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INTRODUCTION |
Human T lymphocytes express two potassium channels, the
voltage-gated K+ channel Kv1.3 and the
calcium-activated K+ channel IKCa1, that are
involved in proliferation and cytokine secretion (1-6). Recently, we
reported (7) that myelin-reactive encephalitogenic rat T cells
expressed unusually high numbers of Kv1.3 channels following eight or
more repeated antigenic stimulations in vitro. Adoptive
transfer of these Kv1.3high T cells into rats induced
experimental autoimmune encephalomyelitis (EAE).1 EAE is an animal
model for multiple sclerosis (MS), a chronic inflammatory disease of
the central nervous system characterized by immune-mediated focal
demyelination and axonal damage resulting in severe neurological
deficits (8-10). Studies with several myelin-specific rat T cell lines
revealed a correlation between encephalitogenicity and the number of
expressed Kv1.3 channels (7, 11). In addition, in vivo Kv1.3
blockade ameliorated adoptive EAE, suggesting a crucial role of Kv1.3
in the pathogenicity of these cells (7, 12). A rapid method to detect
Kv1.3high lymphocytes might therefore facilitate studies of
the role of Kv1.3 in the pathogenesis of MS and its potential as a
therapeutic target for this disease.
The patch clamp technique is widely used to determine functional
Kv1.3 and IKCa1 channel levels in lymphocytes but
requires highly specialized equipment and is time-consuming, allowing
the study of only a few dozen cells per day. Reverse transcriptase-PCR and Western blot analysis provide a measure of channel-transcript or
channel-protein expression in lymphocytes but not the number of
functional channels in the cell membrane. All known Kv1.3-specific antibodies target intracellular epitopes (13) in the channel making it
necessary to permeabilize lymphocytes before they can be stained with
these reagents. In addition, nonspecific staining imposes a further
limitation to the use of antibodies for protein detection. Furthermore,
no reports have yet described any success in using these antibodies for
immunostaining lymphocytes, necessitating the development of novel
tools to identify Kv1.3high lymphocytes in tissues.
Despite the clear need for fluorescently labeled toxins as markers of
channel expression and distribution in intact cells, there have been
only a few reports of channel-binding peptides that have been
successfully tagged with fluorophores. Fluorophore-labeled channel-binding peptides were reported previously for sodium channels (14, 15), voltage-activated calcium channels (16), and
N-methyl-D-aspartic acid receptors (17).
Most recently, a fluorophore-tagged hongotoxin analog was used to
detect Kv1.1 and Kv1.2 channels in rat brain sections and Kv1.3 in
Jurkat cells (18, 19). Several Kv1.3-blocking polypeptides have been
discovered by us and by others (5, 20-24) in scorpion venom and sea
anemone extracts. Because these polypeptides bind with extremely high
affinities to Kv1.3, they might be used in much the same way as
antibodies. Unlike Kv1.3-specific antibodies, these polypeptides bind
to the outer vestibule of Kv1.3 and can therefore reach their binding
pocket in live intact lymphocytes. ShK, a 35-amino acid polypeptide
from the Caribbean sea anemone Stichodactyla helianthus, is
the most potent known inhibitor of Kv1.3 (Kd 11 pM), and once bound to the channel does not wash off easily
(5). ShK binds via high affinity interactions to residues in the outer
vestibule of the channel (5, 25). If suitably tagged with fluorophores,
ShK could be used as a molecular probe to detect Kv1.3high
lymphocytes by flow cytometry.
In this study, guided by the high resolution structure of ShK (26, 27)
and the experimentally verified ShK-Kv1.3-interacting surface (5, 25,
28), we attached fluorescein-6- carboxyl (F6CA) through an
aminoethyloxyethyloxy-acetyl (Aeea) linker to the
-amino group of
Arg1. This residue was chosen because it is located on the
back side of the toxin surface facing away from the channel pore. We
chose the 11-carbon atom linker Aeea to minimize steric effects caused by the aromatic moiety of F6CA in binding and folding of the peptide. The labeled polypeptide selectively blocked Kv1.3 with picomolar affinity. In flow cytometry experiments, chronically activated rat and
human T lymphocytes with >600 Kv1.3 channels/cell were detected by
ShK-F6CA staining, whereas resting and acutely activated lymphocytes
with lower Kv1.3 channel numbers were not visualized by this method.
ShK-F6CA may therefore be a useful tool to detect the presence of
Kv1.3high lymphocytes under normal and diseased conditions.
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EXPERIMENTAL PROCEDURES |
Reagents--
Guinea pig myelin basic protein (MBP),
concanavalin A (Con A), cyclosporin A (CsA), and staurosporine were
from Sigma. S. helianthus toxin (ShK), margatoxin
(MgTX), charybdotoxin (ChTX), and apamin were from Bachem (King of
Prussia, PA). Tetanus toxoid (TT) was a generous gift from Dr. Peter A. Calabresi (University of Maryland).
