Download Caveolin-3 and SAP97 form a scaffolding protein complex that

Am J Physiol Heart Circ Physiol 287: H681–H690, 2004;
10.1152/ajpheart.00152.2004.
Caveolin-3 and SAP97 form a scaffolding protein complex
that regulates the voltage-gated potassium channel Kv1.5
Eduardo J. Folco, Gong-Xin Liu, and Gideon Koren
Bioelectricity Laboratory, Cardiovascular Division, Brigham and Women’s
Hospital, Harvard Medical School, Boston, Massachusetts 02115
Submitted 31 March 2004; accepted in final form 31 March 2004
lipid rafts; potassium channels; caveolae; heart
TARGETING OF PROTEINS to specific membrane regions may have
a profound effect on their function. Membrane-associated
guanylate kinases (MAGUKs) are PDZ domain-containing
proteins that have been proposed to play a role in the targeting
of sodium channels, N-methyl-D-aspartate (NMDA) receptors,
and voltage-gated potassium channels to discrete plasma membrane domains in neuronal cells (5, 8, 22). MAGUKs also
contain other interaction modules, such as the Src homology-3
(SH3) and guanylate kinase (GUK)-like domains. Hence,
MAGUKs serve as a scaffold for the assembly of different
polypeptides into macromolecular signaling complexes (8).
Lipid rafts are membrane microdomains rich in cholesterol
and sphingolipids that are enriched in important signaling
molecules (19). Caveolae represent a subpopulation of lipid
rafts that contain the scaffolding protein caveolin, which can
organize the assembly of macromolecular complexes and regulate protein function within the caveolae. Three caveolin
genes (Cav-1, Cav-2, and Cav-3) have been identified. Cav-1
and Cav-2 have a wide distribution among different cell types,
whereas Cav-3 is expressed exclusively in all types of muscle
(21). Mutations in the human Cav-3 gene are associated with
muscular dystrophy (16, 17), and inactivation of the Cav-3
gene in mice leads to muscle degeneration (7, 11), cardiac
hypertrophy, and progressive cardiomyopathy (27).
Address for reprint requests and other correspondence: G. Koren, Bioelectricity Laboratory, Cardiovascular Div., Brigham and Women’s Hospital, 75
Francis St., Boston, MA 02115 (E-mail: [email protected]).
http://www.ajpheart.org
Very little is known about scaffolding proteins that mediate
the organization of “transducisomes” in the heart in general
and those that contain potassium channels (channelosomes) in
particular. SAP97 is expressed abundantly in the heart, and
recent reports indicate that it can interact with voltage-gated
and inward rectifier potassium channels (14, 26). We recently
reported that Kv1.5 colocalizes with SAP97 in the heart and
interacts with it in heterologous expression systems (18).
Godreau et al. (10) have confirmed these interactions and
reported that Kv1.5 and SAP97 associate in the human atrium.
Martens and colleagues (15) recently reported that in heterologous expression systems, certain voltage-gated potassium
channels are differentially targeted to distinct subpopulations
of lipid rafts: in mouse L cells, Kv2.1 targets to noncaveolar
lipid rafts, whereas Kv1.5 targets to caveolae (15). The mechanisms that regulate these targeting events are unknown.
The observed association of Kv1.5 with SAP97 (18) and its
localization in caveolae (15) led us to hypothesize that SAP97
associates with Cav-3 to form a scaffolding complex that can
recruit ion channels and regulate their function. Here, we report
that Cav-3 interacts with SAP97 in the heart, in heterologous
expression systems, and in vitro. We define the sites of proteinprotein interactions involved in the assembly of this complex
and use model cell systems to demonstrate that it can recruit
Kv1.5 to form a tripartite complex. Furthermore, we show that
the Cav-3/SAP97 complex selectively regulates the expression
of currents encoded by a glycosylation-deficient Kv1.5 mutant.
MATERIALS AND METHODS
Materials. The following antibodies were used: anti-PDZ [previously characterized (18) mouse monoclonal antibody (MAb) that
recognizes SAP97] from Upstate Biotechnology, anti-Cav-3 (mouse
MAb) from BD Transduction Laboratories, anti-Cav-3 (goat pAb)
from Santa Cruz Biotechnology, anti-FLAG epitope (M2, mouse
MAb) from Sigma, and anti-c-Myc (mouse MAb) and anti-green
fluorescent protein (GFP) [rabbit polyclonal antibody (PAb)] from
Clontech. The secondary antibodies were from Zymed Laboratories,
the glutatione-Sepharose 4B was from Amersham Pharmacia Biotech,
and the protein A/G agarose was from Santa Cruz Biotechnology.
DNA cloning and bacterial expression. The construction of rat
SAP97, FL-Kv1.5, and FL-Kv1.5-A cDNAs was described previously
(18). Myc-tagged Cav-3 and Kv2.1 cDNAs were gifts from Drs.
