDNA library (kindly provided by Dr. G. Lemke, Salk
Institute, La Jolla) at low stringency using the S5-S6 region of Kv1.1
as an hybridizing probe (Attali et al., 1993). We also performed a
reverse transcription-PCR (RT-PCR) cloning using degenerate oligonucleotides as described previously (Attali et al., 1997). The
upstream primer (5' AAYGAGTACTTCTTYGAYMG 3') corresponded to a
conserved region NEYFFDR, located upstream of the first transmembrane domain S1. The downstream primer (5' NCCRTANCCNRNNGWNGA 3')
corresponded to the most conserved H5 pore signature sequence TTVGYG.
For semiquantitative RT-PCR, the reverse-transcription was performed as
described previously (Attali et al., 1997). Unique primer pairs
encoding specific 3' coding region of the respective Kv channels were
used for RT-PCR amplification: Kv1.2 sense 5' CACCGGGAGACAGAGGGA 3'
(1249-1266) and Kv1.2 antisense 5' TCAGACATCAGTTAACAT 3' (1479-1497);
and Kv1.5 sense 5' CATCGGGAGACAGACCAC 3' (1834-1851) and Kv1.5
antisense 5'TTACAAATCTGTTTCCCG 3' (2089-2107). A semiquantitative PCR analysis was performed to quantify the input mRNA and related cDNA
of the various samples. The coamplification of an internal control
housekeeping S16 mouse ribosomal protein mRNA was performed using an
upstream primer (S16 sense, 5' AGGAGCGATTTGCTGGTG 3') and a downstream
primer (S16 antisense, 5' CAGGGCCTTTGAGATGGA 3'), which amplified a 102 bp cDNA fragment. Equal aliquots of each PCR reaction were removed and
analyzed by 1.2% agarose gel electrophoresis, Southern blotted onto
nylon membranes, and probed with an unique internal
[32P]-labeled oligonucleotide. Data were
quantified by scanning the labeled bands as above, and the optical
densities of Kv channel bands were normalized to the S16 signal.
Electrophysiology. Cultured SCs, plated on
poly-D-lysine-coated glass coverslips, were placed in a 1 ml recording chamber mounted on the stage of a Zeiss Axiovert 35 inverted microscope. The whole-cell configuration of the patch-clamp
technique (Hamill et al., 1981) was used to record the macroscopic
whole-cell currents at room temperature (22 ± 1°C). Signals
were amplified using an Axopatch 200B patch-clamp amplifier (Axon
Instruments), filtered below 2 kHz via a 4-pole Bessel low-pass filter.
Data were sampled at 5 kHz and analyzed using pClamp 6.0.2 software
(Axon Instruments) and an IBM-compatible 486 computer, with a DigiData
1200 interface (Axon Instruments). The patch pipettes were pulled from
borosilicate glass (fiber-filled) with resistance of 4-8 M and were
filled with (in mM): 120 KCl, 2 MgCl2, 1 CaCl2, 11 EGTA, 10 HEPES, and 11 glucose, pH 7.4. The external solution contained (in mM): 140 NaCl, 5 KCl, 5 CaCl2, 2 MgCl2, 11 glucose, and
10 HEPES, pH 7.4. TEA (10 mM), BaCl (0.5 mM),
4-AP (3 mM), and the other blockers were externally
perfused to block the K+ currents. Series
resistances were within 10-16 M and were compensated by 85-90%.
Traces were leak-subtracted by the Clampfit program of the pClamp 6.02 software and further analyzed by the Axograph 3.0 software (Axon
Instruments). Activation and steady-state inactivation curves were
fitted by the Boltzmann distribution (assuming reversal potential of
85 mV, calculated by Nernst
equation): I/Imax = a/{1+exp[(V50 VK)/s]},
where V50 is the half-maximal
activation (for the steady-state activation protocol) or the voltage at
which half of the steady-state inactivation was removed, and
s is the slope of the curve. The transient and the
sustained components of the K+ currents were
measured at the peak and the end (400 msec) of the depolarizing traces,
respectively. All data were expressed as mean ± SEM.
Statistically significant differences were assessed by Student's
t test.
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RESULTS |
Schwann cells express KA and
KD currents: pharmacology and molecular
characterization of Kv channel subunits
SCs were isolated from P4 mouse sciatic nerves and grown in
vitro for 2-7 d in the presence of serum. Under these conditions, the SCs proliferated actively and expressed the SC marker S-100 (see
Fig. 3A). Macroscopic currents were recorded from cultured cells using the whole-cell configuration of the patch-clamp technique (Hamill et al., 1981). In agreement with previous studies (Hoppe et
al., 1989; Konishi, 1989; Wilson and Chiu, 1990a; Amedee et al., 1991),
the mouse SCs exhibited prominent voltage-gated outward K+ currents that activated after membrane
depolarization (Fig.
1A). Whole-cell
recordings revealed the presence of transient KA
and sustained KD current components (Fig.
