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INTRODUCTION |
Ion channel regulation within neuronal and muscle membranes is an
important determinant of electrical excitability in the nervous and
cardiovascular systems, skeletal muscle, gastrointestinal tract, and
uterus. Voltage-gated K+
(Kv)1 channels play an
important role in setting the resting membrane potential and
determining repolarization in these functionally diverse systems. In
addition, Kv channels are involved in the regulation of membrane
potential in both T-lymphocytes and pancreatic
cells (1, 2). Given
their diverse role, it is not surprising that Kv channels comprise the
largest and most diverse class of ion channels. Multiple Kv channel
gene families (Kv1-Kv9) have been identified that give rise to more
than 40 mammalian isoforms. Most tissues, and even single cells,
express multiple channel types belonging to one or more subfamilies.
Importantly, the subcellular localization of these different channel
isoforms is often necessary for proper cell function and signaling.
Although progress has been made in identifying elements involved in
channel targeting, clustering, and anchoring, it is not yet clear how
the number and location of channel complexes within the plane of the
membrane are determined or how this compartmentalization affects
channel function. Historically, K+ channel targeting and
localization is believed to primarily involve protein-protein
interactions between channels and PDZ domain containing scaffolding proteins or the actin cytoskeleton (3, 4). Recently, we
reported that Kv channels differentially target to specialized microdomains, termed lipid rafts, within the plane of the plasma membrane, suggesting that protein-lipid interactions should be considered as a mechanism of Kv channel localization (5).
The term "lipid raft" is used to describe membrane microdomains
rich in tightly packed cholesterol and sphingolipids. Two defining
characteristics of these lipid microdomains are resistance to detergent
solubilization and a low buoyant density (6). Biochemical isolation of
raft domains has demonstrated that certain proteins target to these
subcellular compartments, whereas others are excluded. The presence or
absence of proteins is used to define different types of lipid rafts
(7). Caveolae represent one well studied subpopulation of lipid rafts
that contain the scaffolding protein, caveolin. Caveolin can bind,
organize, and sometimes functionally regulate proteins within the lipid
raft complex (8, 9). The emerging picture of the coexistence of
multiple raft populations, containing diverse proteins or different
ratios of similar proteins, within a single membrane provides a unique
mechanism of functional regulation based on spatial organization. Here, we demonstrate isoform-specific localization of voltage-gated K+ channels to distinct lipid raft populations with the
finding that Kv1.5 specifically targets to caveolae.
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EXPERIMENTAL PROCEDURES |
Materials--
The polyclonal anti-Kv2.1 antibody was purchased
from Upstate Biotechnology, Inc. (Lake Placid, NY). Anti-caveolin
polyclonal, which recognizes caveolin isoforms 1, 2, and 3, was
obtained from Transduction Laboratories (Lexington, KY). Rabbit
antiserum against the NH2-terminal region of Kv1.5 was
produced in the Tamkun laboratory using a glutathione transferase
fusion protein containing 112 amino acids of the
NH2-terminal Kv1.5 channel sequence (10). Antiserum against
the S1-S2 epitope of Kv1.5 was produced in the Philipson laboratory
using a synthetic peptide, LLRHPPAPHQPPAPAPGANGSGVMA, as previously
described (11). Fumonisin B was purchased from Sigma.
Raft Isolation--
Low density, Triton-insoluble complexes were
isolated as described by Lisanti and co-workers (12) from mouse L-cells
stably expressing either rat Kv1.5 or Kv2.1 channels under the control of a dexamethasone-inducible promoter (13, 14). Briefly, cells from ten
100-mm near-confluent culture dishes were homogenized in 1 ml of 1%
Triton X-100, and sucrose was added to a final concentration of 40%. A
5-30% linear sucrose gradient was layered on top of this detergent
extract followed by ultracentrifugation (39,000 rpm) for 18-20 h at
4 °C in a Beckman SW41 rotor. Gradient fractions (600 µl) were
collected from the top and analyzed by Western blot.