Thomas Michel and Barbara Wible, respectively. The various SAP97
deletion mutants (S1–S5; see schemes in Fig. 2A) were amplified by
PCR in-frame with the FLAG epitope coding sequence at the 5⬘-end
and subcloned into pCDNA3 (Invitrogen). GFP-⌬SAP (see Fig. 2A)
and GFP1.5C (see Fig. 4B) were amplified by PCR and subcloned into
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
0363-6135/04 $5.00 Copyright © 2004 the American Physiological Society
H681
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.3 on August 3, 2017
Folco, Eduardo J., Gong-Xin Liu, and Gideon Koren. Caveolin-3 and SAP97 form a scaffolding protein complex that regulates the
voltage-gated potassium channel Kv1.5. Am J Physiol Heart Circ
Physiol 287: H681–H690, 2004; 10.1152/ajpheart.00152.2004.—The
targeting of ion channels to particular membrane microdomains and
their organization in macromolecular complexes allow excitable cells
to respond efficiently to extracellular signals. In this study, we
describe the formation of a complex that contains two scaffolding
proteins: caveolin-3 (Cav-3) and a membrane-associated guanylate
kinase (MAGUK), SAP97. Complex formation involves the association of Cav-3 with a segment of SAP97 localized between its PDZ2
and PDZ3 domains. In heterologous expression systems, this scaffolding complex can recruit Kv1.5 to form a tripartite complex in
which each of the three components interacts with the other two.
These interactions regulate the expression of currents encoded by a
glycosylation-deficient mutant of Kv1.5. We conclude that the association of Cav-3 with SAP97 may constitute the nucleation site for the
assembly of macromolecular complexes containing potassium channels.
H682
SCAFFOLDING COMPLEX OF CAVEOLIN-3 AND SAP97
RESULTS
Cav-3 interacts directly with SAP97. Kv1.5 has been shown
to interact with SAP97 (18) and to target to caveolae (15) in
heterologous expression systems. These findings prompted us
AJP-Heart Circ Physiol • VOL
to hypothesize that SAP97 and Cav-3 form a scaffolding
complex capable of recruiting Kv1.5 and regulating its function. We first carried out coimmunoprecipitation and pulldown experiments to test whether SAP97 and Cav-3 form a
complex in the mouse heart. After SAP97 was immunoprecipitated from mouse ventricle lysates with the use of anti-PDZ
antibodies, the immunopellets were size separated by SDSPAGE and immunoblotted with anti-Cav-3 antibody. Figure
1A, lane 2, shows that anti-PDZ antibodies could coprecipitate
Cav-3. Control immunoprecipitations with IgG often yielded a
weak background signal corresponding to Cav-3 polypeptide
(Fig. 1A, lane 3). To provide additional evidence that this
complex exists in the heart, we made a cDNA construct
encoding a GST fusion polypeptide that contains the SAP97binding site of Kv1.5 (GST1.5C1; Fig. 1B). The fusion protein
was expressed in E. coli, purified by glutathione-agarose chromatography, and reacted with total mouse heart lysates. Figure
1B shows that GST1.5C1 coprecipitated SAP97 and Cav-3
from whole heart mouse extracts (lane 2), whereas control
GST did not precipitate either polypeptide (lane 3). A direct
association of GST1.5C1 with Cav-3 seems unlikely because
GST1.5C1 was unable to pull down Cav-3 in similar experiments with extracts from Cav-3-transfected COS-7 cells (not
shown). Therefore, the Cav-3 that was pulled down from heart
extracts by GST1.5C1 likely represents the fraction bound to
SAP97.
The interaction between SAP97 and Cav-3 was confirmed in
cotransfection experiments and in vitro. Figure 1C shows that
anti-PDZ antibody specifically coprecipitated Cav-3 (lane 8,
bottom), and anti-Cav-3 antibody specifically coprecipitated
SAP97 (lane 8, top) only from lysates of cotransfected cells but
not from mock-transfected cells (lane 7) or cells transfected
with either Cav-3 (lane 5) or SAP97 (lane 6) alone. We also
tested for direct interaction in an in vitro translation assay: after
T7-directed coexpression of SAP97 and Cav-3 cDNAs in
reticulocyte lysate, both polypeptides were coprecipitated with
anti-Cav-3 antibody (Fig. 1D, lane 4). Control precipitations
showed that SAP97 was not pulled down by anti-Cav-3 antibody in the absence of Cav-3 (lane 5) or when control IgG was
used in the precipitation (lane 6).
Cav-3-binding site of SAP97 resides in a region located
between PDZ2 and PDZ3. We next made a series of SAP97
deletion mutants to map the site of interaction with Cav-3.
Initially, we made three FLAG-tagged cDNA constructs: S1SAP97(451–926), which contained the PDZ3, SH3, and GUKlike domains (Fig. 2A); S2-SAP97(201–504), which contained
the PDZ1 and PDZ2 domains followed by a proline-rich region
and part of PDZ3 (Fig. 2A); and SAP97(1–200), which contained the NH2-terminal multimerization domain (not shown).
Each of these constructs was cotransfected with Cav-3 and
tested for interaction with it in coimmunoprecipitation assays
using anti-FLAG and anti-Cav-3 antibodies. Cav-3 specifically
coprecipitated with S2 (see below) but not with S1 (not
shown), indicating that the central segment of SAP97 is sufficient for binding to Cav-3. The expression of the NH2-terminal
fragment of SAP97 (amino acid residues 1–200) could not be
detected in this system.