1A). Both K+ current components
were activated above a threshold of approximately 50 mV, which is
close to the SC resting potential. Activation curves could be described
by single Boltzmann distributions with V50 = 13.7 ± 0.9 mV and V50 = 18.1 ± 0.8 mV, and slopes s = 8.1 ± 0.5 and
s = 6.5 ± 0.4 for the transient and the
sustained components, respectively (±SEM; n = 16)
(Fig. 1B, left). The steady-state inactivation curves (Fig. 1B, right)
indicated that the transient component inactivated at slightly more
hyperpolarized potentials (by 8.1 mV), relative to the sustained
component with V50 = 17.7 ± 1.5 mV and
V50 = 9.6 ± 0.3 mV, and slopes
s = 16.5 ± 0.6 and s = 16.5 ± 0.6, respectively (n = 7).
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Figure 1.
Activation and inactivation characteristics of
K+ currents in cultured SCs. A,
Left, In these representative whole-cell recordings, the
cells were stepped for 400 msec from a holding potential of 80 to +60
mV in 10 mV increments. Right, For the steady-state
inactivation protocol, the cell membrane was subjected to inactivating
prepulses of 1 sec duration from 70 to +40 mV in 10 mV increments,
before the 400 msec test pulse to +60 mV. B,
Left, The normalized conductance was plotted against
voltage steps for the transient (open triangles) and
sustained (open squares) components. The curves were
fitted using a single Boltzmann distribution with the parameters of
V50 = 13.7 ± 0.9 mV and
V50 = 18.1 ± 0.8 mV, and slopes of
s = 8.1 ± 0.5 and s = 6.5 ± 0.4 (n = 16) for the transient and
sustained components, respectively. Right, The
steady-state inactivation curves of the transient (open
triangles) and sustained (open squares)
components gave the parameters of V50 = 17.7 ± 1.5 mV and V50 = 9.6 ± 0.3 mV, and slopes of s = 16.5 ± 0.6 and
s = 16.5 ± 0.6 (n = 7),
respectively.
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Figure 2 illustrates the pharmacology of
the mouse SC K+ currents. In addition to
broad-spectrum K+ channel blockers (TEA, 4-AP,
quinine, quinidine, Ba2+, and clofilium), we also
used channel toxins, whose spectrum activity is more restricted. For
example, the scorpion toxin CTX is known to block Kv channels
comprising at least one Kv1.3 subunit with high affinity, whereas
the snake toxin DTX specifically inhibits Kv1.1, Kv1.2, and Kv1.6
homomultimeric channels (MacKinnon, 1991; Grissmer et al., 1994; Tytgat
et al., 1995). Likewise, AgTx displays a very high affinity for Kv1.1,
Kv1.2, Kv1.3, and Kv1.6 channels (Garcia et al., 1994). Our results
show that the SC K+ currents are sensitive to block
by 3 mM 4-AP (76 ± 4%; n = 12), 10 mM TEA (65 ± 6%; n = 11), and 0.5 mM Ba2+ (32 ± 14%;
n = 7) as measured by a +50 mV test pulse. These
K+ channel blockers did not discriminate between
KA and KD currents. However, 3 mM 4-AP could totally eliminate
KA, leaving an unblocked fraction
(~30%) of KD (Fig. 2B). The
block of 4-AP and to a lesser extent that of TEA and
Ba2+ were voltage-dependent, with a maximum block in
the voltage range of 20 to 0 mV (Fig. 2D,
left). The 4-AP block decreased with increasing
depolarization, suggesting that 4-AP binding occurs preferentially in
the closed state (Yeh et al., 1976; Wang et al., 1995). Both
KA and KD currents were
potently blocked by 100 µM quinine (76 ± 9%;
n = 8), 100 µM quinidine (100%;
n = 8), and 10 µM clofilium (70 ± 8%; n = 7) as measured by a +50 mV test pulse (data
not shown). The SC K+ currents were totally
insensitive to 100 nM DTX (n = 6), 100 nM CTX (n = 6), and 10 nM AgTx
(n = 6) (Fig. 2D, right).
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Figure 2.
Pharmacology of the SC K+
currents. A, B, Current traces of a
control cell (A, left) and after external
application of 10 mM TEA (A,
right), 0.5 mM BaCl2
(B, left), and 3 mM 4-AP
(B, right). Note that in the 4-AP
experiment shown, the transient component totally disappears after 7 min application (bottom traces) but remained partially
inhibited after 4 min exposure (top traces). The
activation protocol was the same as in Figure 1. C, The
current density (pA/pF)/voltage curves of the transient
(left) and sustained (right) components
are shown for control cells (open squares) and after
application of 10 mM TEA (filled
squares), 0.5 mM BaCl2
(filled circles), and 3 mM 4-AP
(filled triangles). The current density/voltage
curve of the transient component in the presence of 4-AP is not shown,
because 4-AP totally eliminates this transient current.
