Am J Physiol Cell Physiol 299: C1015–C1027, 2010.
First published August 18, 2010; doi:10.1152/ajpcell.00120.2010.
ESCRT-dependent targeting of plasma membrane localized KCa3.1 to the
lysosomes
Corina M. Balut, Yajuan Gao, Sandra A. Murray, Patrick H. Thibodeau, and Daniel C. Devor
Department of Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, Pennsylvania
Submitted 8 April 2010; accepted in final form 13 August 2010
endocytosis; Rab7; lysosomes; endosomal sorting complex required
for transport
Ca2⫹-activated K⫹
channels (KCa2.3 and KCa3.1) are present in vascular endothelial cells and have been shown to play an important role in
the endothelium-derived hyperpolarizing factor (EDHF) response, a powerful regulator of vascular tone (16, 22, 23, 45,
56, 64). EDHF signaling is thought to be initiated by the
activation of KCa2.3 and KCa3.1, leading to hyperpolarization
of the endothelium, followed by hyperpolarization of the adjacent vascular smooth muscle, resulting in relaxation (24, 25,
31). Recent studies demonstrate that the impaired function or
expression of KCa2.3 and/or KCa3.1 abrogate the EDHF
response, resulting in blood pressure elevation (6, 16, 35, 56,
64), suggesting that these channels may be novel targets for the
SMALL- AND INTERMEDIATE-CONDUCTANCE,
Address for reprint requests and other correspondence: D. C. Devor, Dept.
of Cell Biology and Physiology, Univ. of Pittsburgh School of Medicine, S331
BST, 3500 Terrace St., Pittsburgh PA 15261 (e-mail: [email protected]).
http://www.ajpcell.org
development of alternate antihypertensive therapies (23, 65,
70). Numerous activators of KCa3.1 and KCa2.3 have been
identified (20, 21, 57, 58, 62), which have subsequently been
shown to modulate vascular tone and hence blood pressure,
suggesting that this channel may be targeted for therapeutic
benefit (9, 16, 35, 68, 70). An alternate approach to modulating
the activity of individual channels (Po) present in the membrane is to regulate the number of channels (N), as N is
similarly directly proportional to current flow and hence the
physiological response of the cell. The number of channels in
the membrane (N) is regulated by the balance between the
anterograde and retrograde trafficking pathways of the channels. Considerable progress has been made in understanding
the mechanisms involved in the assembly/trafficking of
KCa2.3 and KCa3.1 toward the plasma membrane (18, 40, 41,
51, 63); however, the mechanisms of endocytosis and downstream sorting of these channels are virtually unknown. Deciphering the molecular machinery that governs the (post)endocytic trafficking of KCa2.3 and KCa3.1 is the first step in
advancing our understanding of how the number of channels is
regulated at the plasma membrane, and whether this will result
in an altered EDHF response. In this respect, new therapeutic
strategies that focus on regulation of ion channel density at the
cell surface are emerging. For example, molecules which
correct the trafficking of mutant forms of CFTR (50) and
hERG (60) have recently been described. Additional studies
have focused on the pharmacological regulation of channel
stability within the membrane and their subsequent fate following endocytosis (55).
Very recently, we demonstrated that plasma membrane
KCa2.3 has a long half-life (⬃13 h), whereas KCa3.1 is rapidly
internalized (within 60 –90 min) (28). Furthermore, the long
plasma membrane half-life of KCa2.3 could be attributed to a
dynamic recycling of the channel back to the cell surface in an
RME-1- and Rab35/EPI64C-dependent manner. In contrast,
KCa3.1 does not enter this recycling pathway after being
internalized and is rapidly degraded (28). The degradation of
many cell surface proteins and receptors occurs within the
lysosomal lumen and depends on the function of the multivesicular body (MVB) sorting pathway (32, 43, 47). The endosomal sorting complex required for transport (ESCRT)-0, -I,
-II, and -III plays a critical role in the targeting of proteins for
lysosomal degradation. In the classic model, cargo recognition
is mediated by interactions with the ESCRT-0 (Hrs) complex
as well as with ESCRT-I (tumor susceptibility gene 101,
TSG101). ESCRT-II appears to function downstream or in
parallel to ESCRT-I and facilitates the recruitment and assembly of ESCRT-III (composed of charged multivesicular body
proteins, e.g., CHMP4). Finally, Vps4, a AAA ATPase, dissociates the ESCRT complex and facilitates the fusion of MVBs
with lysosomes, resulting in the degradation of the cargo (17,
0363-6143/10 Copyright © 2010 the American Physiological Society
C1015
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Balut CM, Gao Y, Murray SA, Thibodeau PH, Devor DC.
ESCRT-dependent targeting of plasma membrane localized KCa3.1 to
the lysosomes. Am J Physiol Cell Physiol 299: C1015–C1027, 2010.
First published August 18, 2010; doi:10.1152/ajpcell.00120.2010.—
The number of intermediate-conductance, Ca2⫹-activated K⫹ channels (KCa3.1) present at the plasma membrane is deterministic in any
physiological response. However, the mechanisms by which KCa3.1
channels are removed from the plasma membrane and targeted for
degradation are poorly understood. Recently, we demonstrated that
KCa3.1 is rapidly internalized from the plasma membrane, having a
short half-life in both human embryonic kidney cells (HEK293) and
human microvascular endothelial cells (HMEC-1). In this study, we
investigate the molecular mechanisms controlling the degradation of
KCa3.1 heterologously expressed in HEK and HMEC-1 cells. Using
immunofluorescence and electron microscopy, as well as quantitative
biochemical analysis, we demonstrate that membrane KCa3.1 is
targeted to the lysosomes for degradation. Furthermore, we demonstrate that either overexpressing a dominant negative Rab7 or short
interfering RNA-mediated knockdown of Rab7 results in a significant
inhibition of channel degradation rate. Coimmunoprecipitation confirmed a close association between Rab7 and KCa3.1. On the basis of
these findings, we assessed the role of the ESCRT machinery in the
degradation of heterologously expressed KCa3.1, including TSG101
[endosomal sorting complex required for transport (ESCRT)-I] and
CHMP4 (ESCRT-III) as well as VPS4, a protein involved in the
disassembly of the ESCRT machinery. We demonstrate that TSG101
is closely associated with KCa3.1 via coimmunoprecipitation and that
a dominant negative TSG101 inhibits KCa3.1 degradation. In addition, both dominant negative CHMP4 and VPS4 significantly decrease
the rate of membrane KCa3.1 degradation, compared with wild-type
controls. These results are the first to demonstrate that plasma membrane-associated KCa3.1 is targeted for lysosomal degradation via a
Rab7 and ESCRT-dependent pathway.