Generation of the ShK Conjugates--
Fmoc-amino acids (Bachem
A.G., CH-4416 Bubendorf, Switzerland) included the following:
Ala, Arg(Pmc), Asn(Trt), Asp(OtBu), Cys(Trt), Gln(Trt),
Glu(OtBu), Gly, His(Trt), Ile, Leu, Lys(Boc), Met, Phe, Pro,
Ser(tBu), Thr(tBu), and Tyr(tBu).
Stepwise assembly was carried out starting with 10 g of
Fmoc-Cys(Trt)-resin (0.65 mmol/g) on a Labortec SP4000 peptide
synthesizer. Following final removal of the Fmoc-group from the
N-terminal Arg residue, a resin aliquot was removed, and the
hydrophilic linker Fmoc-Aeea-OH (Fmoc-amino-ethyloxy-ethyloxyacetic acid) was coupled as an HOBT ester. This resin was subsequently divided into four portions for preparation of the biotinyl (for ShK-biotin), fluorescein-6-carboxyl (for ShK-F6CA),
tetramethylrhodamine-6-carboxyl (for ShK-TMR), or the
biotinyl-(Aeea)4 derivatives. Each of these residues was
also coupled as an HOBT ester after deblocking the Fmoc group.
Following N-terminal derivatization, each of the peptides was cleaved
from the resin and simultaneously deprotected with reagent K (29) for
2 h at room temperature. The free peptide was then filtered to
remove the spent resin beads and precipitated with ice-cold diethyl
ether, collected on a fine filter by suction, washed with ice-cold
ether, and finally extracted with 20% AcOH in H2O.
Oxidative folding of the disulfide bonds and its subsequent purification were as described previously (23) with the addition of
25% MeOH to the solution to maintain solubility. Oxidative folding was
facilitated by addition of 1.5 mM reduced glutathione and
0.75 mM oxidized glutathione. Each sample was purified by preparative reverse phase-high pressure liquid chromatography using a
Rainin Dynamax C18 column. High pressure liquid chromatography-pure fractions for each sample were pooled and lyophilized. Structures and
purity of all analogs were confirmed by high pressure liquid chromatography and amino acid and matrix-assisted laser desorption ionization/time of flight analysis.
Cells--
L929, B82, and MEL cells stably expressing
mKv1.1, rKv1.2, mKv1.3,
mKv3.1, and hKv1.5 have been described previously (30) and were maintained in Dulbecco's modified Eagle's medium containing 10% heat-inactivated FCS (Summit Biotechnology, Fort Collins, CO), 4 mM L-glutamine, 1 mM sodium
pyruvate, and 500 µg/ml G418 (Calbiochem). LTK cells expressing
hKv1.4 were obtained from M. Tamkun (University of Colorado,
Boulder). An enhanced green fluorescent protein-C3 construct containing
mKv1.7 (31) was transiently transfected into COS-7 cells
using FuGENE 6 (Roche Molecular Biochemicals) according to the
manufacturer's protocol. Kv1.7 currents were recorded 6-8 h after transfection.
The PAS T cell line was a kind gift from Dr. Evelyne
Béraud (Marseille, France). This long term cell line,
specific for MBP, was generated in Lewis rats and expresses only two
types of K+ channels, Kv1.3 and IKCa1 (7). Once activated
with MBP and injected into naive Lewis rats, PAS T cells induce EAE (7, 12, 32). They were maintained in culture by alternating rounds of
antigen-induced activation and rounds of expansion in
interleukin-2-containing medium. For antigen stimulation, PAS T cells
(3 × 105/ml) were incubated for 2 days with 10 µg/ml MBP and 15 × 106/ml syngeneic irradiated
(2500 rads) thymocytes as antigen-presenting cells (APCs) in RPMI 1640 Dutch modification containing 4 mM glutamine, 1 mM sodium pyruvate, 1% nonessential amino acids, 1% RPMI
vitamins, 100 units/ml penicillin, 100 µg/ml streptomycin, and 50 µM
-mercaptoethanol (basic medium) supplemented with
1% syngeneic rat serum. For the interleukin-2-dependent
growth phase, PAS cells were seeded in basic medium supplemented with
10% FCS and 5% T cell growth factor (TCGF). After 5 days of expansion
in this medium, PAS T cells were restimulated with MBP. TCGF was
produced by activating Lewis rat (5-8 weeks old; Charles River
Laboratories, Wilmington, MA) splenocytes (2 × 106/ml) with 2 µg/ml Con A in basic medium supplemented
with 10% FCS. After 48 h cells were pelleted, and 15 mg/ml
-methylmannoside (Sigma) was added to the supernatant to inactivate
Con A. After thorough mixing the supernatant was passed through a
0.2-µm filter and stored at
20 °C.