To further define the domain in S2 involved in its interaction
with Cav-3, we made a series of deletion mutants in which we
kept PDZ1 and PDZ2 but removed the NH2 terminus (S3), the
COOH terminus (S4), or both (S5) (Fig. 2A). The mutants were
287 • AUGUST 2004 •
www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.3 on August 3, 2017
pEGFP-C3 in-frame with the GFP coding sequence. Kv1.5-N1-FL
(see Fig. 4B) was amplified by PCR in-frame with the FLAG epitope
coding sequence at the 3⬘-end and subcloned into pCDNA3. The
SAP97 deletion mutants S3 and S5 were amplified by PCR and
subcloned into pRSETB (Invitrogen) in-frame with the 6xHis tag
coding sequence. GST1.5C1 (see Fig. 1B) and GST1.5C2 (see Fig.
4B) were amplified by PCR and subcloned into pGEX-4T-1 in-frame
with the glutathione S-transferase (GST) coding sequence.
Cell lines and transfections. COS-7 and Chinese hamster ovary
(CHO) cells were transfected using Fugene 6 (Roche) and harvested
24 h posttransfection. We searched for a suitable expression system
for our biochemical and electrophysiological analyses. We chose
COS-7 cells for the biochemical analyses, because CHO cells express
an endogenous SAP97 that interacts with Kv1.5 and interferes with
coprecipitation assays (9). Thus the use of transfected COS-7 cells for
the biochemical characterization of the tripartite complex simplified
the interpretation of these experiments. Functional assays of transiently transfected COS-7 cells proved impractical, because variable
fractions of the transfected COS-7 cells expressed Kv1.5-encoded
potassium currents (18). By contrast, ⬎90% of the transfected CHO
cells expressed Kv1.5-encoded currents. Thus all functional studies
were done in CHO cells.
Protein expression, pull-down assays, and immunoprecipitations.
The expression of the 6xHis-tagged (S3 and S5) and GST fusion
(GST1.5C1 and GST1.5C2) proteins was carried out in Escherichia
coli strain BL21pLysS (Novagen) and DH5␣, respectively. After
induction of expression by the addition of 0.5 mM isopropyl-␤-Dthiogalactoside (Sigma), fusion proteins were affinity purified with
agarose-bound glutathione (for GST1.5C1 and GST1.5C2) or antiPDZ antibody (for S3 and S5). For pull-down assays and immunoprecipitations, cells were lysed with buffer A [50 mM Tris (pH 7.5),
150 mM NaCl, 1% Igepal, 0.1% SDS, and a mixture of protease
inhibitors] and centrifuged at 15,000 g for 10 min. For the pull-down
assays, cell extracts were precleared for 1 h and incubated overnight
with the corresponding agarose-bound affinity-purified fusion proteins. The beads were washed five times with buffer A, resuspended in
equal volume of 2⫻ sample buffer, boiled for 5 min, and subjected to
SDS-PAGE and immunoblotting. The immunoprecipitation experiments were carried out as described previously (18). For immunoblotting, samples were transferred to polyvinylidene difluoride
(PVDF) membranes, and the blots were probed with the indicated
antibodies. T7-directed cotranscription/cotranslation was carried out
in the TNT reticulocyte lysate expression system (Promega) in the
presence of L-[35S]methionine (Amersham Pharmacia Biotech). After
incubation for 90 min at 30°C, the lysates were diluted 20-fold with
buffer A, subjected to immunoprecipitation as described above, and
analyzed by SDS-PAGE and fluorography.
Electrophysiological studies. Recordings were made with an Axopatch-200B amplifier (Axon Instruments) using the standard whole
cell configuration of the patch-clamp technique (3, 18, 29). Briefly,
the pipette resistances were 2– 4 M⍀ when filled with (in mM) 50
KCl, 65 K-glutamate, 5 MgCl2, 5 EGTA, 10 HEPES, 5 K2-ATP, and
0.2 Tris-GTP (pH 7.2). The extracellular bath solution contained (in
mM) 140 NaCl, 5.4 KCl, 1 CaCl2, 1 MgCl2, 0.33 NaH2PO4, 7.5
glucose, and 5 HEPES (pH 7.4). The currents were recorded at room
temperature (21–23°C). The holding potential was ⫺70 mV; the test
potential ranged from ⫺60 to ⫹60 mV, lasting 400 ms; and the tail
currents were recorded at ⫺30 mV. Data are expressed as means ⫾
SE. ANOVA was applied for analyzing the multigroup data. Student’s
t-test was used to compare unpaired data between two groups, and a
two-tailed P ⬍ 0.05 was taken to indicate statistical significance.
SCAFFOLDING COMPLEX OF CAVEOLIN-3 AND SAP97
H683
tested for interaction with Cav-3 in cotransfection experiments.