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ESCRT-DEPENDENT DEGRADATION OF KCa3.1
METHODS
Molecular biology. Generation of KCa3.1 (also referred to as IK1
or SK4) in pcDNA3.1(⫹) (Invitrogen, Carlsbad, CA) has been previously described (63). The biotin ligase acceptor peptide (BLAP)
sequence (GLNDIFEAQKIEWHE) was inserted into the extracellular
loop between transmembrane domains S3 and S4 of KCa3.1 as
described (28). The COOH-terminal myc epitope-tagged KCa3.1 was
previously described (63). The NH2-terminal, hemagglutinin (HA)tagged full-length TSG101 (pcGNM2/TSG-F) and COOH-terminal
portion of TSG101 (pcGNM2/TSG-3=) expression vectors were generously provided by Dr. E. O. Freed (National Institutes of Health,
Bethesda, MD) and Dr. Z. Sun (Stanford University, Palo Alto, CA),
respectively. The green fluorescent protein (GFP)- and hemagglutinin
(HA)-tagged Rab7 constructs (14) were obtained from Addgene
[Addgene plasmid 12605 for the wild type (WT) and Addgene
plasmid 12660 for the dominant negative (DN) form]. The human
CHMP4B and VPS4B expression vectors were obtained from Open
Biosystems. To convert CHMP4B to a DN form, CHMP4B was
fluorescently tagged by subcloning it into pECFP-N1 vector (BD
Biosciences) using the XhoI and KpnI restriction sites. The ATPasedefective mutant VPS4B(E235Q), which acts as a DN, was generated
using the Stratagene QuikChange site-directed mutagenesis strategy
(Stratagene, La Jolla, CA). The fidelity of all constructs utilized in this
study was confirmed by sequencing (ABI PRISM 377 automated
sequencer, University of Pittsburgh, Pittsburgh, PA) and subsequent
sequence alignment (NCBI BLAST) using GenBank accession
numbers NP_789782.1 for CHMP4B and NP_004860.2 for VPS4B.
Cell culture. Human embryonic kidney (HEK293) cells were obtained from the American Type Culture Collection (Manassas, VA).
The human microvascular endothelial cell line, HMEC-1 (1), was
generously provided by Dr. Edwin Ades [Centers for Disease Control,
Atlanta, GA (CDC)], Mr. Francisco J. Candal (CDC), and Dr. Thomas
Lawley (Emory University, Atlanta, GA). HMEC-1 cells possess
morphological, phenotypical, and functional characteristics of human
AJP-Cell Physiol • VOL
microvascular endothelial cells (1, 5) and are therefore physiologically relevant for our study. The cells were cultured and transfected as
previously described (28).
Antibodies. Polyclonal ␣-streptavidin antibody (Ab) was obtained
from Genscript (Piscataway, NJ). Monoclonal HA (HA.11) and c-myc
(clone 9E10) antibodies were obtained from Covance (Richmond,
CA). Monoclonal ␣-tubulin and monoclonal ␣-Rab7 were obtained
from Sigma-Aldrich (St. Louis, MO). Monoclonal anti-lysosomeassociated membrane protein 2 (Lamp2) directed against the human
epitope (H4B4) (developed by J. Thomas August and James E. K.
Hildreth) was obtained from the Developmental Studies Hybridoma
Bank developed under the auspices of the National Institute of Child
Health and Human Development (Bethesda, MD) and maintained by
the University of Iowa, Department of Biological Sciences (Iowa
City, IA). Rabbit ␣-VPS4A and ␣-VPS4B polyclonal antibodies were
generously provided by Dr. W. I. Sundquist (University of Utah, Salt
Lake City, UT). The monoclonal ␣-TSG101 Ab was obtained from
GeneTex (Irvine, CA).
Biotinylation of KCa3.1 using recombinant biotin ligase. BLAPtagged KCa3.1, heterologously expressed in HEK293 or HMEC-1
cells, was enzymatically biotinylated using recombinant biotin ligase
(BirA), as described (28). BirA was either purchased from Avidity
(Aurora, CO) or expressed from pET21a-BirA (generously provided
by Dr. Alice Y. Ting, Massachusetts Institute of Technology, Cambridge, MA) in Escherichia coli according to previously published
methods (12). Plasma membrane BLAP-tagged KCa3.1 was then
labeled with streptavidin-Alexa 488 or streptavidin-Alexa 555
(Invitrogen), and the cells were either incubated for various periods of
time at 37°C, as indicated in the text, or immediately fixed and
permeabilized (28). Nuclei were labeled with DAPI (Sigma-Aldrich).
Cells were imaged in one of two ways, as indicated in the figure
legends. In some cases, cells were subjected to laser confocal microscopy using an Olympus FluoView 1000 system. To ensure maximal
X-Y spatial resolution, sections were scanned at 1,024 ⫻ 1,024 pixels,
with sequential three-color image collection to minimize cross talk
between the channels imaged. In other experiments, cells were imaged
using a wide-field Olympus IX-81 with motorized stage. Multiple
planes were imaged, deconvolved using a point-spread function, and
presented as a projection image.
Immunofluorescence. To assess colocalization of internalized
KCa3.1 with lysosomes, BLAP-tagged KCa3.1 was labeled with
streptavidin-Alexa 555 as above and the cells were then incubated for
5 h at 37°C, in the presence of the lysosomal protease inhibitors
leupeptin (100 ␮M)/pepstatin (1 ␮g/ml; L/P) (Sigma-Aldrich). The
cells were then fixed/permeabilized as described (41) and the lysosomes labeled with ␣-Lamp2 antibody, followed by labeling with
Alexa 488-conjugated goat anti-mouse IgG antibody. Intracellular
HA-tagged Tsg101 was labeled with ␣-HA antibody, followed by a
goat anti-mouse IgG-Alexa 488 (Invitrogen). Imaging was carried out
as above.
Immunoprecipitations and immunoblots. Our immunoprecipitations (IP) and immunoblot (IB) protocols have been previously
described (28, 29, 40, 41). Briefly, cells were lysed and equivalent
amounts of total protein were precleared with protein G-agarose beads
(Invitrogen) and incubated with the indicated antibody. Normal IgG
was used as negative control. Immune complexes were precipitated
with protein G-agarose beads, and the proteins were resolved by
SDS-PAGE followed by IB. To eliminate interference by the heavy
and light chains of the immunoprecipitating antibody in the IP, mouse
IgG Trueblot ULTRA (eBioscience) was used as a secondary antibody for the detection of immunoprecipitated proteins in the IB.