D, Left, Voltage-dependent block of 10 mM TEA, 0.5 mM BaCl2, and 3 mM 4-AP is shown. The percentage of block is plotted versus
voltage step. Right, Effects of 10 mM TEA
(n = 11), 0.5 mM BaCl2
(n = 7), 3 mM 4-AP
(n = 12), 100 nM DTX
(n = 6), 100 nM CTX
(n = 6), and 10 nM AgTx
(n = 6) were expressed as percentage of control
current elicited by a +50 mV step (400 msec).
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Next, we characterized extensively the molecular identity of the Kv
channel subunits in cultured SCs and the sciatic nerve. For this
purpose, we used RT-PCR cloning, conventional screening of a rat
Schwann cell cDNA library, and immunodetection using a battery of
subunit-specific polyclonal and monoclonal antibodies. From the Schwann
cell cDNA library, we fished out partial and full-length cDNA clones
encoding the Shaker Kv1.4 and Kv1.5 subunits,
respectively. For RT-PCR cloning, mRNA extracts prepared from cultured
mouse SCs (4 d in vitro) were subjected to RT-PCR using
degenerate oligonucleotides encoding conserved domains of Shaker, Shab, Shaw, and
Shal family members (see Materials and Methods; Attali et
al., 1997). The upstream primer corresponded to a conserved region
NEYFFDR, located upstream of the first transmembrane domain S1. The
downstream primer encoded the most conserved H5 pore signature sequence
TTVGYG. After this strategy, Kv1.1, Kv1.2, Kv1.5, Kv1.6, Kv2.1, Kv2.2,
Kv3.1, and Kv3.2 channel subunits were identified. To confirm the
cloning data and to characterize at the protein level the various Kv
channel subunits, we performed immunoprecipitation, immunoblot, and
immunofluorescence analyses using various monoclonal and polyclonal
antibodies (Figs.
3-7). The specificity of antibody staining in SCs was directly checked by
preadsorbing the antisera with their respective antigens (see Materials and Methods; Fig. 3B). When possible, we further
checked the specificity of staining by using antibodies from two
different commercial sources (anti-Kv1.1, Kv1.2, Kv1.4, and Kv1.5
antibodies from Upstate Biotechnology and Alomone Labs) and found an
identical labeling pattern (data not shown). In agreement with the
molecular cloning data, Figure 3B shows that the crude
membranal fractions of cultured SCs contained the immunoreactive
proteins: Kv1.1 (~75 kDa), Kv1.2 (~85 kDa), Kv1.4 (~115 kDa),
Kv1.5 (two bands of 65 and 90 kDa), Kv2.1 (doublet of 105 and 115 kDa),
Kv3.1b (~126 kDa), and Kv3.2 (~130 kDa). The RT-PCR cloning did not
detect Kv1.3 mRNA, nor did the immunoblot analysis show Kv1.3
immunoreactive protein. A very faint immunostaining was observed for
Kv1.6 and Kv2.2 subunits, suggesting that cultured SCs expressed
relatively low levels of these Kv channel proteins (data not
shown).
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Figure 3.
Immunodetection of Kv channel subunits in
cultured SCs. A, Cultured cells were subjected to
immunofluorescence analysis with anti-S-100 antibody to confirm SC
identity. Indirect immunofluorescence with anti-rabbit FITC was used
for detection. Scale bar, 20 µM. B,
Immunoblot analysis of Schwann cell cultures with antibodies to Kv
channel subunits. Membrane fractions of mouse primary cultured SCs
were subjected to immunoblot analysis with subunit-specific antibodies
to Kv1.1, Kv1.2, Kv1.4, Kv1.5, Kv2.1, Kv3.1b, and Kv3.2 subunits.
To check the specificity of antibody labeling toward SC membrane
extracts, each antiserum was preincubated for 1 hr at room temperature
in the presence (+) or absence (-) of its respective antigen, as
indicated in Materials and Methods. HRP-conjugated secondary antibodies
and ECL were used for detection.
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Figure 4.
The heteromultimeric association of Kv1.2, Kv1.4,
and Kv1.5 subunits in cultured Schwann cells. A,
Coimmunoprecipitation of Kv1.5 with other Shaker-like
subunits. SC membranal fractions were subjected to immunoprecipitation
with anti-Kv2.1, anti-Kv1.2, anti-Kv1.4, or anti-Kv1.5 antibodies, and
blots were probed with anti-Kv1.5 antibodies. To verify the presence of
channel proteins, blots were stripped and reprobed with the respective
anti-Kv antibodies (data not shown). Nonspecific bands corresponding to
immunoglobulin heavy chain (IgG) are also observed.