Western Blot Analysis and Immunoisolation of
Caveolae--
Sucrose gradient fractions were fractionated on a 10%
polyacrylamide SDS gel. After electrophoretic transfer to
nitrocellulose, the membrane was incubated with the Kv1.5, Kv2.1, or
caveolin antibodies (1:500, 1:500, and 1:1000 dilutions, respectively). Bound primary antibody was detected with a 1:3,000 dilution of alkaline
phosphatase-conjugated goat anti-rabbit IgG and the Renaissance Western
blot chemiluminescence reagent according to manufacturer's protocol
(PerkinElmer Life Sciences). Immune purification of caveolae was
performed as previously described with a few modifications (15).
Briefly, Triton X-100 was used to solubilize Kv1.5-containing rafts
from stably expressing L-cells. Following sucrose gradient sedimentation, floating membranes were collected and pooled. Sample was
diluted with Mes-buffered saline (25 mM Mes, pH 6.5, 0.15 M NaCl) containing 0.05% Triton X-100 and precleared with
25 µl of protein A-Sepharose beads (Sigma) for 2 h at 4 °C
with gentle mixing. The beads were then removed by centrifugation at
200 × g for 2 min at 4 °C. The sample was then
incubated overnight with anti-caveolin antibodies (5 ng/ml) at 4 °C
with gently mixing. 25 µl of protein A-Sepharose were then added to
each sample for 2 h at 4 °C. The beads were then removed by
centrifugation at 200 × g for 2 min at 4 °C, washed
three times in phosphate-buffered saline (pH 7.5), and
resuspended in 50 µl of SDS sample buffer.
Immunostaining and Antibody-induced Patching--
Immunostaining
of cells was performed using Kv1.5 antiserum and/or anti-caveolin
antibodies as described previously (10). Briefly, cells grown on
gelatin-coated coverslips were fixed with methanol and incubated with
primary antibody followed by biotin-conjugated goat anti-rabbit IgG
(Jackson Immunoresearch Laboratories, West Grove, PA). The bound Kv1.5
antibody was detected with BODIPY-conjugated streptavidin, and the
anti-caveolin antibody was detected with CY3-conjugated streptavidin.
Fluorescent signals were collected using a Nikon E800 microscope
equipped with standard epifluorescence and a Princeton Instruments CCD
camera. Demecolcine (0.1 µg/ml; Sigma) was dissolved in the culture medium.
For antibody-induced patching experiments, gelatin-coated coverslips
with nonfixed cells were incubated in their culture dishes with the
S1-S2 Kv1.5 antiserum (diluted 1:1000 in HEPES-based culture
medium) for 1 h at room temperature. The cells were taken through three 2-min washes in phosphate-buffered saline and then incubated for 1.5 h with biotin-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) diluted 1:500 in
culture medium. Another series of washes with phosphate-buffered saline was followed by incubation for 30 min with CY3-conjugated streptavidin (Jackson ImmunoResearch Laboratories) diluted 1:500 in
culture medium. Following three 2-min washes in
phosphate-buffered saline, cells were fixed with 4% paraformaldehyde
for 5 min and stained with monoclonal caveolin-1 antibodies. A
BODIPY-conjugated goat anti-mouse IgG was used for visualization.
Electrophysiology--
Electrophysiological recordings and data
analysis were made as described previously using the whole cell
configuration of the patch clamp technique (13). The steady-state
inactivation was measured using a 10-s conditioning pulse to various
potentials followed by 250-ms test steps to +50 mV. Because Kv1.5 shows
incomplete inactivation, data were normalized after subtraction of the
non-inactivating fraction at the test potential (16). Additional
details are presented in the figure legends.
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RESULTS |
Kv1.5- and Kv2.1-containing Lipid Rafts Show Differential
Sensitivity to Increasing Triton X-100 Concentrations--
A defining
characteristic of lipid rafts is their resistance to solubilization in
nonionic detergents and relative buoyancy because of an enriched lipid
content (6). We isolated low density, Triton X-100-insoluble complexes
from mouse Ltk
cells (L-cells) stably expressing Kv1.5
channel protein. Western blot analysis of sucrose gradient fractions
probed with anti-Kv1.5 antibodies demonstrates that Kv1.5 floats in a
low density, 1% Triton-insoluble fraction together with caveolin (Fig.