Figure 2B shows that the deletion of the COOH terminus in S4
and S5 abrogated the interactions (lanes 7 and 8), whereas S2
and S3 interacted with Cav-3 with similar efficiency (lanes 5
and 6). These results indicate that the 99-amino acid segment
COOH terminal to PDZ2 (residues 406 –504; ⌬SAP) is necesAJP-Heart Circ Physiol • VOL
sary for the association of SAP97 and Cav-3. The sequence
between the COOH-terminal end of PDZ2 and the NH2terminal end of PDZ3 contains 10 serine and 2 threonine
residues, with at least 4 putative phosphorylation sites. Deletion of these sites (S4) eliminated the multiple bands observed
(Fig. 2B, compare lanes 1 and 2 vs. lanes 3 and 4).
287 • AUGUST 2004 •
www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.3 on August 3, 2017
Fig. 1. SAP97 interacts with caveolin-3 (Cav-3) in vivo and in vitro. A: coimmunoprecipitation assays from heart lysates. Extracts
from mouse ventricle (lane 1) were incubated with anti-PDZ (lane 2) or control IgG (lane 3), and the bound proteins were analyzed
by immunoblotting with anti-PDZ (top) and anti-Cav-3 (bottom). IP, immunoprecipitation. B: pull down of Cav-3 and SAP97 from
heart lysates using GST1.5C1, the GST-Kv1.5C-terminal fusion polypeptide shown in top scheme. Lysates from mouse ventricle
(lane 1) were incubated with agarose-bound GST1.5C1 (lane 2) or control glutathione S-transferase (GST; lane 3), and the bound
proteins were analyzed by immunoblotting with anti-PDZ (top blot) and anti-Cav-3 (middle blot). Bottom, Coomassie blue staining
of the recombinant proteins used in the assay. TM, transmembrane segment; P, pore. C: coimmunoprecipitation assays in COS-7
cells. Cells were cotransfected with different combinations of Cav-3, SAP97, and pCDNA3 vector (⫺) cDNAs. One-half of the cell
extracts were reacted with anti-Cav-3 (top right) and one-half with anti-PDZ (bottom right). The bound proteins were analyzed by
immunoblot with anti-PDZ (top, lanes 5– 8) or anti-Cav-3 (bottom, lanes 5– 8). Lanes 1– 4 are total cell extracts. The
immunoreactive endogenous polypeptide with a molecular weight similar to SAP97 (lanes 1 and 3, top) did not interact with Cav-3
(lane 5). D: coimmunoprecipitation assays from in vitro coexpressed Cav-3 and SAP97. Indicated cDNAs were transcribed and
translated in vitro in the presence of [35S]methionine, and the resulting polypeptides were immunoprecipitated with anti-Cav-3
(lanes 4 and 5) or control IgG (lane 6). Bound proteins were separated by SDS-PAGE and visualized by fluorography. Lanes 1–3
depict total reticulocyte lysates programmed with the indicated cDNAs.
H684
SCAFFOLDING COMPLEX OF CAVEOLIN-3 AND SAP97
We further confirmed the mapping of the Cav-3 binding site
of SAP97 in pull-down experiments with bacterially expressed
6xHis-tagged S3 and S5 deletion mutants (Fig. 2A). The
recombinant polypeptides were isolated from E. coli lysates by
immunoprecipitation with anti-PDZ antibody (Fig. 2C, lanes 1
and 2) and then reacted with equal amounts of extracts from
cells transfected with Cav-3. Consistent with our observations
in transfected cells, only S3, but not S5, could pull down Cav-3
(Fig. 2C, lanes 4 and 3, respectively).
To establish that ⌬SAP is necessary and sufficient for
binding to Cav-3, we made a cDNA construct encoding a
fusion protein in which ⌬SAP was linked to the COOH
terminus of GFP (GFP-⌬SAP, see scheme in Fig. 2A). Cotransfection experiments showed that GFP-⌬SAP coprecipiAJP-Heart Circ Physiol • VOL
tated with Cav-3 (Fig. 2D, lane 4), whereas GFP did not (lane
3), indicating that ⌬SAP is sufficient for binding to Cav-3.
Both lysates contained equal amounts of Cav-3 and GFP
polypeptides (Fig. 2D, lanes 1 and 2).
Kv1.5 is recruited to the Cav-3/SAP97 scaffolding complex.