B, Reciprocal coimmunoprecipitation of Kv1.2 and Kv1.4
with Kv1.5. Left, SC membrane proteins were
immunoprecipitated with either Kv1.5, Kv2.1, or Kv3.1 antibodies, and
blots were probed with anti-Kv1.2. Right, SC membrane
proteins were immunoprecipitated with either Kv1.4, Kv1.5, or preimmune
antibodies (P.I.), and blots were probed with
anti-Kv1.4. An aliquot of SC membranes corresponding to ~10% of the
proteins used in immunoprecipitation (SC input) was used
as positive control. C, SCs were double labeled with
mouse monoclonal anti-Kv1.5 (C1, C3) and
rabbit polyclonal anti-Kv1.2 (C2,
C4). Indirect immunofluorescence with anti-mouse
FITC and anti-rabbit TRITC was used for detection. Scale bar:
C1-C2, 35 µM; C3-C4, 17 µM.
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Figure 5.
The heteromeric association of Kv1.2 and Kv1.5
channel subunits in sciatic nerve. Reciprocal coimmunoprecipitation
of Kv1.5 and Kv1.2 in P4 sciatic nerve is shown. A,
Homogenates of acutely isolated sciatic nerves from P4 mice were
subjected to immunoprecipitation with anti-Kv1.2, anti-Kv1.5,
anti-Kv3.1, and preimmune antibodies. The Kv1.2 and Kv1.5 subunits were
depleted from sciatic nerve extracts with their respective antibodies,
and unbound proteins were subjected to a second round of
immunoprecipitation with anti-Kv1.2 (Kv1.5 depl. Kv1.2)
and anti-Kv1.5 (Kv1.2 depl. Kv1.5), respectively. Blots
were probed with anti-Kv1.5. B, Reciprocal
coimmunoprecipitation of Kv1.2 with Kv1.5 in P4 sciatic nerve. The blot
shown in A was stripped and reprobed with
anti-Kv1.2.
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Figure 6.
Expression of Kv1.2 and Kv1.5 channel subunits
in mouse sciatic nerve during postnatal development. A,
Left, Membrane fractions of sciatic nerves from P1, P4,
P8, and P40 mice were subjected to SDS-PAGE and immunoblot analysis
with anti-Kv1.5 antibodies. To estimate and compare total protein
inputs in each lane, blots were stained with Ponceau S before
immunoblot analysis (data not shown). Right, RT-PCR,
followed by Southern blot analysis of Kv1.5 transcripts in sciatic
nerves from P1 and P40 mice. B, Left,
Immunoblot analysis of Kv1.2 on postnatal sciatic nerve as in
A. Right, RT-PCR and Southern blot
analysis of Kv1.2 as in A. Primer pairs to the specific
3' coding regions of either Kv1.5 (A) or Kv1.2
(B) amplified PCR fragments of 273 and 248 bp,
respectively. The bottom band represents the S16
ribosomal protein PCR fragment (102 bp), which was used to estimate the
starting input RNA. C, Quantitation of the developmental
downregulation of Kv1.2 and Kv1.5 proteins from P1 to P40 sciatic nerve
as illustrated in A and B. Data of
densitometric scanning were normalized to values of P40 and represent
mean ± SEM of three independent experiments.
*p < 0.05, differs significantly from P40.
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Figure 7.
Postnatal developmental profile of Kv1.4, Kv2.1,
and Kv3.1b in the sciatic nerve. A, Immunoblot analysis
of sciatic nerve membranal extracts from P4, P8, and P40 mice with
anti-Kv1.4, anti-Kv2.1, and anti-Kv3.1b antibodies. B,
Quantitation of the developmental changes were performed as described
in Figure 6. Data of densitometric scanning were normalized to values
of P40 and represent mean ± SEM of three independent experiments.
*p < 0.05, differs significantly from P40.
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Kv1.5, Kv1.2, and Kv1.4 form heteromultimeric complexes in cultured
mouse SCs and sciatic nerve
In view of the large and complex molecular repertoire of Kv
channel subunits expressed in SCs, we addressed the question of
whether some of the subunits could be involved in heteromultimeric association. We focused our study on Shaker subunits,
because this Kv channel subfamily was the most diversely expressed in SCs. Using a reciprocal immunoprecipitation-immunoblot analysis, we
showed that the Kv1.5 subunits were specifically
coimmunoprecipitated from SC membranes by anti-Kv1.2 and anti-Kv1.4
antibodies (Fig. 4A). Preimmune antibodies did not
precipitate Kv1.5 molecular complexes (Fig. 5). The 65 kDa Kv1.5
protein was the main molecular species to be immunoprecipitated in
cultured SCs under these experimental conditions. In contrast to the
data obtained with the sciatic nerve (see below; Fig. 5A) no
detectable 90 kDa species could be observed. As a control, the 65 kDa
Kv1.5 subunit could be immunoprecipitated by anti-Kv1.5 antibodies
(Fig. 4A). No heteromultimeric complexes of Kv1.5
with Kv1.1 could be detected in SCs and sciatic nerve (data not shown).
Likewise, neither Kv1.5 nor Kv1.2 formed heteromultimers with Kv
channel subunits from other families (Figs.