Rat and human mononuclear cells were isolated from the spleens of Lewis
rats or from the blood of healthy volunteers, and enriched for T cells
by nylon wool purification for rat T cells or with CD3+
RosetteSep (StemCell Technologies, Vancouver, British Columbia, Canada)
for human T cells. Rat T cells were activated with 5 µg/ml Con A;
human T cells were activated with 50 ng/ml anti-CD3 Ab (Biomeda) in the
presence of autologous irradiated (2500 rads) peripheral blood
mononuclear cells (PBMC) as APCs for 48 h. Human TT or
MBP-specific T cells were generated from PBMCs from a healthy volunteer. Cells (2 × 108) were stimulated with 10 µg/ml TT or MBP. After 2 days 5% TCGF was added, and the cells were
expanded for 5 days. Cells were restimulated in regular 7-day cycles
with TT or MBP in the presence of autologous irradiated PBMCs.
Experiments were performed after the 9th stimulation when 95% of cells
expressed an effector memory phenotype.
Electrophysiological Analysis--
All experiments were carried
out in the whole-cell configuration of the patch clamp technique with a
holding potential of
80 mV. Pipette resistances averaged 1.5 megohms,
and series resistance compensation of 80% was employed when currents
exceeded 2 nA. Kv1.3 currents were elicited by repeated 200-ms pulses
from
80 to 40 mV, applied every 30 s. Kv1.3 currents were
recorded in normal Ringer solution with a calcium-free pipette solution
containing (in mM): 145 KF, 10 HEPES, 10 EGTA, 2 MgCl2, pH 7.2, 300 mOsm. Whole-cell conductances were
calculated from the peak current amplitudes at 40 mV, and Kv1.3 channel
numbers per cell were calculated by dividing the whole-cell conductance
by the single-channel conductance (12 pS). Kv1.1, Kv1.2, Kv1.4, Kv1.5,
Kv1.7, Kv3.1 and IKCa1 currents were measured as described previously
(7, 30, 31, 33).
Cell Staining with ShK Conjugates and Flow Cytometry
Analysis--
Adherent L929 (Kv1.1 and Kv1.3), B82 (Kv1.2), and LTK
(Kv1.4) cells were detached from culture flasks with trypsin-EDTA.
Detached cells or suspension cells (lymphocytes or MEL cells expressing Kv1.5) were washed twice with PBS and incubated in the dark at room
temperature with 10 nM ShK-conjugate in PBS + 2% goat
serum (Sigma) for 30 min and then washed 3× with PBS + 2% goat serum before flow cytometry analysis. For ShK-biotin and
ShK-(Aeea)4-biotin staining, this primary step was followed
by a 30-min incubation with 2 µg/ml streptavidin-phycoerythrin
(Pharmingen) or streptavidin-Alexa Fluor 488 (Molecular Probes) and 3 washes with PBS + 2% goat serum. For competition experiments cells
were preincubated with 100 nM unlabeled ShK, 1 µM MgTX, 1 µM ChTX, or 5 µM
apamin before addition of 10 nM ShK-F6CA. Stained cells
were analyzed by flow cytometry on a BD Biosciences FACScan. Data were
further analyzed using CellQuest software.
Pharmacological Analysis of the Pathways Involved in the
Up-regulation of Kv1.3 Channels--
Rested PAS T cells were incubated
with 100 nM CsA, 10 nM staurosporine, or 100 nM MgTX for 1 h. MBP and APCs were added for a further
20-30 h to activate the cells before patch clamp and flow cytometry
analysis (see above). Statistical analysis was carried out using the
non-parametric Mann-Whitney U test.
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RESULTS |
ShK-F6CA, ShK-Biotin, and ShK-TMR Are Potent Kv1.3
Inhibitors--
By using the polypeptide ShK as a template, we
generated a novel fluoresceinated peptide toxin for use in flow
cytometry to rapidly detect T cells that exhibit high levels of Kv1.3
channels. The parent peptide toxin ShK blocks Kv1.3 with picomolar
affinity by binding to the outer vestibule of the channel, one
polypeptide molecule per Kv1.3 tetramer (5, 25). ShK was first
assembled stepwise through solid phase synthesis (23). After removal of a protecting group, Arg1 of ShK was reacted with the
hydrophilic linker Aeea and then coupled through this linker with F6CA
(ShK-F6CA), biotin (ShK-biotin), or tetramethylrhodamine (ShK-TMR). The
respective ShK-derivatives were cleaved from the resin and folded as
described previously (23).