Having established the formation of the Cav-3/SAP97 scaffolding complex, we next tested whether Kv1.5 could be
recruited to the complex by examining the formation of a
tripartite complex containing Kv1.5, Cav-3, and SAP97. After
performing triple transfections, we used specific antibodies to
immunoprecipitate each of the three polypeptides from total
cell extracts. Analysis of the immunoprecipitates by SDSPAGE and Western blotting revealed that each of the three
components of the putative tripartite complex coprecipitated
287 • AUGUST 2004 •
www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.3 on August 3, 2017
Fig. 2. Mapping of the Cav-3-binding site of SAP97. A: schematic representation of the various SAP97 mutants used. B:
coimmunoprecipitation of Cav-3 and several SAP97 truncation mutants. Cells were cotransfected with Cav-3 and S2 (lanes 1 and
5), S3 (lanes 2 and 6), S4 (lanes 3 and 7), or S5 (lanes 4 and 8) cDNAs. One-half of the cell lysates were reacted with anti-Cav-3
(top, lanes 5– 8) and one-half with anti-PDZ (bottom, lanes 5– 8), and the bound proteins were analyzed by immunoblotting with
anti-PDZ (top) or anti-Cav-3 (bottom). Lanes 1– 4 are total cell extracts. **Position of IgG light chain. C: pull down of Cav-3 from
cell lysates by recombinant S3. S3 and S5 were isolated from E. coli lysates by immunoprecipitation with anti-PDZ and then
incubated with equal portions of lysates from cells transfected with Cav-3 cDNA. Lanes 1 and 2 are recombinant polypeptides,
precipitated and blotted with anti-PDZ. Lanes 3 and 4 are immunoblot analyses using anti-Cav-3 of the polypeptides pulled down
with recombinant S5 and S3, respectively. D: coimmunoprecipitation of Cav-3 and GFP-⌬SAP. Cells were cotransfected with
Cav-3 and green fluorescent protein (GFP; lanes 1 and 3) or Cav-3 and GFP-⌬SAP (lanes 2 and 4), and the cell lysates were
immunoprecipitated with anti-GFP. Lanes 3 and 4 are immunoblot analyses using anti-Cav-3 of the polypeptides coprecipitated
with GFP or GFP-⌬SAP, respectively. Lanes 1 and 2 represent the immunoblot analysis of total lysates using anti-Cav-3 (top) and
anti-GFP (bottom).
SCAFFOLDING COMPLEX OF CAVEOLIN-3 AND SAP97
with the other two polypeptides (Fig. 3A, lanes 1–3). No signal
was detected in a control reaction with nonimmune mouse IgG
(Fig. 3A, lane 4), indicating that the observed coimmunoprecipitations were specific.
The T-D-L sequence located at the COOH terminus of
Kv1.5 is critical for its association with SAP97, because this
interaction was abolished in FL-Kv1.5-A, which has a mutation in the three COOH-terminal amino acid residues of Kv1.5
(T-D-L to A-A-A) (18). We reasoned that if SAP97, Cav-3,
and FL-Kv1.5-A were all present in a tripartite complex,
FL-Kv1.5-A might coimmunoprecipitate with SAP97 even
though the direct interaction between the two proteins was
AJP-Heart Circ Physiol • VOL
abrogated. Triple transfection experiments showed that this
was indeed the case (Fig. 3B): FL-Kv1.5-A coprecipitated with
SAP97 in the presence (lane 7) but not in the absence (lane 6)
of coexpressed Cav-3. Lanes 5 and 8 of Fig. 3B show the
positive (FL-Kv1.5) and negative (no SAP97) controls of the
experiment, respectively. This result further confirms the formation of the tripartite complex in this system and suggests
that Kv1.5 may bind directly to both SAP97 and Cav-3.
Kv1.5 interacts with Cav-3 through a region located at the
NH2 terminus. We next designed a series of cotransfection
experiments to confirm the specific association of Cav-3 with
Fl-Kv1.5. Cotransfection of Cav-3 with FL-Kv1.5 (Fig. 4A,
lanes 1–7) resulted in coprecipitation of Cav-3 and the channel
using antibodies to both Cav-3 (lane 4) and FLAG epitope
(lane 6). By contrast, cotransfection of Cav-3 with Myc-Kv2.1,
a channel known to target to noncaveolar lipid rafts, did not
result in coprecipitation using anti-Cav-3 antibodies (Fig. 4A,
lane 10). We therefore concluded that Kv1.5 specifically associates with Cav-3.
We made a series of Kv1.5 mutants to delineate the site of
its interaction with Cav-3 (Fig. 4B). The long NH2- and
COOH-terminal regions of Kv1.5 reside in the cytoplasmic
side of the plasma membrane and are therefore candidates for
the site of interaction with Cav-3, which localizes to the inner
leaflet of the plasma membrane. Several lines of evidence
indicated that the COOH-terminal cytoplasmic region of Kv1.5
is not involved in the interaction with Cav-3. First, in cotransfection experiments, a deletion mutant encoding a truncated
FL-Kv1.5 polypeptide lacking the 86 COOH-terminal amino
acids (TrFL-Kv1.5, Fig. 4B) was able to bind Cav-3 as efficiently as full-length FL-Kv1.5. Second, a cotransfected fusion
protein containing the entire COOH-terminal region of Kv1.5
linked to GFP (GFP1.5C, Fig. 4B) failed to bind Cav-3.
Finally, a GST fusion protein containing the entire COOHterminal region of Kv1.5 (GST1.5C2, Fig. 4B), expressed in E.
coli and purified by glutathione-agarose affinity chromatography, failed to pull down Cav-3 from lysates of transfected cells
(results not shown).