Determination of degradation rate for plasma membrane localized
KCa3.1. The degradation rate for endocytosed membrane KCa3.1 was
determined as described (28). Briefly, the channel was specifically
biotinylated using BirA and labeled with streptavidin, as above. Next,
cells were incubated for various periods of time at 37°C, as indicated.
The cells were then lysed and equivalent amounts of total protein were
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48). However, recent studies have revealed that ESCRT-independent routes to lysosomes exist. For example, lysosomal
targeting of the activated PAR1 receptor requires sorting nexin 1
(SNX1), but it is independent of ESCRT-0 (Hrs) and ESCRT-I
(TSG101) function (33). Also, the ␦-opioid G protein-coupled
receptor (DOR) does not require TSG101 for agonist-induced
lysosomal degradation (36). In other studies, cargo engagement
directly with ESCRT-III, via binding to Alix/Bro1 (independent of ESCRT-I or ESCRT-II), has been implicated (26, 67).
Moreover, it has been suggested that ESCRT-dependent and
ESCRT-independent mechanisms of MVB biogenesis exist in
mammalian cells (61). These data emphasize the intricate
mechanism of recruitment and function of different ESCRT
components for lysosomal targeting of membrane proteins.
Thus, we have undertaken studies to elucidate the molecular
mechanisms involved in the retrograde trafficking of KCa3.1
from the plasma membrane.
In this study, we demonstrate that, subsequent to endocytosis, KCa3.1 is targeted to the lysosomes for degradation, and
that this process follows an ESCRT-dependent pathway, with
components of the ESCRT family of proteins acting at early,
intermediate, and later stages of channel sorting into lysosomes. Furthermore, we demonstrate a role for the smallmolecular-weight guanine nucleotide-binding protein Rab7 as
a regulator of the lysosomal degradation of KCa3.1. To our
knowledge, the present results are the first to demonstrate that
membrane KCa3.1 is targeted for degradation through a MVB
pathway.
ESCRT-DEPENDENT DEGRADATION OF KCa3.1
RESULTS
Membrane KCa3.1 is targeted to the lysosomes for degradation. To follow the fate of KCa3.1 channels subsequent to
endocytosis, HEK293 and HMEC-1 cells were transfected with
BLAP-tagged KCa3.1 (29). This construct will be referred to
as KCa3.1 for simplicity throughout the remainder of the
manuscript. Previous patch-clamp data demonstrate that this
channel maintains the functional characteristics of the WT
channel (29). KCa3.1 was enzymatically biotinylated at the cell
surface using BirA and labeled with streptavidin-Alexa 488 in
HEK cells (Fig. 1A, top) and streptavidin-Alexa 555 in
HMEC-1 cells (Fig. 1A, bottom). Cells were incubated at 37°C
AJP-Cell Physiol • VOL
for the indicated periods of time, and the fate of the endocytosed channel was assessed by confocal microscopy. At time 0
h, the labeled channel is present at the cell surface, as predicted
for our labeling protocol. Note that, for HMEC-1 cells, which
are only ⬃2 ␮m thick, the images display also the top of the
cells, with a discontinuous punctuate appearance of the labeled
channels at the cell surface. Importantly, no labeling was
observed if we express KCa3.1 with no BLAP tag and carried
out the labeling procedure as above. Also, if the BirA-dependent biotinylation step was omitted, streptavidin exposure
alone failed to label KCa3.1 (data not shown), confirming the
specificity of the labeling observed. After 1-h incubation at
37°C, almost all the channel present at the plasma membrane
was endocytosed in both HEK293 and HMEC-1 cells. After 5
h at 37°C, some KCa3.1-containing vesicles are still visible
inside the cells, but significant channel has been presumably
degraded as assessed by the reduced fluorescence signal. These
results show that KCa3.1 is rapidly internalized from the
plasma membrane and targeted for degradation, in good agreement with our previous report (29).
As lysosomes are the final destination to terminate the fate of
many membrane proteins, we hypothesized that membrane
KCa3.1 degradation also occurs in the lysosomes. To confirm
this, we performed colocalization studies between the endocytosed KCa3.1 and the lysosomal marker Lamp2. The channel
was expressed in HEK293 (Fig. 1B, top) and HMEC-1 cells
(Fig. 1B, bottom) and labeled at the cell surface with streptavidin-Alexa 555. Next, the channel was allowed to be endocytosed for 5 h at 37°C, in the presence of the lysosomal
protease inhibitors L/P. Cells were fixed and processed for
double label indirect immunofluorescence, to detect the lysosomal marker Lamp2 (labeled with Alexa 488, see METHODS).
As is apparent in the overlay panels, there is a high degree of
colocalization (yellow) between KCa3.1 and Lamp2 in both
HEK and HMEC-1 cells, indicating that internalized KCa3.1 is
targeted to the lysosomes.
To obtain a direct structural correlate to our IF results, we
determined the localization of KCa3.1 following endocytosis
using transmission electron microscopy (TEM). For these studies, HEK293 cells were transfected with KCa3.1 and the
channel was labeled at the cell surface with streptavidin conjugated with QuantumDot-655, followed by incubation at 37°C
in the presence of L/P for various periods of time. As shown in
Fig. 1C, the channel is rapidly internalized from the plasma
membrane in to endocytic vesicles (top). After 5 h, the channel
is localized in the lysosomes, as shown by the pronounced
QDots staining of these organelles (bottom).
We next confirmed the lysosomal targeting and proteolysis
of membrane KCa3.1 by evaluating the degradation of the
channel in cells treated with L/P. HEK293 and HMEC-1 cells
were transfected with KCa3.1, and the channel at the cell
surface was labeled with streptavidin-Alexa 488 in HEK cells
(Fig. 1D, top) and streptavidin-Alexa 555 in HMEC-1 cells
(Fig. 1D, bottom). Cells were incubated at 37°C for 5 h in the
presence or absence of L/P, and the fate of the endocytosed
channel was assessed by confocal microscopy. At time 0 h, the
labeled channel is present at the cell surface. At the end of the
incubation period, in cells treated with L/P for 5 h at 37°C,
channel degradation is inhibited and KCa3.1 accumulates inside the cells in a vesicular compartment with a perinuclear
distribution, as compared with control cells, where the channel
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separated by SDS-PAGE, followed by IB for streptavidin. As streptavidin remains tightly coupled to the channel during SDS-PAGE, this
provides a direct correlate to the immunofluorescence (IF) studies
detailed above. Bands were quantified by the densitometry function of
Quantity One software (Bio-Rad, Hercules, CA). Data were corrected
for background and nonspecific labeling (measured on cells treated
only with streptavidin, in the absence of biotinylation). The obtained
band intensities for the various time points were normalized relative to
the intensity at time 0 and are reported. The blots were also probed for
tubulin, as a protein-loading control.