1A). As previously reported,
this is also true for L-cells stably expressing Kv2.1, a member of the
Shab family of voltage-gated K+ channels (5). To
facilitate comparison, these data have been reproduced in the
lower two panels of Fig. 1A. Although cells stably expressed channel protein, the extent of cell surface expression varied between individual cells. In the case of the Kv1.5 cell line,
channel expression was often very high and resulted in the intracellular accumulation of protein (Fig. 1B, left
panel). The percentage of channel in the nonraft fractions at the
bottom of the gradient most likely represents overexpressed
intracellular protein. The percentage of nonraft channel varied between
clonal cell lines and induction times and correlates with the amount of
intracellular protein as determined by immunostaining (Fig. 1B, top panels). However, cell surface channel
expression was easily detectable, and nanoampere currents could be
recorded from both the Kv1.5- and Kv2.1-expressing cell lines (Fig.
1B, bottom panels). The localization of Kv1.5 and
caveolin to light membrane fractions was also confirmed using a
detergent-free method of raft isolation (data not shown and Ref.
17).

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Fig. 1.
Kv1.5 channel targets to lipid rafts in
stably transfected fibroblasts. A, detergent-based
isolation of lipid rafts. Sucrose density gradient centrifugation of
1% Triton X-100-solubilized extracts from cells stably expressing Kv
channel protein were analyzed by Western blot. The immunoblots show low
density, raft-associated, distribution of Kv1.5, caveolin, and Kv2.1.
B, immunostaining of mouse L-cells stably expressing Kv1.5
channel with NH2-terminal antibody (left panels)
to show both cell surface and intracellular localization of channel.
Immunostaining of L-cells stably expressing Kv2.1 shows predominantly
cell surface distribution of channel protein (right panels).
Below are representative currents recorded from cell surface during 10 mV step depolarizations (holding potential, 80 mV) to +60 mV in mouse
Ltk cells stably expressing Kv1.5 or Kv2.1, respectively.
C, sucrose density gradient fractions of 2.5% Triton
X-100-solubilized extracts from cells stably expressing Kv channel
protein.
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The detergent-to-protein ratio affects recovery of raft-localized
proteins (18) and has been exploited to describe the nature of raft
association (19). We examined the effect of increasing Triton X-100
concentrations on Kv1.5 and Kv2.1 raft association. As shown in Fig.
1C, Kv1.5 remains raft-associated together with caveolin
following extraction with 2.5% Triton X-100. Even though the signals
were detected in a greater number of floating fractions, the two
protein peaks were symmetrical within the sucrose gradient. In
contrast, the Kv2.1 channel protein is partially solubilized by the
increased detergent concentration and no longer cofractionates with
caveolin (Fig. 1, compare A and C). The
differential sensitivity to detergent extraction suggests that the two
proteins may exist in different raft populations.
Kv1.5 Localizes to Caveolae--
We recently reported that Kv2.1
associates with a noncaveolar lipid raft (5). In that report, it was
shown that Kv2.1 channel protein does not internalize with caveolin
after microtubule disruption. In Fig. 2,
we show that Kv1.5 channel protein redistributes together with caveolin
after treatment with colcemide (100 ng/ml). Fig. 2C shows a
DIC image of morphologically altered L-cells following disruption of
the microtubules. In these cells, the Kv1.5 was retracted from the cell
surface (Fig. 2D). This was also true for the caveolin
protein (Fig. 2H) in which colcemide treatment disrupted the
normal cell surface distribution and resulted in internalization of the
protein. Although this experiment suggests an association of Kv1.5 and
caveolae, it may simply reflect a dependence of both proteins on
microtubule organization. Therefore, additional experiments were
performed to demonstrate association.

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Fig. 2.
Kv1.5 channel protein follows caveolin
redistribution after microtubule disruption. Immunofluorescence
localization of Kv1.5 shows punctate cell surface distribution
(B) that is altered with colcemide (0.1 µg/ml) treatment
for 24 h at 37 °C (compare B and D).
Similarly, the normal punctate cell surface distribution of caveolin
(F) was retracted from the cell surface following
microtubule disruption (compare F and H).
A, C, E, and G show the
corresponding phase images. Note colcemide treatment caused cells to
change morphology and become rounded.