4A,B, 5), such as Kv2.1
(Shab) or Kv3.1b (Shaw), confirming that only
intrafamily Kv channel association can occur (Covarrubias et al.,
1991). In reciprocal experiments, we demonstrated that Kv1.2 and Kv1.4
subunits were coimmunoprecipitated by anti-Kv1.5 but not by
anti-Kv2.1 or anti-Kv3.1b antibodies (Figs. 4B, 5).
Two Kv1.2 broad molecular complexes of ~85 and 95 kDa were
specifically precipitated by anti-Kv1.5 antibodies, reflecting a
microheterogeneity of Kv1.2 molecules (Fig. 4B,
left). A 115 kDa Kv1.4 molecular species could be
precipitated by anti-Kv1.5 antibodies (Fig. 4B,
right). Double-immunofluorescence labeling indicated
that Kv1.5 and Kv1.2 were stained rather uniformly on all SC somata and
processes, with a slightly sharper staining of Kv1.2 on processes.
Overall, the staining patterns of Kv1.5 and Kv1.2 primarily overlapped
in SCs (Fig. 4C). These patterns differ from that of Kv2.1,
which exhibits membranal clusters around the cell soma (Sobko et al.,
1998).
Next, we verified that the occurrence of heteromultimeric complexes of
Shaker subunits was not an artifact of culture in vitro and could be also found in vivo in the sciatic
nerve from P4 mice. Figure 5 shows that, as in cultured SCs, Kv1.5 and
Kv1.2 could be specifically and reciprocally coimmunoprecipitated from the sciatic nerve in vivo. The 65 kDa Kv1.5 species was very
weakly immunoprecipitated. Rather, the 90 kDa and a higher molecular weight species of ~110 kDa were the primary Kv1.5 molecular complexes immunoprecipitated by both anti-Kv1.5 and anti-Kv1.2 antibodies. This
might reflect differential posttranslational processing of Kv1.5 in P4
sciatic nerve compared with other postnatal stages (see below; Fig. 6)
and SCs in culture. Using sequential rounds of immunoprecipitation
(Shamotienko et al., 1997), we depleted Kv1.5 subunits from the P4
sciatic nerve extracts with anti-Kv1.5 antibodies. This depletion
totally prevented the subsequent immunoprecipitation of Kv1.5 with
anti-Kv1.2 antibodies, thus demonstrating the specificity of the
precipitation protocol (Fig. 5). When Kv1.2 subunits were depleted
from the nerve extracts with anti-Kv1.2 antibodies, a substantial
fraction of Kv1.5 protein could still be subsequently immunoprecipitated with anti-Kv1.5 antibodies, indicating that the pool
of Kv1.5 subunits is not involved exclusively in heteromultimeric association with Kv1.2 subunits. Interestingly, when Kv1.5 subunits were depleted from the P4 sciatic nerve extracts with anti-Kv1.5 antibodies, no Kv1.2 subunit could subsequently be immunoprecipitated by anti-Kv1.2 antibodies, suggesting that most of the Kv1.2 subunits are involved in heteromultimeric association with Kv1.5 subunits.
Developmental profile of Kv channel subunits in the postnatal
sciatic nerve
Physiological studies have shown that KD
and KIR channels are downregulated in the soma
as myelination proceeds and proliferation ceases (Konishi, 1990; Wilson
and Chiu, 1990a). Because of the diversity of Kv channel
proteins, which could contribute to KD channel
activity, it was important to analyze the developmental profile of the
various Kv channel subunits in the developing sciatic nerve.
Semiquantitative RT-PCR showed that there was a significant (42 ± 9 and 68 ± 7%) downregulation (n = 4;
p < 0.01) of Kv1.5 and Kv1.2 mRNAs, respectively, in
the mouse sciatic nerve from P1 to P40 (Fig.
6A,B, right panels).
Similarly, Western blot analysis indicated that there was a 38 ± 8 and 68 ± 8% downregulation (n = 3;
p < 0.05) of the Kv1.5 and Kv1.2 immunoreactive
proteins, respectively, from P1 to P40 (Fig.
6A,B,C).
Interestingly, the 90 kDa Kv1.5 species was expressed exclusively at P4
and subsequently disappeared at later developmental stages. Immunoblot
data indicated that there was also an abrupt drop in Kv1.2 of >70%
from P8 to P40 (n = 3). Likewise, there was a marked
decrease in Kv2.1 (47 ± 6%; n = 3) and Kv3.1b
(64 ± 10%; n = 3) immunoreactive proteins from
P4 to P40, with an early downregulation at P8 for Kv3.1b (Fig. 7). A
contrasting pattern was obtained with the Kv1.4 immunoreactive protein,
whose levels did not change substantially throughout the postnatal
period (Fig. 7). In addition to the 115 kDa species found in cultured
SCs, a 94 kDa Kv1.4 immunoreactive protein was also specifically
detected at all postnatal stages in the sciatic nerve.