Short-interfering RNA treatment. Smartpool short-interfering RNA
(siRNA) constructs against the coding region of either human Rab7,
TSG101, or the two isoforms of VPS4 (A and B) were obtained from
Dharmacon Research (Chicago, IL). Each siRNA smartpool comprised a mixture of four oligonucleotide duplexes. The siGENOME
Non-Targeting siRNA Pool no. 2 (Dharmacon Research), was used as
control. HEK293 cells were plated at ⬃50% confluence and transfected with 50 nM siRNA duplexes (for Rab7) and up to 100 nM
siRNA for TSG101 and VPS4, using DharmaFECT 1, according to
the manufacturer’s directions. Experiments were carried out 72-h
posttransfection.
Transmission electron microscopy of quantum dot-labeled KCa3.1.
For an ultrastructural analysis of channel localization following endocytosis, HEK293 cells stably expressing KCa3.1 were grown on
plastic coverslips for 24 h before labeling. Next, the channel was
enzymatically biotinylated at the cell surface as described above, then
labeled with streptavidin conjugated with QuantumDot-655 (Invitrogen).
These QDots can be visualized in electron micrographs as dense
geometric structures (19). Following incubation at 37°C for different
periods of time, the cells were fixed in 2.5% glutaraldehyde in PBS for
1 h at room temperature. Samples were rinsed three times for 5 min
in PBS and postfixed in 1% osmium tetroxide containing 1% potassium ferricyanide for 1 h at room temperature and then rinsed three
times in PBS. The cells were dehydrated in ascending grades of
ethanol (30%, 50%, 70%, 90%) 10 min each, and three changes of
absolute alcohol for 15 min at room temperature. Samples were
infiltrated three times with epon (1 h each). Samples were embedded
in epon overnight at 37°C and then 48 h at 65°C. Ultrathin sections
were mounted on grids and stained in 2% uranyl acetate in methanol
and then 1% lead citrate before imaging with a JEOL 1011 (Joel,
Tokyo, Japan) electron microscope operated at an accelerating voltage
of 80 kV.
Chemicals. All chemicals were obtained from Sigma-Aldrich, unless otherwise stated.
Statistical analysis. All data are presented as means ⫾ SE, where
n indicates the number of experiments. Statistical analysis was performed using a Student’s t-test, to compare the IB band intensities at
time 0 for KCa3.1 expressed together with either the WT or DN form
of a certain construct. For the normalized values of band intensities,
statistical analysis was performed using the nonparametric KruskalWallis test. A value of P ⬍ 0.05 was considered statistically significant and is reported.
C1017
C1018
ESCRT-DEPENDENT DEGRADATION OF KCa3.1
is extensively degraded. Taken together, these data confirm the
lysosomal nature of the vesicles where the channel accumulates.
To quantify the degradation rate of membrane KCa3.1 in
HEK293 cells under normal conditions and conditions where
the lysosomal proteolysis is inhibited, we used biotinylation as
previously described (29). KCa3.1 stably expressed in HEK
cells was biotinylated at the cell surface with BirA, then
labeled with streptavidin, in the same way as for the IF
measurements. Next, the channel was allowed to be internal-
Time 5 hrs
KCa3.1
Lamp2
Overlay
C
Time 10 min
Time 1hr
Time 5 hrs
HMEC-1
Time 0 hrs
Time 5 hrs
Time 5 hrs + L/P
HEK293
D
HMEC-1
HMEC-1
HEK293
B
E
Ctrl1 Ctrl2
0
1
3
5
8
12
KCa3.1
KCa3.1+L/P
hrs
100
75
KCa3.1
50
Tubulin
100
75
KCa3.1 + L/P
Tubulin
50
*
1.0
Relative Protein
Mol. Mass
(kD)
0.8
*
0.6
0.4
0.2
0.0
T0
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Time 0 hrs
HEK293
A
ized for various periods of time at 37°C with or without L/P
treatment. Subsequently, the cells were lysed and the complex
KCa3.1/streptavidin still present inside the cells was probed by
IB, with antibodies against streptavidin. To control for nonspecific labeling, HEK cells not expressing KCa3.1 were labeled as above (Ctrl1 lane in Fig. 1E) or KCa3.1-expressing
cells were labeled with streptavidin in the absence of enzymatic biotinylation (Ctrl2 lane in Fig. 1E). As is apparent, no
channel is detected in these controls, confirming the specificity
of our labeling protocol. As shown in Fig. 1E, subsequent to
ESCRT-DEPENDENT DEGRADATION OF KCa3.1
(n ⫽ 3) of the initial value. This value is significantly lower
than the amount of protein detected after 8 h at 37°C in control
cells, not transfected with Rab7 (38 ⫾ 9%, n ⫽ 3; P ⬍ 0.05),
suggesting that, as previously reported (7), overexpression of
Rab7 WT might induce a dominant active phenotype, accelerating the rate of channel degradation. In contrast, DN Rab7
dramatically inhibits the rate of degradation. After 8-h incubation at 37°C, the levels of membrane KCa3.1 still present
inside the cells measured 69 ⫾ 2% (n ⫽ 3; P ⬍ 0.05) of the
original values.
To confirm a role for Rab7 in the degradation of KCa3.1, we
utilized an siRNA approach. As above, the channel was
streptavidin-labeled at the cell surface and the degradation rate
was measured for untransfected cells, cells transfected with
control siRNA, and cells transfected with Rab7 siRNA. As
shown in Fig. 2D (bottom IB), 72 h after transfection with
Rab7 siRNA duplexes, endogenous Rab7 is not detected by IB,
whereas the scrambled control siRNA had no effect on Rab7
levels in HEK cells. Moreover, after 8-h incubation at 37°C,
the channel is extensively degraded in untransfected cells,
being only 31 ⫾ 5% (n ⫽ 3) of the initial membrane protein,
as well as in siRNA control-transfected cells (32 ⫾ 11%, n ⫽
3, P ⬎ 0.05). In contrast, in Rab7 siRNA-transfected cells,
channel degradation is significantly inhibited, with 71 ⫾ 1%
(n ⫽ 3; P ⬍ 0.05) of membrane KCa3.1 still present.