The NMR structure of ShK (26) and the AM-1 modeled structures of
Aeea-F6CA, Aeea-biotin, and Aeea-TMR are shown in Fig. 1. ShK-Arg1, the residue of
which these linkers are attached (highlighted in orange) is
on the "back side" of ShK (5, 25, 28). Lys22, the
critical residue on the channel-binding surface of ShK that occludes
the Kv1.3 channel pore (5, 25, 28), is highlighted in cyan.
The fluorophores were attached to Arg1 in order to minimize
the likelihood of interference with the interaction of polypeptide with
the channel. Our strategy of labeling ShK during synthesis avoided the
possibility of accidental labeling the crucial
-amino group of
Lys22.

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Fig. 1.
Generation of ShK conjugates.
F6CA (highlighted in green), biotin (highlighted in
yellow), or TMR (highlighted in red) were
conjugated to ShK (left) through a linker attached on its N
terminus (residue Arg1, in orange). The
Lys22, required for channel blockade, is highlighted in
cyan. The molecular model of ShK is based on the published
NMR structure (5, 26); the structures of F6CA-, biotin-, and TMR-Aeea
were modeled with AM1 in Hyperchem.
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ShK-F6CA, ShK-biotin, ShK-TMR, and native ShK were tested for their
ability to block mouse Kv1.3 channels stably expressed in L929 cells.
Kv1.3 currents were elicited by 200-ms depolarizing steps from a
holding potential of
80 mV to 40 mV (Fig.
2, A-E). Native ShK blocked
these currents at picomolar concentrations (Fig. 2A), and
the dose-response curve shown in Fig. 2F revealed a
Kd of 10 pM. ShK-F6CA
(Kd = 48 ± 4 pM) and ShK-TMR
(52 ± 5 pM) were 4-fold less potent than ShK (Fig. 2, B, E, and F). Although the affinity of
ShK-biotin for Kv1.3 (Kd = 11 ± 2 pM) was comparable with that of ShK, it was completely ineffective when pre-assembled with phycoerythrin-conjugated
streptavidin (Fig. 2, C, D, and F),
probably because the complex was too large to reach the binding site in
the channel pore. Attachment of biotin via a longer linker (33 Å in
length) to ShK dramatically reduced its affinity for Kv1.3
(Kd >100 nM; not shown in graph). All
these polypeptides blocked Kv1.3 with a Hill coefficient of 1. These
results demonstrate that ShK retains its ability to block the Kv1.3
channel at picomolar concentrations after the attachment of a
fluorophore to Arg1.

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Fig. 2.
ShK conjugates block Kv1.3 channels at
picomolar concentrations. Effect of ShK (A), ShK-F6CA
(B), ShK-biotin alone (C), or preincubated with
streptavidin (D), and ShK-TMR (E) on Kv1.3
currents in stably transfected L929 cells. F,
dose-dependent inhibition of Kv1.3 currents by ShK ( ,
Kd = 10 ± 1 pM,
nH = 1.15), ShK-F6CA ( , Kd = 48 ± 4 pM, nH = 0.98),
ShK-biotin ( , Kd = 11 ± 2 pM,
nH = 1.05), ShK-biotin preincubated with
streptavidin ( ), and ShK-TMR ( , Kd = 52 ± 5 pM, nH = 0.99). Each data point
is the mean of three determinations.
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ShK-F6CA Is a Specific Inhibitor of Kv1.3 Channels--
ShK is
reported to block Kv1.3 and the neuronal channel Kv1.1 with equivalent
potency (5). We therefore examined the ability of ShK-F6CA, ShK-TMR,
ShK-biotin, and native ShK to block mouse Kv1.1 channels stably
expressed in L929 cells. We used the classical K+ channel
blocker, TEA as a control, because Kv1.1 is one of the most
TEA-sensitive channels. As expected, Kv1.1 was potently blocked by ShK
(Kd = 25 ± 3 pM) (Fig.
3A) and exhibited low
micromolar sensitivity to TEA (Fig. 3B). Surprisingly,
ShK-F6CA was 160-fold less effective (Kd = 4.0 ± 0.3 nM) than ShK (Fig. 3A) and showed 83-fold
greater affinity for Kv1.3 over Kv1.1 (compare Fig. 2, A and
F, with Fig. 3A). In contrast, ShK-TMR and
ShK-biotin blocked Kv1.1 at picomolar concentrations (ShK-TMR,
Kd = 397 ± 36 pM; ShK-biotin,
Kd = 114 ± 8 pM) and only exhibited 7-10-fold higher affinity for Kv1.3 than Kv1.1 (compare Fig.