Proliferation of mouse SCs is blocked by broad-spectrum
K+ channel blockers but not by specific Kv1.1,
Kv1.2, Kv1.3, and Kv1.6 channel toxin blockers
To compare the ability of various K+ channel
blockers to depress the SC K+ currents with their
capacity to inhibit SC proliferation, we measured
[3H]thymidine incorporation in proliferating SCs
in the absence and presence of broad-spectrum K+
channel blockers, as well as more selective channel toxins. SCs grown
in the presence of 10% FCS exhibited a high level of
[3H]thymidine incorporation (Fig.
8A). Serum deprivation
(DMEM-F-12) inhibited SC proliferation by >80%. SCs grown in
serum-free defined medium, such as SATO1 (medium containing insulin and
transferrin), had substantial proliferative capacity, reaching a level
of ~60% of that obtained in the presence of 10% serum.
Interestingly, when growth factors, such as PDGF (5 ng/ml) and bFGF (5 ng/ml), were added to the defined medium (SATO2), SC proliferation was further enhanced, reaching values ~85% of that found with 10% serum
(Fig. 8A).
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Figure 8.
Effect of Kv channel blockers on Schwann cell
proliferation. A, [3H]Thymidine
incorporation (0.2 µCi/well) was assayed in SCs cultured as
indicated, in serum-containing medium, in serum-deprived medium
(DMEM-F-12), or in serum-free defined medium supplemented with either
5 µg/ml insulin, 5 µg/ml transferrin, and 5 ng/ml sodium selenite
(SATO1) or with SATO1 containing 5 ng/ml PDGF and 5 ng/ml bFGF (SATO2). B, Proliferation was
assayed in cultured SCs exposed for 24 hr to various channel blockers
at the indicated concentrations in the presence of 10% FCS. The
results were expressed as percentage of maximal serum-stimulated
proliferation. Data points represent the mean ± SEM of four
independent experiments, each performed in triplicate.
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SC proliferation, measured in the presence of 10% serum, was generally
sensitive to blockade by broad-spectrum K+ channel
blockers, such as 10 mM TEA, 0.5 mM
Ba2+, 100 µM quinine, 3 mM
4-AP, 100 µM quinidine, and 10 µM clofilium with 48, 45, 98, 57, 87, and 74% block, respectively
(n = 4) (Fig. 8B). In contrast,
K+ channel toxins, such as CTX (10 nM),
DTX (10 nM), and AgTx (10 nM), did not affect
SC proliferation (n = 4) (Fig. 8B).
Similar results were obtained when the channel toxin concentration was raised to 100 nM (data not shown).
| |
DISCUSSION |
In the neonatal and early postnatal periods (P1-P8), mouse SCs
are known to express KIR,
KA, and KD channel
activities (Konishi, 1990; Amedee et al., 1991; Verkhartsky et
al., 1991a,b). A direct comparison of the biophysical and
pharmacological properties of native currents with those of cloned
channels expressed in heterologous systems is always difficult, often
because of overlapping characteristics of Kv channel subunits,
heteromultimeric association, interaction with  subunits, and
cell-specific posttranslational modifications (e.g., phosphorylation).
Recent studies have begun to address the molecular identity of
K+ channels in SCs. It was found that the
Shaker-like delayed-rectifier Kv1.1, Kv1.2, and Kv1.5 and
the inward-rectifier IRK1 and IRK3 subunits are expressed in SCs of
the rat sciatic nerve (Chiu et al., 1994; Mi et al., 1995, 1996;
Rasband et al., 1998). The present work confirms and extends these
previous studies. Interestingly, we have isolated cDNAs and identified
proteins, such as Kv2.1, Kv3.1b, and Kv3.2, which belong to
Shab- and Shaw- families of noninactivating
K+ channel subunits. This finding suggests that
the predominant KD current expressed in SCs is
not exclusively encoded by Shaker-like subunits but
could be also accounted for by the activity of Shab-related
(Kv2.1) and Shaw-related (Kv3.1b, Kv3.2) channel complexes.
As for the KA current, we show here that the
Kv1.4 subunit is expressed in SCs and sciatic nerve. In the molecular screening process, we were unable to identify any other inactivating Kv
channel subunits, such as Kv1.3, Kv3.4, or Kv4.2. Although we
cannot exclude the presence of such subunits in SCs, these experiments
indicate that KA current is accounted for by the
activity of a channel complex comprising Kv1.4 subunits, either as
homomultimers or/and heteromultimers. Importantly, we found that Kv1.4
could form heteromultimers with Kv1.5 subunits; such heteromultimers may generate an inactivating K+ current. Indeed,
when expressed in Xenopus oocytes in a 1:1 ratio, heteromultimers of Kv1.5 and Kv1.4 were found to generate a transient K+ current with inactivation time constants ( ,
~40 msec) similar to those found in SCs (Po et al., 1993).
Inactivation of KA current could also be
regulated by the presence of subunits in the channel complex.