Because Rab proteins are known to associate with cargo
proteins (46, 54), we determined whether KCa3.1 is associated
with Rab7 by coimmunoprecipitation (co-IP). HEK cells were
cotransfected with myc-tagged KCa3.1 and either the WT or
the DN form of the GFP-HA-tagged Rab7. As shown in Fig.
2E, we observed an association of myc-KCa3.1 with both WT
and DN forms of Rab7. These findings are consistent with
Rab7 being a key regulatory molecule in the degradation
pathway of KCa3.1.
KCa3.1 is targeted to MVB via the ESCRT pathway—role of
TSG101. Because KCa3.1 is targeted for lysosomal degradation, we determined the role of the ESCRT family of proteins
in this process. Initially, we focused on TSG101, a core
component of the ESCRT-I complex (17, 48). We coexpressed
KCa3.1 with either WT, full-length TSG101 (TSG-F), or its
NH2-terminally truncated form (TSG-3=). Previous studies
have shown that overexpression of TSG-3= disrupts the cellular
endosomal sorting machinery (39). Plasma membrane KCa3.1
was labeled at the cell surface with streptavidin-Alexa 555,
after which the cells were incubated at 37°C for the indicated
Fig. 1. Membrane Ca2⫹-activated K⫹ channel KCa3.1 is targeted to the lysosomes for degradation. A: cells were transfected with biotin ligase acceptor peptide
(BLAP)-KCa3.1, and the channel at the cell surface was labeled with streptavidin-Alexa 488 in human embryonic kidney (HEK) cells (top) and
streptavidin-Alexa 555 in human microvascular endothelial cells (HMEC-1) (bottom). The fate of the channel inside the cells was addressed by immunofluorescence (IF) upon incubation at 37°C for the indicated periods of time. At time 0, the channel is localized at the plasma membrane. After 1 h at 37°C, nearly
all the channel is endocytosed in both HEK and HMEC-1 cells. Following 5 h at 37°C, the endocytosed KCa3.1 is extensively degraded in both cell types, as
evidenced by the reduced fluorescence signal. Nuclei were labeled with DAPI (blue). HEK cells are shown as single confocal sections, while HMEC-1 cells are
shown as projection images from multiple z-sections. B: the endocytosed BLAP-KCa3.1 (labeled in red) colocalizes with the lysosomal marker lysosomeassociated membrane protein 2 (Lamp2; labeled in green) in both HEK and HMEC-1 cells. A confocal optical section imaged through a representative cell is
shown. C: ultrastructural analysis using transmission electron microscopy confirms that membrane KCa3.1 is delivered to the lysosomes, following endocytosis.
D: cells were transfected with BLAP-KCa3.1 and the channel at the cell surface was labeled as in A. Cells were incubated for 5 h at 37°C either in the presence
or absence of lysosomal protease inhibitors leupeptin (100 ␮M)/pepstatin (1 ␮g/ml; L/P). At time 0, the channel is localized at the plasma membrane. At the
end of the incubation period, the endocytosed KCa3.1 is extensively degraded in control cells, whereas in cells treated with L/P, this process is inhibited, as shown
by the strong fluorescent signal still present. E: degradation rate for plasma membrane localized BLAP-KCa3.1 was evaluated in HEK cells by specifically
biotinylating the channel using recombinant biotin ligase (BirA). Subsequently, the channel was labeled with streptavidin and the cells were incubated at 37°C
for 0, 1, 3, 5, 8, or 12 h in the presence or absence of L/P. Representative blots are shown at left; 20 ␮g protein was loaded per lane. The data were quantified
by densitometry and are plotted as shown (n ⫽ 3; *P ⬍ 0.05). T, time; Mol, molecular; Ctrl, control.
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endocytosis, membrane KCa3.1 is extensively degraded within
12 h in control cells. That is, only 12 ⫾ 1% (n ⫽ 3) of the
membrane protein was still detected at the end of the incubation period. In contrast, in L/P-treated cells, channel degradation is significantly inhibited, with 49 ⫾ 4% (n ⫽ 3; P ⬍ 0.05)
of membrane KCa3.1 still present. Taken together, these data
indicate that lysosomes are the final destination of the endocytosed membrane KCa3.1 channel.
Rab7 is required for the degradation of KCa3.1 by the
lysosomes. Recent studies demonstrate that Rab7 controls intracellular vesicle traffic to late endosomes/lysosomes and
lysosome biogenesis in mammalian cells (7, 59). Thus, we
investigated the role of Rab7 as a regulator of the distal stages
of the KCa3.1 endocytic pathway. HEK293 cells were cotransfected with KCa3.1 and either the WT or a DN form of
GFP-HA-tagged Rab7. The plasma membrane-localized channel was labeled with streptavidin-Alexa 555 and allowed to
internalize for either 5 or 8 h at 37°C. In agreement with
previous reports (7), WT GFP-Rab7 primarily localized to
vesicles with a perinuclear distribution (Fig. 2A, middle).
Overexpression of WT Rab7 did not affect the plasma membrane localization of KCa3.1, as shown by the strong labeling
of the channel, at time 0 h (Fig. 2A, left). As is also seen for
these cells, the endocytosed KCa3.1 is extensively degraded
after 8-h incubation at 37°C (Fig. 2A, left). In contrast, overexpression of DN GFP-Rab7 caused a diffuse, widespread
signal and the accumulation of large vacuoles (Fig. 2B, arrow,
middle), as previously reported (3). At time 0 h, KCa3.1 is also
localized to the plasma membrane, similar to WT Rab7expressing cells. Quantification of band intensities at time 0
obtained from IB (see below; compare lane 2 for WT and DN
Rab7 in Fig. 2C) revealed that DN Rab7 does not affect cell
surface expression of KCa3.1, as compared with WT (n ⫽ 3;
P ⬎ 0.05). However, DN Rab7 had a strong inhibitory effect
on membrane KCa3.1 degradation, as shown by the clear
fluorescent signal associated with the channel at the end of 8-h
incubation period (Fig. 2B, left).
To quantify the rate of KCa3.1 degradation in HEK cells
expressing either the WT or DN Rab7, we biotinylated, then
labeled the channel at the cell surface with streptavidin, as
above. The cells were incubated at 37°C for either 5 or 8 h, and
the amount of KCa3.1 present was assessed by immunoblotting. As shown in Fig. 2C, right, in the presence of WT Rab7
the channel is extensively degraded, the detected amount of
protein at the end of the incubation period being only 11 ⫾ 1%
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ESCRT-DEPENDENT DEGRADATION OF KCa3.1
indicative of TSG101 and KCa3.1 being minimally in the same
endosomal compartment following endocytosis. These results
indicate a role for TSG101 in recruiting membrane KCa3.1
into MVB.