2F and Fig. 3, B and C). The enhanced
specificity of ShK-F6CA for Kv1.3 over Kv1.1 might be explained by the
differences in charge of F6CA, TMR, and biotin: F6CA is negatively
charged; TMR is positively charged; and biotin is neutral. In a recent
experimentally determined docking configuration of ShK in the Kv1.3
vestibule (28), ShK-Arg1 was found to be in the vicinity of
Asp376-Asp377-Pro377-Ser378-Ser379
on the channel. If ShK sits in the Kv1.1 vestibule with the same geometry as it does in Kv1.3, the presence of three glutamates in the
corresponding sequence in Kv1.1
(Glu350-Glu351-Ala352-Glu353-Ser354)
could electrostatically repel the negatively charged F6CA, while not
affecting ShK-biotin and possibly strengthening the interaction with
ShK-TMR. Negatively charged channel residues in neighboring loops
(e.g. S3-S4 and S1-S2) may also contribute to the
diminished potency of ShK-F6CA for Kv1.1.

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Fig. 3.
Conjugating ShK to F6CA, but not to TMR or
biotin, reduces its affinity for Kv1.1 channels. Kv1.1 currents
were elicited by 200-ms depolarizing steps from a holding potential of
80 to 40 mV. Effect of ShK and ShK-F6CA (A), ShK-TMR and
TEA (B), and ShK-biotin (C) on Kv1.1 currents is
expressed by stably transfected cells.
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We further tested the specificity of ShK-F6CA for Kv1.3 by screening it
against a panel of additional KV channels that were either
stably expressed in mammalian cells (mKv1.2, hKv1.4, hKv1.5, and
mKv3.1) or transiently transfected (mKv1.7). ShK-F6CA, like ShK, did
not block mKv1.2, hKv1.5, mKv1.7, and mKv3.1 at concentrations up to
100 nM (Table I) as expected
from published reports (5). Surprisingly, neither peptide blocked human
Kv1.4 (Table I), although mouse Kv1.4, transiently expressed in
Xenopus oocytes, has been reported to be blocked in the
mid-picomolar range by ShK (5). The reason for this difference in
results is not clear. Collectively, our data indicate that ShK-F6CA,
but not ShK-TMR or ShK-biotin, is a specific inhibitor of Kv1.3
channels.
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Table I
Selectivity of ShK, ShK-F6CA, ShK-TMR, and ShK-biotin on KV
channels
The Kd values from three independent determinations
are given in pM ± S.D. Statistical analysis was carried
out using the analysis of variance test.
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ShK-F6CA Staining and Flow Cytometry Detects Kv1.3 Channels in
Mammalian Cells--
ShK-F6CA stained L929 cells stably expressing
roughly 2000 Kv1.3 channels/cell, the fluorescence signal in these
cells being clearly distinguishable from that in unstained cells (Fig.
4A, left). The
addition of an anti-fluorescein Ab conjugated to Alexa-488 (Molecular
Probes) did not increase the intensity of the stain, probably because
the size of the Ab prevented it from reaching ShK-F6CA docked in the
channel vestibule (data not shown). ShK-TMR also stained these cells
although less brightly (data not shown), possibly because TMR is a less
bright fluorophore with a lower quantum yield than F6CA. An excess of
unlabeled Kv1.3 inhibitors (ShK, MgTX, and ChTX) competitively
inhibited ShK-F6CA staining, whereas an inhibitor of small-conductance
calcium-activated K+ channels (apamin) had no effect (Fig.
4B). The specificity of ShK-F6CA for cells with Kv1.3
channels was further confirmed by the lack of staining of L929 cells
stably expressing an equivalent number of Kv1.1 (Fig. 4A,
right) or Kv3.1 (not shown) channels.

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Fig. 4.
ShK-F6CA specifically stains cells expressing
Kv1.3 channels. A, flow cytometry profiles of cells
stably expressing Kv1.3 (left) or Kv1.1 channels
(right) unstained (black line) or stained with 10 nM ShK-F6CA (shaded). B, competition
of ShK-F6CA staining with 100 nM unlabeled ShK, 1 µM MgTX, 1 µM ChTX, or 5 µM
apamin (red filled). Control unstained cells are shown with
black lines; cells stained with 10 nM ShK-F6CA
are shown as the shaded region, and competitions are shown
in red.
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Identification of Kv1.3high T Cells by ShK-F6CA
Staining--
Our success in identifying Kv1.3-bearing cells by
ShK-F6CA staining and flow cytometry encouraged us to screen rat and
human lymphocytes for Kv1.3 expression. Fig.