To test for interaction of the NH2 terminus with Cav-3, we
made a cDNA construct encoding a membrane-anchored
polypeptide (1) containing the entire NH2-terminal cytoplasmic region of Kv1.5, followed by the first transmembrane
segment (TM1), 18 amino acid residues corresponding to the
first extracellular loop, and a COOH-terminal FLAG tag
(Kv1.5N1-FL, Fig. 4B). Cotransfection of Cav-3 with
Kv1.5N1-FL (Fig. 4C) resulted in coprecipitation of Cav-3 and
the truncated channel with antibodies to both Cav-3 (Fig. 4C,
top, lane 6) and FLAG epitope (Fig. 4C, bottom, lane 7),
indicating that the Cav-3-binding site of Kv1.5 resides in the
NH2-terminal portion of the molecule.
Cav-3/SAP97 scaffolding complex selectively regulates the
function of a glycosylation-deficient mutant of FL-Kv1.5. The
above results indicate that multiple interactions are involved in
the formation of the Kv1.5-SAP97-Cav-3 complex, in which
each component binds the other two. We next designed a series
of electrophysiological experiments to functionally validate
our biochemical findings. An examination of the subcellular
localization of the expressed polypeptides in triple transfection
experiments in CHO cells revealed an extensive colocalization
of Cav-3 with both Kv1.5 and SAP97, primarily at the level of
the plasma membrane (not shown). Figure 5A shows that
287 • AUGUST 2004 •
www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.3 on August 3, 2017
Fig. 3. Formation of a tripartite complex containing SAP97, Cav-3, and
FL-Kv1.5. A: coimmunoprecipitation of SAP97, FL-Kv1.5, and Cav-3. Lysates from two 100-mm dishes of triple-transfected cells were pooled, divided
in 4 portions, and immunoprecipitated with anti-PDZ, anti-FLAG, anti-Cav-3,
or nonimmune control IgG. Bound proteins were analyzed by immunoblotting
with anti-PDZ (top), anti-FLAG (middle), and anti-Cav-3 (bottom). Lane 5 is
a Western blot of the total cell extract. B: coimmunoprecipitation of SAP97
and FL-Kv1.5-A in the presence of Cav-3. Cells were cotransfected with
different combinations of SAP97, FL-Kv1.5, FL-Kv1.5-A, and pCDNA3
vector (⫺) cDNAs. Cell extracts were reacted with anti-PDZ (lanes 5– 8), and
the bound proteins were analyzed by immunoblotting with anti-PDZ (top),
anti-FLAG (middle), and anti-Cav-3 (bottom). Lanes 1– 4 are immunoblots of
total cell extracts. The immunoreactive endogenous polypeptide with a molecular weight similar to SAP97 (lanes 4 and 8, top) did not interact with
FL-Kv1.5 or Cav-3 (lane 8).
H685
H686
SCAFFOLDING COMPLEX OF CAVEOLIN-3 AND SAP97
FL-Kv1.5 transfected into CHO cells codes for rapidly activating outward currents.
The cotransfection of Cav-3 with FL-Kv1.5 did not lead to
changes in current density (not shown). During our studies on
the effect of protein glycosylation on Kv1.5 function, we
created a Ser292-to-Gly mutation that codes for a glycosylationdeficient mutant of the channel (FL-Kv1.5Glyc⫺). Transfection
of FL-Kv1.5Glyc⫺ revealed that it coded for one-half the
current density of FL-Kv1.5 (Fig. 5, A–C), with a shift to the
Fig. 4. Kv1.5 interacts with Cav-3 through a region located at the NH2
terminus. A: coimmunoprecipitation of Cav-3 with FL-Kv1.5 coexpressed in
transfected COS-7 cells. In lanes 1–7, cells were cotransfected with different
combinations of Cav-3, FL-Kv1.5, and pCDNA3 vector (⫺) cDNAs. One-half
of the cell extracts were reacted with anti-Cav-3 (lanes 4 and 5) and one-half
with anti-FLAG (lanes 6 and 7). Bound proteins were analyzed by immunoblotting with anti-FLAG (top) or anti-Cav-3 (bottom). Lanes 1–3 are total cell
extracts. In lanes 8 –11, cells were cotransfected with Myc-Kv2.1 and Cav-3
(lanes 8 and 10) or Myc-Kv2.1 and pCDNA3 vector (⫺) (lanes 9 and 11)
cDNAs. Cell extracts were reacted with anti-Cav-3, and the bound proteins
were analyzed by immunoblotting with anti-Myc (lanes 10 and 11). Lanes 8
and 9 are total cell extracts. B: schematic representation of the various Kv1.5
fusion and truncation mutants. C: coimmunoprecipitation of Kv1.5N1-FL and
Cav-3. Cells were cotransfected with different combinations of Kv1.5N1-FL,
Cav-3, FL-Kv1.5, and pCDNA3 vector (⫺) cDNAs. One-half of the cell
extracts were reacted with anti-Cav-3 (top, lanes 5–7) and one-half with
anti-FLAG (bottom, lanes 5–7). Bound proteins were analyzed by immunoblot
with anti-FLAG (top) or anti-Cav-3 (bottom). Lanes 1– 4 are total cell extracts.
AJP-Heart Circ Physiol • VOL
287 • AUGUST 2004 •
www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.3 on August 3, 2017
right of the voltage dependence of steady-state activation (Fig.