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Overexpression of channel protein often results in a uniform
distribution of cell surface protein, which is often difficult to
resolve with conventional microscopic techniques. To improve resolution, we performed antibody-induced patching experiments using an
antibody directed against an extracellular epitope of the Kv1.5
channel. L-cells stably expressing Kv1.5 channel protein were incubated
at room temperature with polyclonal anti-Kv1.5 antibody directed
against the S1-S2 linker region for 1 h. Cross-linking was
performed using biotin-conjugated goat anti-rabbit secondary antibody
and detected with fluorophor-conjugated streptavidin. Punctate cell
surface patches of Kv1.5 protein were observed (Fig. 3B). The cells were then fixed
and immunostained with monoclonal anti-caveolin antibodies (Fig.
3C). An overlay of the two images (Fig. 3D) shows
complete overlap of Kv1.5 and caveolin. The enlarged area displayed in
Fig. 3E shows the extent of colocalization of the two
proteins (yellow).

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Fig. 3.
Caveolin copatches with Kv1.5 following
antibody induced cross-linking of channel protein. Images have
been pseudo-colored to facilitate comparison. A, phase image
of fibroblast. B, immunohistochemical localization of cell
surface Kv1.5 (green) using the S1-S2 antibody which
recognizes an extracellular epitope on the channel. Antibody binding
was performed prior to fixation and permeablization as described under
"Experimental Procedures." C, immunohistochemical
localization of caveolin (red) following fixation with 4%
paraformaldehyde. An overlay of the two images is shown in
D (yellow signifies colocalization). The
white box represents enlarged area shown in
E.
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Immunoprecipitation experiments using membranes from stably transfected
L-cells solubilized with n-octyl-glucoside failed to
demonstrate direct protein-protein interaction between Kv1.5 and
caveolin (data not shown). This is not surprising because Kv1.5
lacks a caveolin-binding motif (20). Therefore, we performed immunoprecipitations under nonsolubulizing conditions (see
"Experimental Procedures") with anti-caveolin antibodies as
described by other investigators (21-23). Raft-containing gradient
fractions from L-cells stably expressing Kv1.5 protein were pooled and
incubated with polyclonal anti-caveolin antibodies. Protein A-Sepharose beads were then used to isolate caveolar rafts. After extensive washing, bound material was eluted with SDS sample buffer. Samples were
then analyzed for Kv1.5 content via Western blot analysis. Western
analysis of membranes probed with Kv1.5 antiserum from nontransfected
and stably transfected L-cells (Fig. 4,
lanes 1 and 2) were used as controls for the
Kv1.5 signal. Lane 3 of Fig. 4 shows the presence of Kv1.5
in the starting raft fraction from which immunoisolation was performed,
and lane 4 shows Kv1.5 immunoprecipitation with the caveolin
antibody. The arrow points to bands representing Kv1.5.
Lipid rafts containing Kv1.5 did not nonspecifically bind to the beads
as indicated in lane 5, where the anti-caveolin antibody was
omitted. In addition, the caveolin antibodies specifically detected
raft-associated caveolin protein (data not shown). Note the absence of
the high molecular weight band in the starting material. This was
characteristic of all experiments performed and suggests that
post-translational processing of the channel protein in the fibroblasts
affects caveolae association. These results suggest that although there
does not exist a direct protein-protein interaction between Kv1.5 and
caveolin, the channel protein does immunoisolate with protein-lipid
complexes that contain caveolin. This result raises the issue initially
put forth by Johnson (4) and confirmed here experimentally that what
are now considered direct protein interactions should be assumed to
include the possibility of a lipid intermediary where direct binding
has not been clearly demonstrated.

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Fig. 4.
Immunoisolation of caveolae shows Kv1.5 is
associated with caveolin-containing lipid rafts. Lipid raft
fractions prepared by Triton X-100 extraction and buoyant density
centrifugation were subject to immunoprecipitation with anti-caveolin
antibodies under nonsolubulizing conditions (see "Experimental
Procedures"). Western analysis of membranes probed with Kv1.5
antiserum from nontransfected and stably transfected L-cells
(lanes 1 and 2) were used as control for Kv1.5
signal. Lane 3 shows the presence of Kv1.5 in the starting
raft fraction from which immunoisolation was performed. Lane
4 shows Kv1.5 immunoprecipitation with the caveolin antibody. The
arrow points to bands representing Kv1.5. Lipid rafts
containing Kv1.5 did not nonspecifically bind to the beads as indicated
in lane 5. The arrowhead indicates the position
of the primary IgG antibody (AB) from the immune
purification as detected with the goat anti-rabbit secondary antibody.