5 shows Kv1.3 currents and flow cytometry
profiles of ShK-F6CA staining in rat (left panel) and human
(right) T lymphocytes. Rat T lymphocytes freshly isolated
from spleen contained barely detectable Kv1.3 currents, corresponding
to about 5 Kv1.3 channels/cell. The Kv1.3 current amplitude increased
48 h after activation with the mitogen Con A (5 µg/ml),
averaging about 200-300 channels/cell (Fig. 5, middle left
panels). Neither resting nor activated rat T cells showed detectable ShK-F6CA staining, presumably because the Kv1.3 channel number in these cells is below the level of detection. In contrast, chronically stimulated MBP-reactive encephalitogenic rat T cells (7,
12, 32) exhibit, after activation, large Kv1.3 currents (~2000
channels/cell) and small IKCa1 currents (~100 channels/cell) and are
brightly stained by ShK-F6CA (Fig. 5, left bottom panels) 48 h after the last antigenic stimulation. Upon stimulation, rat T
lymphocytes preferentially up-regulate either IKCa1 or Kv1.3 channels
depending on the number of encounters with antigen (7). The first three
stimulations induce an up-regulation of IKCa1 channels, but if the
cells undergo more rounds of stimulation (seven or more), they
down-regulate IKCa1 and up-regulate Kv1.3 channels. ShK-F6CA therefore
only stains activated T cells that have been repeatedly stimulated and
rely preferentially on Kv1.3 channels for their proliferation.

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Fig. 5.
ShK-F6CA only stains rat and human T
lymphocytes expressing high numbers of Kv1.3 channels. Kv1.3
currents expressed by three populations (resting, one-time activated,
and chronically activated) of rat or human T cells. Flow cytometry
profiles of the same rat or human T cell populations unstained
(black lines) or stained with 10 nM ShK-F6CA
(shaded).
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We extended these findings to human T lymphocytes that correspond to
the three groups of cells examined in rats. Freshly isolated unstimulated peripheral blood T lymphocytes expressed about 300-400 channels per cell and did not stain with ShK-F6CA (Fig. 5, top right panel). The Kv1.3 current amplitude increased modestly after 48 h of activation through the T cell receptor with anti-CD3
antibody, but the 500-600 Kv1.3 channels/cell in these activated cells
only produced faint ShK-F6CA staining (Fig. 5, middle right
panels). As with rat cells, repeatedly stimulated human T cells
specific for the vaccine antigen TT expressed large Kv1.3 currents
(~1500 channels/cell) and exhibited reproducible ShK-F6CA staining
48 h after the ninth round of antigenic stimulation (Fig. 5,
bottom right panels). Similar results were obtained with
human myelin-specific T cells that had been repeatedly stimulated with
MBP (data not shown). ShK-F6CA staining thus clearly differentiates
chronically activated rat and human T cells with ~1500-2000 Kv1.3
channels/cell from lymphocytes with channel numbers below 600/cell.
ShK-F6CA Staining Parallels Up-regulation of Kv1.3 Expression
during Antigenic Activation of Rat T Cells--
To study the time
course of Kv1.3 up-regulation in chronically activated cells, we
measured Kv1.3 expression and ShK-F6CA staining in a long term
MBP-specific encephalitogenic rat T cell line, PAS, before and at
various times after activation with MBP. PAS cells that had been
"rested" in TCGF medium for 5 days after the last antigenic
stimulation expressed small Kv1.3 currents and increased Kv1.3
expression dramatically after MBP stimulation, peaking in about 15 h (Fig. 6A). The channel
levels remained elevated for the next 48 h during which time PAS
cells are at their peak of encephalitogenicity (11, 32). Following the
addition of TCGF at the 48th h, Kv1.3 levels progressively declined to
a base line of <500 channels/cell on day 8 paralleling the decrease in encephalitogenicity (11, 32). Representative Kv1.3 currents measured 3, 4, 6, and 7 days post-activation are superimposed in Fig. 6B
and demonstrate this time-dependent reduction in channel expression from the peak reached on days 1-3. The other K+
channel in these cells, IKCa1, remained at ~100
channels/cell throughout the activation cycle (data not
shown).

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Fig. 6.
ShK-F6CA staining intensity correlates with
up- and down-regulation of Kv1.3 channels by encephalitogenic rat
memory T cells. A, average numbers of Kv1.3
channels/cell (n = 13 to 29 ± S.E.) are plotted
versus time after antigen-induced activation of PAS T cells.
Arrows below the plot mark the time of
stimulation with MBP and of addition of TCGF. B,
representative Kv1.3 currents expressed by PAS T cells on days 3, 4, 6, and 7 after activation. C, flow cytometry profiles
(unstained controls: black lines; cells stained with
ShK-F6CA: shaded) of PAS T cells acquired on days 3, 4, 6, and 7 after antigen-induced activation.
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ShK-F6CA staining intensity changed in parallel with the up- and
down-regulation of functional Kv1.3 expression. On days 1 to 4 post-stimulation, cells with 1000-2000 Kv1.3 channels/cell (Fig.