KCa3.1 is targeted to MVB via the ESCRT pathway—role of
CHMP4B. We next determined whether CHMP4B, a member
of the ESCRT-III complex (34, 69), is involved in the lysosomal targeting of KCa3.1. It has been previously shown that
fluorescently tagged CHMPs behave as dominant negatives,
disturbing the regulation of membrane trafficking from endosomes to lysosomes (42). Thus, we cotransfected HEK cells
with KCa3.1 and CHMP4B-cyan fluorescent protein (CFP),
labeled cell surface KCa3.1 with streptavidin-Alexa 555, and
allowed the channel to internalize for 5 h at 37°C. At time 0 h,
the channel is localized at the plasma membrane in these cells,
as shown in Fig. 4A (top). Moreover, quantification of band
intensities at time zero obtained from IB (see below, compare
lane 2 for WT and DN CHMP4B in Fig. 4B) showed that
overexpression of DN CHMP4B does not affect cell surface
expression of KCa3.1, compared with the WT CHMP4B (n ⫽
3; P ⬎ 0.05). As shown in Fig. 4A (middle), expression of
CHMP4B-CFP induces formation of enlarged endosomal
structures, indicative of the dominant negative effect of the
fused CFP-CHMP4B. Moreover, endocytosed KCa3.1 accumulates in the CHMP4B-CFP-induced structures, as indicated by
the clear colocalization (arrow in the bottom overlay, Fig. 4A).
The effect of CHMP4B-CFP expression on the degradation
rate of membrane KCa3.1 was quantified using surface biotinylation as above. As shown in Fig. 4B, after 8-h incubation at
37°C, CHMP4B-CFP expression significantly inhibited channel degradation (70 ⫾ 9% channel still present inside the cells,
n ⫽ 4), compared with the CHMP4B WT-expressing cells
(33 ⫾ 8%, n ⫽ 4; P ⬍ 0.05). These data emphasize the
importance of a functional CHMP4B for proper sorting of
internalized KCa3.1 into the late endosomes.
Lysosomal proteolysis of KCa3.1 is dependent on VPS4
activity. One of the essential steps in MVB biogenesis and
function is the recruitment of the AAA ATPase SKD1/VPS4,
which drives dissociation of the ESCRT machinery and allows
the intraluminal release of vesicles formed by endosomal
membrane invagination (52). It has been shown that while
VPS4 transiently associates with late endosomes, the ATPasedefective VPS4 mutant (VPS4B E235Q) fails to dissociate
from these membranes and severely impairs MVB sorting (27).
To test this effect on KCa3.1 degradation, HEK and HMEC-1
cells were doubly transfected with KCa3.1 and either the WT
VPS4B or VPS4B E235Q. KCa3.1 was labeled at the cell
Fig. 2. Rab7 is required for the degradation of KCa3.1 by the lysosomes. A and B: HEK cells were doubly transfected with BLAP-KCa3.1 and either wild-type
(WT) green fluorescent protein (GFP)-Rab7 (A) or dominant negative (DN) GFP-Rab7 (B). The channel was labeled at the cell surface with streptavidin-Alexa
555 and cells incubated at 37°C for either 5 or 8 h. Transient expression of DN Rab7 results in endosomal accumulation of KCa3.1, compared with the wild-type
Rab7. C: to quantify the effect of Rab7 activity on the degradation rate of membrane KCa3.1, HEK cells were transfected with BLAP-KCa3.1 and either
GFP-Rab7 construct. Cells were enzymatically biotinylated with BirA, labeled with streptavidin, and then blotted for streptavidin at the indicated periods of time.
A representative blot is shown, together with the results obtained from densitometry quantification (n ⫽ 3; *P ⬍ 0.05). D: HEK cells stably expressing
BLAP-KCa3.1 were either mock-transfected, transfected with short interfering RNA (siRNA) control, or Rab7-specific siRNA. At 72-h posttransfection, the
channel was streptavidin-labeled at the cell surface and incubated at 37°C for the indicated periods of time. The degradation rate of the channel was assessed
by immunoblotting (IB). A representative blot is shown, together with the results obtained from densitometric quantification (n ⫽ 3; *P ⬍ 0.05).
E: coimmunoprecipitation of myc-KCa3.1 with either hemagglutinin (HA)-tagged WT or DN Rab7 was carried out in HEK cells as described in METHODS.
KCa3.1 was immunoprecipitated using either an anti-myc Ab (lanes 1–3, 5, and 6) or an anti-V5 Ab as IgG control (lanes 4 and 7) and subsequently IB for Rab7
using an anti-HA Ab. When only a single construct was transfected (myc or HA), the empty vector was included to maintain the plasmid at the same final
concentration. Rab7 was detected by IB in lanes 3 and 6, confirming association between KCa3.1 and Rab7.
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periods of time (Fig. 3A). As shown in Fig. 3A, top, TSG-F
displayed a highly punctate, putatively endosomal expression
pattern. In contrast, transfection of cells with TSG-3= (Fig. 3A,
bottom) resulted in the formation of large vacuolar structures,
as reported (30, 39). As shown in Fig. 3A for time 0 h, the
channel is expressed at the cell surface in both TSG-F or
TSG-3=-transfected cells. Moreover, quantification of band
intensities at time 0 obtained from IB (see below, compare lane
2 for TSG-F and TSG-3= in Fig. 3B) revealed that the truncated
form of TSG101 does not affect cell surface expression of
KCa3.1, compared with the full-length TSG101 (n ⫽ 3; P ⬎
0.05). Within 1-h incubation at 37°C, the channel was almost
completely endocytosed in the presence of TSG-F (Fig. 3A,
top) as well as TSG-3= (Fig. 3A, bottom), similar to what we
observed for control, nontransfected cells (Fig. 1A). After 5-h
incubation at 37°C, TSG-3=-expressing cells show a pronounced staining associated with the channel, indicative of a
reduced degradation rate, compared with the control.
The effects of full-length and mutant TSG101 constructs on
the rate of KCa3.1 degradation were quantified using surface
biotinylation as above (Fig. 3B). After 8 h at 37°C, the level of
membrane KCa3.1 still detected inside the cells was 43 ⫾ 14%
for the TSG-F-expressing cells and 72 ⫾ 7% for the truncated
TSG-3= construct (n ⫽ 3; P ⬎ 0.05). Although suggesting an
inhibitory effect on channel degradation rate in cells expressing
TSG101–3= relative to the WT TSG-F, the results are not
significantly different, owing to the variability obtained for the
TSG-F construct. This observation is not uncommon, because
previous studies have reported that overexpression of the WT
TSG101 might slow the degradation rate and hence induce
accumulation of EGF receptor in HeLa cells (30). To obtain a
better estimate for the effect of truncated TSG101 on the rate
of channel endocytosis, we compared TSG-3= cells with control cells (transfected only with KCa3.1 and the empty vector).