Although a recent study showed that the 2 subunits are not expressed
in rat SCs (Rasband et al., 1998), the status of other subunits in
SCs remains to be examined.
Kv1.2 transcripts were detected previously in SCs; however, protein
expression has not been documented so far (Chiu et al., 1994; Mi et
al., 1995; Rasband et al., 1998). Using the reciprocal coimmunoprecipitation strategy, we show here that the Kv1.5 subunits could form heteromultimeric complexes with Kv1.4 and Kv1.2 in both
cultured SCs and sciatic nerve. Furthermore,
immunodepletion-immunoprecipitation experiments performed in the P4
sciatic nerve indicate that most of the Kv1.2 channel subunits seem to
be involved in heteromultimeric association with Kv1.5, whereas the
pool of Kv1.5 subunits is not exclusively involved in
heteromultimeric association with Kv1.2 subunits. Obviously, this
feature found in the P4 sciatic nerve may not apply entirely for
cultured SCs in vitro. Although Kv1.2 clearly forms
heteromultimers with Kv1.5 in both cultured SCs and the sciatic nerve,
the ratio of Kv1.2/Kv1.5 heteromultimers over the total pool of Kv1.2
may be different in cultured SCs compared with the P4 sciatic nerve.
The immunostaining pattern of Kv1.5 and Kv1.2 seems to overlap in
cultured SCs; however, resolutive confocal studies, especially in the
sciatic nerve, are clearly needed to address the colocalization issue.
Interestingly, although Kv1.1 is expressed in both SCs and sciatic
nerve, we could not detect heteromultimeric complexes of Kv1.5 with
Kv1.1. Our findings are in line with previous studies showing a
differential subcellular immunolocalization of Kv1.1 and Kv1.5
proteins, thus providing evidence against their heteromultimeric
coassembly (Mi et al., 1995; Rasband et al., 1998).
DTX blocks homomultimeric Kv1.1, Kv1.2, and Kv1.6 channels with high
affinity (Grissmer et al., 1994). This feature implies that there are
very few, if any, homomultimeric DTX-sensitive Kv1.1 and Kv1.2
functional channel complexes in SCs. Despite the rather abundant
expression of Kv1.2 subunits in mouse SCs, the insensitivity of
K+ currents to DTX suggests at least two possible
explanations. First, assuming that native Kv1.2/Kv1.5 heteromultimeric
channels are DTX-sensitive, then one should consider that this channel complex has a minor contribution to SC KD
currents. For example, the Kv1.2/Kv1.5 channels may not be functional.
In this regard, we showed that Kv2.1 and Kv3.1 are also expressed in
SCs and probably account for a substantial part of
KD currents (Sobko et al., 1998; present study).
Second, assuming that the coassembly of Kv1.2 with the DTX-insensitive
Kv1.5 subunit results in DTX-insensitive channels, then the SC
KD currents would be accounted for by the concerted activity of essentially DTX-insensitive channel complexes of
the Kv1, Kv2, and Kv3 families. In support of this explanation, previous studies showed that the association rate of DTX depends on
the number of DTX-sensitive subunits in a channel complex, suggesting that all four DTX-sensitive subunits must interact with
the toxin to produce a high-affinity binding site (Tytgat et al.,
1995). A similar mechanism of additive contributions has been also
characterized for TEA on Shaker-type channels (Heginbotham and MacKinnon, 1992; Liman et al., 1992). However, other studies showed
that the heteromultimeric assembly of Kv1.2 with the DTX-insensitive Kv1.4 subunit does not follow an additivity model but rather a single
subunit model by conferring the DTX sensitivity of the channel complex
(Ruppersberg et al., 1990; Hopkins, 1998). In our hands, equimolar
expression of Kv1.2 and Kv1.5 subunits in Xenopus oocytes
results in K+ currents that are insensitive to 30 nM DTX (B. Attali and A. Peretz, unpublished data).
The nature of the toxin-insensitive subunit is probably a crucial
determinant of the heteromultimeric channel behavior. For example,
Russel et al. (1994) showed that the assembly of the CTX-sensitive
Kv1.2 subunit with Kv1.5 surprisingly results in a CTX-insensitive
channel complex that follows a dominant negative behavior rather than a
single subunit model (MacKinnon, 1991). This feature is in line with
our data, because the mouse SC K+ currents were
insensitive to CTX (100 nM). Furthermore, we did not detect
any Kv1.3 mRNA or protein in SCs and sciatic nerve. Regarding the
pharmacology of the broad-spectrum K+ channel
blockers, with few exceptions, most of the Kv channel subunits we
identified display relatively high affinities for TEA and 4-AP (Pongs,
1992).