6A) stained brightly with ShK-F6CA (Fig. 6C,
top panels). Weak ShK-F6CA staining was observed on day 6 (Fig. 6C, lower left panel) when the cells
express ~750 channels/cell, whereas staining was undetectable on day
7 when the cells express <500 channels/cell (Fig. 6C,
lower right panel). These results indicate that the increase
and decrease of Kv1.3 functional expression during the activation
process are due to changes in the number of Kv1.3 tetramers present in
the cell membrane, each tetramer binding one molecule of ShK-F6CA. Our
data also establish the detection limit for ShK-F6CA staining at about
600 Kv1.3 channels/cell (Fig. 6C). By using this cut off, it
is feasible to distinguish encephalitogenic Kv1.3high
myelin antigen-activated T cells from non-encephalitogenic
Kv1.3low rested myelin-reactive cells or normal lymphocytes.
Calcium and Protein Kinase C (PKC)-dependent Pathways
Are Required for Kv1.3 Up-regulation--
Stimulation through the T
cell receptor activates two major signaling pathways, the first
involving calcium and the second PKC (34-36). We used a
pharmacological approach to determine whether one or both these
pathways were responsible for the up-regulation of Kv1.3 channel
expression following antigenic activation of PAS T cells. Kv1.3 current
amplitudes and ShK-F6CA staining, 20-30 h after MBP activation and in
the presence or absence of pharmacological agents, are shown in Fig.
7, A and B,
respectively. MBP activation of PAS cells augmented functional Kv1.3
expression to a maximum of 2000 ± 121 channels/cell (S.E.,
n = 62 cells) from a base line of 408 ± 39 Kv1.3
channels/cell (n = 19 cells, p < 0.001) in cells resting in TCGF medium for 4 days. ShK-F6CA staining
also increased corresponding to the increased Kv1.3 level. CsA (100 nM), at a concentration that completely inhibits
calcium-calcineurin-dependent nuclear factor of activated T
cells activation, and staurosporine (10 nM), a PKC
inhibitor, significantly suppressed MBP-induced Kv1.3 up-regulation
(CsA, 1174 ± 115 channels/cell, n = 23, p = 0.003; staurosporine, 1062 ± 133 channels/cell, n = 13, p < 0.001) and
decreased the intensity of ShK-F6CA staining. We could not study the
effect of simultaneous inhibition of both pathways because cells
exposed concurrently to CsA and staurosporine died. Neither agent had a
direct effect on Kv1.3 channels as demonstrated by an absence of effect
when either CsA or staurosporine was applied in the bath during
whole-cell patch clamp (data not shown). Blockade of Kv1.3 channels by
100 nM MgTX during activation also suppressed MBP-triggered
augmentation of functional Kv1.3 expression (MgTX, 637 ± 124 channels/cell, n = 19, p < 0.001) and
ShK-F6CA staining intensity. Reduced Kv1.3 numbers in these cells were
not due to direct blockade of the channel by residual MgTX, because the
cells were washed extensively before patching and staining, and we had ascertained in other experiments that this procedure was sufficient to
wash out MgTX (data not shown). ShK or ShK-F6CA could not be used for
this experiment because it was not possible to wash them out completely
(5). Suppression of Kv1.3 up-regulation by MgTX is probably due to
attenuation of the calcium-signaling cascade upstream to the point of
interruption by CsA (4, 37). Taken together, these results indicate
that Kv1.3 up-regulation requires the activation of both the calcium-
and PKC-dependent signaling pathways.

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Fig. 7.
Essential role of the pathways leading to the
activation of nuclear factor of activated T cells in the up-regulation
of Kv1.3 channels by rat T cells. A, average numbers of
Kv1.3 channels/cell (n = 13 to 62 ± S.E.) are
shown for each culture condition. PAS T cells were stimulated with MBP
in the presence of either 100 nM CsA, 10 nM
staurosporine, or 100 nM MgTX and were cultured for 20-30
h before determination of Kv1.3 channel numbers by patch clamp. The
dashed line indicates the limit of detection of Kv1.3
channels by ShK-F6CA (~600 channels/cell). B, PAS T cells
stimulated in the presence of the pharmacological agents listed in
A and stained with 10 nM ShK-F6CA 20-30 h later
(shaded; unstained controls are shown as black
lines).
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DISCUSSION |
T cells expressing unusually high levels of the Kv1.3 channel have
recently been implicated in an animal model for MS (7), and we now
describe a tool for the detection of Kv1.3high cells in
tissues to facilitate the study of these cells in MS pathogenesis. We attached F6CA to ShK-Arg1 so as not to
interfere with the ShK-Kv1.3-interacting surface, and this fluorescent
toxin, ShK-F6CA, exhibited low picomolar affinity for Kv1.3 and
>80-fold selectivity for Kv1.3 over closely related KV
channels. When used in flow cytometry, the 1:1 stoichiometry of
interaction between ShK-F6CA and Kv1.3 resulted in a small but
reproducible signal in flow cytometry with a detection limit of ~600
channels per cell. Repeatedly activated rat and human memory T cell
lines with channel numbers above the detection limit were clearly
identified by ShK-F6CA staining, whereas resting or acutely activated
lymphocytes with lower Kv1.3 channel expression were below the
detection limit using flow cytometry. ShK-F6CA might therefore have use
in the rapid identification of Kv1.3high cells in normal
and diseased tissues.