5D). These results suggest that FL-Kv1.5Glyc⫺, similar to the
Shaker and Kv1.1 channels, may have lower membrane expression than FL-Kv1.5. This mutant was recruited to the
Cav-3/SAP97 scaffolding complex similarly to FL-Kv1.5
(Fig. 5E).
In striking contrast with the lack of effect of Cav-3 on the
wild-type channel, cotransfection of increasing amounts of
Cav-3 cDNA inhibited up to 90% of the FL-Kv1.5Glyc⫺encoded currents (from 312.3 ⫾ 22.6 to 33.1 ⫾ 6.1 pA/pF, P ⬍
0.001; Fig. 6A). Moreover, Cav-3 shifted the voltage dependence of the steady-state activation curve of the channel to the
left (Fig. 6B). These results suggest that the decrease in the
macroscopic Kv1.5-encoded currents due to the coexpression
with Cav-3 (Fig. 6A) was not due to a modification of the
gating properties of Kv1.5, because such a modification would
have led to an increase in current. Rather, the marked reduction
of the outward currents most likely reflects a decrease in the
number of functional Kv1.5 channels on the cell surface.
We next tested whether cotransfection of exogenous SAP97
could abrogate the suppressive effect of Cav-3 on FLKv1.5Glyc⫺-encoded currents. Figure 6C shows that the cotransfection of SAP97 with FL-Kv1.5Glyc⫺ and Cav-3 abolished the inhibitory effect of Cav-3. We therefore hypothesized
that SAP97, through its high-affinity interactions with the
COOH terminus of Kv1.5 and Cav-3, competed with an endogenous PDZ domain-containing protein that mediated the
inhibition and rescued the basal current level. To test the
hypothesis that SAP97 interactions with Cav-3 are critical for
the rescue, we coexpressed FL-Kv1.5Glyc⫺ with SAP97,
Cav-3, and GFP-⌬SAP, the GFP fusion protein that contains
the segment of SAP97 that interacts with Cav-3 (see Fig. 2A).
GFP-⌬SAP abolished the rescue by SAP97 (Fig. 6D), providing functional proof that this segment is important for SAP97/
Cav-3 association and that these interactions are crucial for
abrogating the inhibitory effect of Cav-3.
We next mutated the COOH-terminal T-D-L of FLKv1.5Glyc⫺ to A-A-A to confirm that an endogenous MAGUK
protein was involved in the inhibition of channel function by
Cav-3. This mutant (FL-Kv1.5Glyc⫺A) binds normally to
Cav-3 but does not interact with PDZ-containing proteins (not
shown). In contrast to the observed inhibition of FLKv1.5Glyc⫺ activity by Cav-3, the cotransfection of Cav-3 with
FL-Kv1.5Glyc⫺A did not cause any decrease in the FL-
SCAFFOLDING COMPLEX OF CAVEOLIN-3 AND SAP97
H687
Kv1.5Glyc⫺A⫺-encoded currents (Fig. 6E). This result confirms that the observed inhibition of FL-Kv1.5Glyc⫺ by Cav-3
was mediated by an endogenous MAGUK protein, and that the
interaction with Cav-3 alone was not sufficient to inhibit
channel activity. This conclusion is further supported by the
finding that the coexpression of FL-Kv1.5Glyc⫺ and Cav-3 with
GFP1.5C, a GFP fusion protein containing the whole COOHterminal cytoplasmic tail of Kv1.5 (see scheme in Fig. 4B), also
abolished the inhibitory effect of Cav-3 on FL-Kv1.5Glyc⫺
(Fig. 6F). This fusion protein likely competed with FLAJP-Heart Circ Physiol • VOL
Kv1.5Glyc⫺ for the endogenous PDZ-containing protein that
mediates the inhibition. It is important to note that the interaction of Kv1.5 with the endogenous MAGUK did not exert a
direct effect on Kv1.5 activity, because the mutation of the
COOH terminus of Kv1.5 to A-A-A had no apparent effect on
the overall channel-encoded currents [Fig. 6, compare FLKv1.5Glyc⫺ (A) and FL-Kv1.5Glyc⫺A (E)]. Collectively, these
results support the notion that the Cav-3/SAP97 complexes can
recruit Kv1.5 to form tripartite complexes that may regulate
channel function.
287 • AUGUST 2004 •
www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.3 on August 3, 2017
Fig. 5. Expression of FL-Kv1.5- and FL-Kv1.5Glyc⫺-encoded currents. A and B: representative current traces from
Chinese hamster ovary cells transfected with either 0.1 ␮g
FL-Kv1.5 (A) or 0.1 ␮g FL-Kv1.5Glyc⫺ (B). C: currentvoltage (I-V) relationships at the end of the test pulse. The
glycosylation-deficient mutant encodes smaller currents
(697.6 ⫾ 80.1 vs. 312.3 ⫾ 22.6 pA/pF at ⫹60 mV, P ⬍
0.001). D: steady-state activation of FL-Kv1.5 and
FL-Kv1.5Glyc⫺ determined by tail current analyses [E, voltage at half-maximal activation (V1/2) ⫺2.8 ⫾ 1.3 mV, n ⫽
15; F, V1/2 3.18 ⫾ 0.6 mV, n ⫽ 10; P ⬍ 0.001]. The
mutation shifted the voltage dependence of steady-state
activation to the right. Data are expressed as means ⫾ SE.