The mobility of the molecular mass standards is
indicated.
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Depletion of Raft Lipid Alters Kv1.5 Function--
The structure
of lipid rafts is dependent on the tight packing of cholesterol and
sphingolipids (24). Agents that bind and/or extract raft lipids have
been shown to alter the function of raft-associated proteins (24). We
have shown that depletion of cellular cholesterol modifies noncaveolar,
raft-associated Kv2.1 channel function but does not affect Kv4.2, which
is not localized to lipid rafts (5). Therefore, we tested whether
disruption of raft lipids affects caveolar-associated Kv1.5 function.
Treatment of cells stably expressing Kv1.5 with 2%
2-hydroxypropyl-
-cyclodextrin, a cholesterol-modifying agent, for
2 h altered the steady-state activation and inactivation properties of Kv1.5 (Fig. 5). Although
current density was not affected, the activation and inactivation
curves were shifted in the hyperpolarizing direction. The
V1/2 for activation (
17.8 ± 1.3 mV,
k = 11.4 ± 1.1) and inactivation (
27.8 ± 0.8 mV, k =
9.1 ± 0.7) under control conditions
were shifted by ~10 mV (
27.8 ± 1.1 mV, k = 8.0 ± 0.9, and 37.2 ± 0.5 mV, k =
6.0 ± 0.4, respectively) following cyclodextrin treatment. Treatment with fumonisin B, which inhibits steps in the biosynthetic pathway of sphingomyelin, has been used to deplete sphingolipids (25).
Treatment of cells for 72 h with 25 µM fumonisin B
produced results similar to those observed with cholesterol depletion
(Fig. 5). The V1/2 for steady-state activation was
shifted from
16.2 ± 1.3 mV, k = 11.9 ± 1.1 to
26.9 ± 1.1 mV, k = 8.7 ± 1.0 after
treatment with fumonisin B. Similarly, the steady-state inactivation
curve was also shifted in the hyperpolarizing direction from
V1/2 equal to
28.3 ± 0.7 mV,
k =
8.8 ± 0.6 to
40.4 ± 0.4 mV,
k =
5.4 ± 0.3 with depletion of sphingolipids.
Acute application of either cyclodextrin or fumonisin B in the bath
solution did not affect channel function. In addition, neither
intervention affected the properties of Kv4.2, a nonraft associated
channel (data not shown). These data show that altering raft structure
affects the function of caveolae-associated Kv channels.

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Fig. 5.
Depletion of raft lipid alters Kv1.5 channel
function. A, representative current recordings ( 80 mV
holding potential; 10 mV steps to +60 mV) from control cell stably
expressing Kv1.5 (left panel), cell treated with 2%
2-hydroxypropyl- -cyclodextrin for 2 h at 37 °C (center
panel), and cell treated with 25 µM fumonisin B at
37 °C for 72 h (right panel). B,
steady-state activation and inactivation of cyclodextrin treated cells.
Left panel, plot showing the voltage dependence of Kv1.5
current activation, as determined from the magnitude of the tail
currents, for control ( , n = 10) and cyclodextrin
treated ( , n = 8) cells. Right panel,
plot showing the voltage dependence of Kv1.5 current inactivation
determined using the double pulse protocol described under
"Experimental Procedures" (control , n = 10;
cyclodextrin-treated , n = 8). Data are plotted as
the percentages of inactivation (relative to peak current) at the
indicated voltage. Increasing the concentration of cyclodextrin or
extending the incubation time made the cells very difficult to patch
clamp. C, steady-state activation and inactivation of
fumonisin-treated cells. Left panel, plot showing the
voltage dependence of Kv1.5 current activation, for control ( ,
n = 10) and fumonisin-treated ( , n = 5) cells. Right panel, plot showing the voltage dependence
of Kv1.5 current inactivation (control , n = 10;
fumonisin-treated , n = 5).
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Kv1.5 Raft Association Does Not Involve COOH-terminal
Residues--
A role for the Kv channel COOH terminus in functional
cell surface expression has been suggested (26, 27). Recently, a COOH-terminal motif that influences processing and cell surface expression of Kv1.5 was identified at residues 593-598 (27). We tested
the importance of the Kv1.5 COOH terminus in lipid raft association
using a deletion mutant in which the last 57 COOH-terminal amino acids
were removed. Thus, this mutant lacks both the PDZ binding motif and
the motif at residues 593-596. Stable expression of the
57 Kv1.5
mutant in mouse L-cells produced nanoampere currents with wild-type
kinetics and amplitude (28). As predicted, the electrophoretic mobility
of this truncation mutant (Fig.