It has been suggested that in rodent SCs K+ channels
play a role in buffering activity-dependent K+
accumulation during early myelinogenesis via a spatial buffering mechanism or via K+ siphoning (Konishi, 1990; Chiu,
1991). The localization found previously for Kv1.5 in the outer SC
membrane near the node indicates that it is not ideally suited for this
function (Mi et al., 1995). The inward-rectifier K+
channels IRK1 and IRK3 found in the nodes on SC microvilli were proposed as better candidates for K+ buffering
activity (Mi et al., 1996). The other important function proposed for
SC K+ channels is their role in SC proliferation.
KD channels are thought to be linked to SC
proliferation during development and after Wallerian degeneration of
sciatic nerves (Chiu and Wilson, 1989; Konishi, 1989; Wilson and Chiu,
1990a). Mitogenic factors, such as axon and myelin fragments or glial
growth factor, were shown to increase KD channel
activity and rat SC proliferation (Wilson and Chiu, 1993). More
recently, we found that KD channels are constitutively activated by a Src family tyrosine kinase in SCs, further suggesting their role in SC proliferation (Sobko et al., 1998).
In the present work, we show that there is a substantial correlation
between the responsiveness of the K+ currents and SC
proliferation to particular channel blockers. Whereas the
broad-spectrum K+ channel blockers were efficient,
with quinidine, quinine, clofilium, and 4-AP being the most potent, the
more selective channel toxins were totally ineffective on both assays,
in agreement with the biochemical data.
Physiological studies performed on freshly dissociated SCs of the
developing sciatic nerve showed that a noninactivating outward K+ current is downregulated during SC development
and differentiation (Wilson and Chiu, 1990a; Konishi, 1990). This
decrease in KD current expression has been
linked to the diminished ability of SCs to proliferate as
differentiation proceeds and myelination begins (Wilson and Chiu,
1990a; Konishi, 1990). The present data are consistent with these
developmental studies. We show that all Kv channel subunits
expressed in the sciatic nerve and encoding noninactivating
delayed-rectifier K+ channels are markedly
downregulated from P1 to P40. These include Kv1.2, Kv1.5, Kv2.1, and
Kv3.1b. The unique exception to this developmental profile is Kv1.4,
whose expression remains approximately constant during the same
postnatal period. This pattern of development at the protein level
contrasts with that found for most Kv channel subunits expressed in
the developing brain in which there is a progressive upregulation from
early to late postnatal stages (Maletic-Savatic et al., 1995). It is
worth noting that the 90 kDa species for Kv1.5 was observed primarily
at P4, whereas the 65 kDa isoform was detected throughout postnatal
developmental periods. Although the significance of this 90 kDa species
is unclear, it may reflect a Kv1.5 posttranslational modification
specific to a premyelinating stage of SCs. This 90 kDa isoform is
consistently observed also in cultured SCs (Sobko et al., 1998; present
study). Our data are similar to those of Chiu et al. (1994), showing a decrease in Kv1.1 and Kv1.2 mRNA levels in the rat sciatic nerve from
P15 to P90. In the developmental analysis presented in this work, the
relative contributions of Kv channels originating from SCs and those
belonging to axonal membranes are not known. With respect to Kv1.5, its
contribution essentially originates from SCs, because no expression was
found in sciatic axonal membranes (Mi et al., 1995). For the other
subunits, we found a similar downregulation pattern at the mRNA level,
indicating that the transcripts and the proteins primarily originate
from SCs. However, a substantial source of Kv channel protein could
belong to axonal membranes (e.g., Kv1.2; see Rasband et al., 1998). In
future studies, it will be important to determine the subcellular
localization of this complex repertoire of Kv channel subunits
along the sciatic nerve.
In summary, this study indicates that a diverse and complex repertoire
of Kv channel subunits is expressed in cultured mouse SCs and
sciatic nerve. We have detected heteromultimeric channel complexes
consisting of either Kv1.5 and Kv1.2 or Kv1.5 and Kv1.4. In P4 sciatic
nerve, most of the Kv1.2 channel subunits appear to be involved in
heteromultimeric association with Kv1.5. We also showed that all Kv
channel subunits encoding KD channel activity are markedly downregulated in the developing sciatic nerve,
reaching lowest levels of protein expression at P40. Our results
suggest that in SCs developmentally regulated patterns of defined
K+ channel complexes underlie
KD currents and confirm that
KD channels are important for SC proliferation.
| |
FOOTNOTES |
Received Aug. 3, 1998; revised Sept. 25, 1998; accepted Oct. 2, 1998.
This research was supported by European Economic Community Grant
BIO-CT97-2207 and the Minerva Foundation (to B.A.). A.P. was supported
by a Weizmann Institute postdoctoral fellowship of the Koret
foundation. B.A. is an incumbent of the Philip Harris and Gerald Ronson
Career Development chair. We thank Dr. G. Lemke for kindly providing
the rat Schwann cell cDNA library and Emily Levine for careful reading
of this manuscript.
Correspondence should be addressed to Dr. Bernard Attali, Department of
Neurobiology, The Weizmann Institute of Science, Rehovot 76100, Israel.
| |
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Abstract
Introduction