Myelin-specific T cells in MS patients are reported to exhibit
properties of memory T cells (38-40), and such cells could potentially contribute to the pathogenesis in MS because they traffic directly to
inflamed tissues and release copious amounts of inflammatory cytokines
such as interferon-
and tumor necrosis factor-
.
Kv1.3high expression may therefore be a functional marker
for such pathogenic myelin-reactive memory cells. In keeping with this
idea, we have reported previously (7) that MBP-specific rat memory T
cells express higher levels of Kv1.3 channels than naive rat T cells and induce severe EAE following adoptive transfer into rats. The level
of Kv1.3 expression in these rat MBP-specific memory T cell lines
correlates with their encephalitogenic potential, further highlighting
the possible role of Kv1.3 in EAE pathogenesis. Furthermore, in rats
that have received Kv1.3high encephalitogenic T cells Kv1.3
blockade in vivo significantly ameliorates adoptive EAE (7,
12). If pathogenic myelin-specific T cells in MS patients exhibit the
Kv1.3high channel phenotype found in their encephalitogenic
rat T cell counterparts, it may be feasible to identify such cells
using ShK-F6CA staining. One could imagine an assay in which T cells freshly isolated from the blood of MS patients are activated by a
mixture of myelin antigens for 48 h, stained with ShK-F6CA and memory T cell markers, and subjected to flow cytometry. The detection of increased numbers of myelin antigen-activated ShK-F6CA+
memory T lymphocytes in MS patients might have clinical utility as a
surrogate marker for disease activity.
Generalization of this approach of tagging polypeptide inhibitors of
ion channels with fluorophores could lead to the development of novel
reagents for flow cytometry. For example, primary acute myeloid
leukemia and hematopoietic cell lines aberrantly express increased
levels of HERG K+ channels, which have been suggested to
regulate proliferation in these cells (41, 42). A fluorophore-labeled
version of the selective HERG channel blocker BeKm-1 (43) might be a
valuable tool to detect these tumor cells. A limitation of our approach is the sensitivity of detection; cells with less than ~600 channels per cell are invisible. The use of other fluorophores to label the
toxin or more sensitive methods may enable detection to the level of
individual channels.
We investigated the intracellular signaling pathways that lead to
up-regulated Kv1.3 expression in chronically activated memory T cells,
using a combination of whole-cell recording and ShK-F6CA. The parallel
increase in Kv1.3 channel numbers and ShK-F6CA staining indicates that
enhanced functional Kv1.3 expression is the consequence of increased
Kv1.3 tetramers in the membrane. Our results validate the utility of
using fluorescently tagged Kv1.3 to investigate channel up-regulation
and help to define a calcium- and PKC-dependent pathway
that leads to the high level of expression seen in memory T cells.
Naive T cells differ from memory T cells by augmenting IKCa1 levels
upon activation instead of Kv1.3 (6, 7). IKCa1 up-regulation in these
cells is mediated by PKC-, AP1-, and Ikaros-2-dependent transcription with no involvement of the calcium-signaling pathway (6).
Functional consequences resulting from these differences in
K+ channel expression are evident in the responsiveness of
naive and memory cells to specific IKCa1 and Kv1.3 inhibitors. IKCa1 but not Kv1.3 blockers suppress the activation of lymphocytes with
up-regulated IKCa1 levels, whereas Kv1.3 but not IKCa1 inhibitors suppress the activation of Kv1.3high memory cells (7). The
increased tendency of lymphocytes with up-regulated IKCa1 to exhibit
oscillatory calcium signals (44, 45) also points to a potential
functional difference with Kv1.3high cells. Up-regulated
Kv1.3 expression in memory cells may promote cell adhesion and
migration via the reported interaction between Kv1.3 and
integrins
(46). The channel may also participate in signaling at the
immunological synapse through possible interactions with PKC and
p56lck (47). Through the use of tools such as ShK-F6CA,
it may be feasible to visualize fluorescently tagged Kv1.3 channels at
the immunological synapse during antigen presentation. Fluorescent probes that directly mark functional channels in the membrane may
therefore find utility as diagnostic tools that can distinguish between
subsets of cells with varying channel phenotypes and as experimental
tools to locate channels within the cell during locomotion or
immunological synapse formation.