6A, lane 1) is
altered compared with wild-type Kv1.5 (Fig. 6A, lane
2). Importantly, the COOH-terminal truncation did not affect lipid raft association (Fig. 6B). These results suggest that the
signal for Kv1.5 raft association does not reside in the COOH terminus of the channel.

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Fig. 6.
COOH-terminal truncation does not affect
Kv1.5 raft association. A, Western blot probed with the
NH2-terminal Kv1.5 antibody confirms that the channel
protein was synthesized with the predicted 57 COOH-terminal amino acid
deletion. Lane 1 shows membranes from Kv1.5 57C mutant.
Lanes 2 and 3 contain membranes from wild-type
Kv1.5 and sham transfected cells, respectively. B, Western
blot of sucrose density gradient fractions from 1% Triton
X-100-extracted lysates of cells expressing the Kv1.5 COOH-terminal
deletion. The truncated Kv1.5 floats in low density lipid raft
fractions.
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DISCUSSION |
Previously, we demonstrated that Kv2.1, a member of the
Shab family of voltage-gated K+ channels,
targeted to noncaveolar lipid rafts, whereas Kv4.2 is not raft
associated in mouse L-cells. Here we report for the first time that a
member of the mammalian Shaker family, Kv1.5, targets to
caveolar rafts in this same cell type. Biochemical and
immunohistochemical evidence demonstrate that Kv1.5 is localized in
caveolae in the transfected fibroblasts. Kv1.5 cofractionates with
caveolin in low density Triton X-100 insoluble raft fractions, but
unlike Kv2.1 expressed in the same cell system, the symmetrical peak of
Kv1.5 and caveolin proteins is maintained with increased stringency of
membrane solubilization. In addition, Kv1.5 colocalizes with caveolin
on the cell surface and redistributes with caveolin following
microtubule disruption. Finally, immunoisolation of caveolae using
caveolin antibodies demonstrates that Kv1.5 channel protein copurifies
with intact caveolae.
Potential Mechanisms of Channel/Raft Association--
Our results
suggest that within a single cell type there exist mechanisms for the
differential sorting of Kv channel isoforms to unique lipid
microdomains. Given that the protein content of a lipid raft is
currently used to distinguish between raft subtypes, it follows that
protein-protein interactions may be a mechanism of isoform-specific
recruiting of proteins to distinct raft populations. Caveolin, for
example, is a scaffolding protein that can bind and assemble multiple
signaling molecules in a large complex (9). One recent example is the
caveolin-mediated association of Ca2+-signaling proteins
and the human Trp1 channel (29). However, to the best of our knowledge,
no work has demonstrated that caveolin binding is a prerequisite for
lipid raft association. Regardless, the results of our immunoisolation
experiments indicate that Kv1.5 is associated with intact caveolae, but
we cannot demonstrate a direct protein-protein interaction between
caveolin and channel protein (see "Results"). In addition, Kv1.5
sequence does not contain any known caveolin binding motifs (20).
Therefore, it is unlikely that the caveolin protein is responsible for
targeting Kv1.5 to caveolae.
Much of the past work on the spatial organization of ion channels has
focused on interactions between ion channels and scaffolding proteins.
Great emphasis has been placed on the role of PDZ domain-containing membrane-associated guanylate kinases (MAGUK), such as PSD-95, in the
targeting and localization of ion channels (30). Recently, it was
suggested that palmitoylated PSD-95 and PSD-93 may function as a lipid
raft scaffolding protein, similar to caveolin (31). The fact that Kv1.5
sequence contains a consensus PDZ binding domain, but the Kv2.1 channel
does not, makes the possibility of channel binding to endogenous PDZ
proteins an attractive candidate for the isoform-specific localization
of the two channels to different lipid microdomains. However, the Kv1.5
COOH-terminal truncation shown in Fig. 6 removes the only known
PDZ-binding site on the channel but does not affect lipid raft
association. Therefore, PDZ proteins are not likely to be responsible
for raft localization. In addition, this truncation removes the 5-amino
acid COOH-terminal motif identified in Kv1 subunits that reportedly
influences membrane localization (27). Together, these results suggest
that the mechanism of lipid raft association is independent of this
amino acid sequence.