In the latter case, the amount of channel still present inside the
cells at the end of 8-h incubation was 39 ⫾ 4% (n ⫽ 3; P ⬍
0.05). Unfortunately, siRNAs directed against TSG101 (100
nM for 72 h) resulted in only an ⬃40% knockdown of
TSG101, as assessed by IB, such that the role of TSG101
knockdown on KCa3.1 degradation was not further explored.
Given that TSG101 is known to directly interact with cargo
proteins destined for lysosomal degradation (2), we determined
whether endocytosed KCa3.1 associates with TSG101. HEK
cells were transfected with myc-tagged KCa3.1 and either the
full-length or the 3=-truncated form of HA-tagged TSG101. As
shown in Fig. 3C, KCa3.1 coimmunoprecipitated with
TSG101-F, and also with the truncated form TSG101–3=,
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Fig. 3. Effects of overexpressing the full-length and the NH2-terminally truncated form of TSG101 on KCa3.1 degradation. A: subcellular localization of TSG-F
and NH2-terminally truncated TSG-3= in HEK cells cotransfected with BLAP-KCa3.1. The channel was labeled at the cell surface with streptavidin-Alexa 555.
After incubation at 37°C for the indicated times, the cells were fixed, permeabilized, and stained for TSG101 using anti-HA Ab and visualized by confocal
microscopy. At time 0 h, BLAP-KCa3.1 is localized at the plasma membrane. After 5-h incubation at 37°C, cells expressing the dominant negative form of
TSG101 show a suppression of channel degradation, and its accumulation in vesicles. Images are single confocal sections. Nuclei were labeled with DAPI.
B: the degradation rate for plasma membrane-localized BLAP-KCa3.1 was evaluated in HEK cells by specifically biotinylating the channel using BirA (see
METHODS). The data were quantified by densitometry and plotted as shown (n ⫽ 3; *P ⬍ 0.05). C: coimmunoprecipitation of myc-KCa3.1, with either HA-tagged
TSG-F or TSG-3=, was carried out in HEK cells as described in METHODS. KCa3.1 was immunoprecipitated using either an anti-myc Ab (lanes 1–3, 6, and 7)
or an anti-V5 Ab as IgG control (lanes 4 and 8) and subsequently IB for TSG101 using an anti-HA Ab. When only a single construct was transfected (myc or
HA), the empty vector was included to maintain the plasmid at the same final concentration. TSG101 was detected by IB in lanes 3 and 7, confirming association
between KCa3.1 and TSG101. Note the difference in the molecular weight between the full-length and the truncated form.
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surface either with streptavidin-Alexa 555 or plain streptavidin, then incubated at 37°C for 5 and 8 h. The rate of membrane
KCa3.1 degradation was assessed with both confocal microscopy
and IB.
At time 0 h, the labeled channel is expressed at the cell
surface as expected, in both HEK cells (Fig. 5A, left) and
HMEC-1 cells (Fig. 5B, left). Importantly, as obtained form IB
quantification of the band intensities at time 0(see below,
compare lane 2 for WT and DN VPS4B in Fig. 5C), expression
of VPS4B E235Q does not affect plasma membrane KCa3.1
compared with the WT VPS4B (n ⫽ 3; P ⬎ 0.05). However,
as shown by the large accumulation of KCa3.1-related fluorescent vesicles inside the cells, the protein degradation rate for
mutant VPS4B E235Q-expressing cells is dramatically decreased compared with the VPS4B WT-expressing cells, in
both HEK (Fig. 5A) and HMEC-1 cells (Fig. 5B). Quantitative
immunoblotting (Fig. 5C) showed that after 8-h incubation at
37°C, the channel is not degraded in cells expressing VPS4B
E235Q, with 86 ⫾ 5% (n ⫽ 3; P ⬍ 0.05) of membrane KCa3.1
still present. Wild-type VPS4B has no effect on KCa3.1 degAJP-Cell Physiol • VOL
radation, such that, after 8 h, only 44 ⫾ 11%, (n ⫽ 3) of the
channel remained. These results indicate a role for VPS4 in the
correct targeting and lysosomal degradation of KCa3.1. As
above for TSG101, siRNAs directed against VPS4A and
VPS4B in combination (100 nM for 72 h) resulted in only an
⬃60% knockdown of VPS4A/B, as assessed by IB, such that
the role of VPS4 knockdown on KCa3.1 degradation was not
further explored.
DISCUSSION
Endothelial small- and intermediate-conductance Ca2⫹-activated K⫹ channels (KCa2.3 and KCa3.1) play a key role in
mediating the agonist-induced activation of endothelium-derived hyperpolarizing factor (EDHF) response, one of the main
regulators of vascular tone (8, 15, 23–25, 65). For cells to
appropriately respond to external agonists, they must maintain
the proper protein complement at the cell surface. This can be
achieved by both regulating the delivery and removal of the
channels to and from the cell surface when necessary. Al-
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Fig. 4. Effect of CHMP4B-cyan fluorescent protein (CFP) overexpression on the distribution and degradation of endocytosed KCa3.1. A: HEK cells were
cotransfected with BLAP-KCa3.1 and a CFP-tagged, CHMP4B. KCa3.1 was labeled at the cell surface with streptavidin-Alexa 555 (red), then allowed to be
internalized for 5 h at 37°C. In cells overexpressing CHMP4B-CFP, the channel is not degraded, as shown by the strong intracellular signal (left). Moreover,
the channel is accumulated in the large CHMP4B-CFP containing intracellular structures (arrow, overlay). Images are single confocal sections. Nuclei were
labeled with DAPI. B: the degradation rate for plasma membrane-localized BLAP-KCa3.1 was evaluated in HEK cells by specifically biotinylating the channel
using BirA (see METHODS). The data were quantified by densitometry and plotted as shown (n ⫽ 4; *P ⬍ 0.05).