Functional Significance of Lipid Raft-Channel
Localization--
The phosphorylation of ion channels is thought to be
one of the key regulatory mechanisms of membrane excitability. Not
surprisingly, channel complexes are basally and transiently
phosphorylated/dephosphorylated by a variety of modulatory enzymes
(32). Because rafts localize a number of signaling proteins such as
protein kinase C, tyrosine kinases, nitrogen-oxide synthase, Ha-Ras,
mitogen-activated protein kinase, and G-proteins (33), channel/raft
association could serve primarily to cluster signaling molecules with
their K+ channel substrates. The isoform-specific targeting
of Kv channels to distinct lipid raft subpopulations, with unique
protein compositions, would allow for differential regulation while
preventing cross-talk between competing pathways. Several recent
reports emphasize the importance of compartmentalization of ion
channels and signaling molecules (34, 35). For example, An et
al. (34) have reported that in developing cardiac muscle, the
electrophysiological consequences of
2-adrenergic
receptor-induced stimulation of cAMP is dependent on
compartmentalization with L-type Ca2+ channels. Although
not yet shown for the cardiac Ca2+ channel, both
2-adrenergic receptors and adenylyl cyclase localize to
caveolae in cardiac myocytes (36). Clearly, the partitioning of
K+ channels and regulatory signaling elements within the
same subcellular microdomains provides for efficient coupling and rapid
signaling. It is possible that the functional effects of raft
disruption reported here are a consequence of altered signaling,
i.e. kinase/phosphatase disruption. Although fumonisin B has
been used in the raft literature to deplete sphingolipids, its
mechanism of action is to inhibit ceramide synthase. Lipid
rafts/caveolae are believed to be highly localized sites for the
initiation of ceramide signaling pathways (37). In fact, it has been
reported that in fibroblasts ~50% of cellular ceramide is localized
in caveolae fractions (38). Interestingly, ceramide has been shown to
decrease Kv1.3 channel current in T-lymphocytes (39). Therefore, it is
possible that the observed functional effects on Kv1.5 are mediated
through caveolae-associated ceramide signaling pathways. Furthermore, tyrosine kinase signaling cascades are also believed to initiate from
lipid raft domains (37). Not surprisingly, Kv1.5 channel activity is
modulated by tyrosine phosphorylation in heterologous expression
systems and cultured Schwann cells (40, 41).
Alternatively, the functional changes illustrated in Fig. 5 could be
due to direct effects of altered lipid on channel activity, for charged
lipids produce a surface potential. Disruption of raft lipid via
cholesterol depletion or inhibition of sphingolipid synthesis could in
theory alter the surface charge and affect channel function. However,
the magnitude of an effect of altered bilayer charge on
potential-dependent channel parameters remains unknown.
Given that the Debye length for electrostatic interactions under
physiological conditions (estimated at 9 Å) is shorter than the
predicted dimensions of the channel protein (42, 43), the gating
charges of the channel appear to be partially insulated from
surrounding bilayer charge. Therefore, the channel may not sense the
full value of the surface potential in the charged lipid. According to
Arhem, a pure surface charge effect predicts that the
t1/2(V), time to half-maximum current
versus potential, and the G(V),
conductance versus potential, curves shift equally (44).
This was not the case for the effects of cyclodextrin on Kv1.5 (data
not shown). Regardless of the mechanism, the effects are raft-specific,
for neither cyclodextrin nor fumonisin B had functional effects on
Kv4.2, which is not raft-localized. Future experiments will address the
mechanism of lipid disruption on the function of raft-associated channels.
In conclusion, we show for the first time localization of a
voltage-dependent K+ channel, Kv1.5, to
caveolar membranes. These results, together with our previous published
work (5), demonstrate that voltage-dependent K+
channels from different families target to distinct lipid microdomains. The differential targeting of Kv channel subtypes to caveolar and
noncaveolar rafts within the same membrane represents a mechanism of
isoform-specific compartmentalization, thus allowing modulation of
K+ channel function within well defined membrane microdomains.