C1024
ESCRT-DEPENDENT DEGRADATION OF KCa3.1
A
B
HEK293
Time 5 hrs
Time 0 hrs
Time 8 hrs
Time 8 hrs
+ VPS4B E235Q
+ VPS4B WT
+ VPS4B E235Q
Mol. Mass
(kD)
Ctrl
0
5
8
KCa3.1+vector
KCa3.1+VPS4B WT
KCa3.1+VPS4B E235Q
hrs
100
50
Tubulin
100
75
50
KCa3.1 + VPS4B E235Q
Relative Protein
75
*
1.0
KCa3.1 + VPS4B WT
*
0.8
0.6
0.4
0.2
0.0
Tubulin
T0
T5h
T8h
Fig. 5. Lysosomal proteolysis of KCa3.1 is dependent on Vps4 activity. A: HEK cells were doubly transfected with BLAP-KCa3.1 and either the WT or a DN
form (E235Q) of VPS4B. Channel was fluorescently labeled at the cell surface and then incubated at 37°C for the indicated periods of time. As shown by the
large accumulation of channel-related vesicles inside the cells, the protein degradation rate for mutant VPS4BE235Q cells is much slower than the one for the
wild-type-expressing cells. Images are single confocal sections. B: the degradation rate for plasma membrane-localized BLAP-KCa3.1 was quantified in HEK
cells by specifically biotinylating the channel using BirA (see METHODS). A representative blot is shown. The data were quantified by densitometry and normalized
to the starting values for each case (n ⫽ 3; *P ⬍ 0.05).
though there is a clear consensus that KCa2.3 and KCa3.1
together are responsible for initiating the EDHF signaling (16,
22, 35), these channels strikingly differ in their fate subsequent
to membrane insertion, as we have recently shown (28). That
is, KCa2.3 has a long residency time at the plasma membrane,
attributed to a dynamic recycling of the channel back to the cell
surface. In contrast, as we present here, KC3.1 is rapidly
internalized and targeted for degradation in both HEK and
HMEC-1 cells. The striking difference in the way these channels are regulated at the cell surface might be regarded as a
“fingerprint” of their physiological role in endothelial cells.
In this study, we characterize, for the first time, the molecular mechanisms responsible for KCa3.1 downstream trafficking and degradation. Using a biotin-ligase acceptor peptidetagged KCa3.1 recently engineered in our lab, we rapidly
biotinylate and streptavidin labeled the channel at the cell
surface. This allows us to follow the channels fate subsequent
to endocytosis and determine its degradation rate, using a
combined imaging and biochemical approach. Our data indicate a degradation pathway in which the channel is delivered to
lysosomes. The first evidence supporting this conclusion came
from colocalization studies between endocytosed KCa3.1 and
AJP-Cell Physiol • VOL
the lysosomal marker Lamp-2 in both HEK and endothelial
HMEC-1 cells (Fig. 1B) and was confirmed using TEM (Fig.
1C). Additionally, in the presence of lysosomal protease inhibitors L/P, KCa3.1 accumulates inside the cells in a vesicular
compartment with a perinuclear distribution, whereas in control cells the protein is mostly degraded over the observed
period of time, as indicated by the lack of fluorescence signal
associated with the channel (Fig. 1D). Biochemical quantification of the degradation rate of membrane KCa3.1 upon
prolonged incubation either in the presence or absence of L/P
indicate that the endocytosed channel is degraded within 12 h
in control cells, whereas for L/P-treated cells the channel
degradation is significantly inhibited (Fig. 1E), conclusively
demonstrating that lysosomes are the final destination for the
internalized channel.
There is a defined role for the small GTPase Rab7 in
regulating the later stages of the endocytic pathway for a
number of proteins (10). Moreover, it has been shown that
Rab7 acts as a key regulatory protein for proper aggregation
and fusion of late endocytic structures in the perinuclear
region, and consequently for the biogenesis and maintenance
of the lysosomal compartment (7, 66). Herein, we define a role
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C
Time 5 hrs
+ VPS4B WT
Time 0 hrs
HMEC-1
ESCRT-DEPENDENT DEGRADATION OF KCa3.1
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attributed to a disturbed endosome-lysosome degradation pathway, based on recent data showing that overexpression of DN
forms of these proteins generates deformed and functionally
impaired MVBs (27, 42).
In total, we demonstrate that membrane KCa3.1 is targeted for lysosomal degradation, subsequent to endocytosis,
and that this process requires components of the ESCRT
machinery. Recent studies demonstrate that other potassium
channels undergo lysosomal degradation, i.e., the inwardrectifier potassium channel Kir2.1 (38), the ATP-sensitive
potassium channel KATP (44), or the HERG channel (11).
However, to the best of our knowledge, this is the first study
to show the role of ESCRT proteins on lysosomal degradation of a potassium channel. Understanding the molecular
mechanisms that govern the downstream sorting and trafficking of KCa3.1 channels in endothelial cells is relevant
for cardiovascular pathological conditions. Importantly, our
data indicate qualitatively similar mechanisms for channel
trafficking and lysosomal degradation in both HEK and
HMEC-1 cells. Thus, inhibition of lysosomal proteolysis
with L/P induced the accumulation of KCa3.1 in both cell
types (Fig. 1D). Also, a functional VPS4B is required for
channel degradation in either cell type (Fig. 5, A and B). In
line with this observation, we have previously shown that
internalized KCa2.3 is recycled back at the plasma membrane via the same molecular machinery (including RME-1,
Rab35, and the Rab GAP, EPI64C) in both HEK and
HMEC-1 cells (28). This suggests that general mechanisms,
governed by similar molecular components, are responsible
for the behavior of these channels in both cell types. Hence,
our results in HMEC-1 cells, which closely resemble the
morphological and functional characteristics of human microvascular endothelia, are relevant for understanding the
trafficking of KCa3.1 and KCa2.3 in intact endothelia.
However, future studies are required, to confirm these results on endogenously expressed KCa3.1 in native endothelia.
ACKNOWLEDGMENTS
The authors gratefully acknowledge Beth Nickel for technical assistance in
TEM. We also acknowledge Dr. Kirk Hamilton for critically reading the
manuscript and for many helpful discussions.
GRANTS
This work was supported by National Institutes of Health Grants HL083060
and HL092157 (to D. C. Devor) and by an American Heart Fellowship
(0825542D; to C. M. Balut).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
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for Rab7 in the degradation of KCa3.1. That is, KCa3.1
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copolymerize at sites of action, delineating and generating
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indicates that ESCRT-III subunits of the CHMP4 family can
form circular filaments that can be engaged to deform the
membrane to which they are attached (34). For the process to
be completed and the cargo delivered into the lumen of
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membrane, and this step is controlled by the AAA ATPase
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well as an ATP-defective DN mutant of VPS4B in HEK and
HMEC-1 cells (Fig. 5), the degradation rate of membrane
KCa3.1 was potently inhibited. The observed effect might be
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