Student’s t-test and ANOVA were used to evaluate the
statistical significance. E: coimmunoprecipitation assays
from COS-7 cells cotransfected with Cav-3, SAP97, and
FL-Kv1.5Glyc⫺. Anti-FLAG antibody precipitated the tripartite complex including SAP97 and Cav-3 only in the
presence of the channel (lane 4) but not in its absence (lane
3). Immunoblots with anti-PDZ antibody (top), anti-FLAG
antibody (middle), and anti-Cav-3 (bottom) are shown.
H688
SCAFFOLDING COMPLEX OF CAVEOLIN-3 AND SAP97
DISCUSSION
In neuronal and epithelial cells, the polarized expression of
membrane proteins relies largely on scaffolding proteins,
which dictate the rules for the organization of large complexes
involving specific interactions. Scaffolding proteins often interact with one another, thus providing multiple binding sites
for complex assembly. Examples of these interactions are the
association of the PDZ-containing proteins CASK, Mint1, and
Velis at the synaptic junction (4) and the recruitment of SAP97
to the lateral surface of epithelial cells through its interaction
with CASK (13). Similar principles possibly govern the assembly of macromolecular complexes in the heart. We propose
that the interaction of Cav-3 with SAP97 may form the nucleation site for the assembly of a macromolecular complex that
contains ion channels, receptors, and signaling molecules involved in electrical impulse propagation. Our results provide
evidence for the recruitment of Kv1.5 to the Cav-3/SAP97
AJP-Heart Circ Physiol • VOL
scaffolding complex in heterologous expression systems and
suggest that protein interactions within this complex can regulate channel function.
We mapped the Cav-3 binding site of SAP97 to a segment
located COOH terminal to PDZ2. The results of a BLAST
search that used this fragment as query revealed that it contains
a serine- and proline-rich region (amino acid residues 430 –
447) that has a significant degree of sequence similarity with
two caveolar enzymes: ceramidase (24) and adenylyl cyclase type V (25). Thus this segment may represent a
previously unrecognized caveolin-binding domain present
in various proteins. In a molecular model of SAP97 created
by Wu et al. (28), the Cav-3-binding site is predicted to be
a ␤-turn readily accessible from the solvent, facing the
exterior of the molecule. It is interesting that this ␤-turn
forms a lid over the PDZ2 peptide-binding pocket, suggesting that the channel interaction with PDZ2 and the binding
287 • AUGUST 2004 •
www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.3 on August 3, 2017
Fig. 6. I-V curves of FL-Kv1.5Glyc⫺. A:
Cav-3 inhibits FL-Kv1.5Glyc⫺-encoded currents in a dose-dependent manner. Control
transfection of 0.1 ␮g FL-Kv1.5Glyc⫺
(312.3 ⫾ 22.6 pA/pF, n ⫽ 18) and cotransfection of 0.1 ␮g FL-Kv1.5Glyc⫺ with 0.01
␮g Cav-3 (356.9 ⫾ 32.6 pA/pF, n ⫽ 18), 0.1
␮g Cav-3 (106.5 ⫾ 17.4 pA/pF, n ⫽ 20), or
1.0 ␮g Cav-3 (33.1 ⫾ 6.1 pA/pF, n ⫽ 21)
(P ⬍ 0.001) are shown. B: Cav-3 shifts the
voltage activation curve to the left. FLKv1.5Glyc⫺ alone (0.1 ␮g) (n ⫽ 10) and 0.1
␮g FL-Kv1.5Glyc⫺ with 0.1 ␮g Cav-3 (n ⫽
12) (V1/2 of ⫺2.0 ⫾ 1.5 vs. 3.2 ⫾ 0.6 mV;
P ⬍ 0.05) are shown. C: SAP97 abrogates
the inhibitory effect of Cav-3. FLKv1.5Glyc⫺ alone (0.1 ␮g) (312.3 ⫾ 22.6
pA/pF, n ⫽ 18), 0.1 ␮g FL-Kv1.5Glyc⫺ cotransfected with 0.1 ␮g Cav-3 (106.5 ⫾ 17.4
pA/pF, n ⫽ 20), and 0.1 ␮g FL-Kv1.5Glyc⫺
cotransfected with 0.1 ␮g Cav-3 and 1.0 ␮g
SAP97 [317.8 ⫾ 41.5 pA/pF, n ⫽ 24; P ⫽
not significant (NS) vs. control] are shown.
D: SAP97 did not abolish Cav-3 inhibition
in the presence of GFP-⌬SAP. FLKv1.5Glyc⫺ alone (0.1 ␮g) (312.3 ⫾ 22.6
pA/pF, n ⫽ 18); 0.1 ␮g FL-Kv1.5Glyc⫺ cotransfected with 2.0 ␮g GFP-⌬SAP
(261.7 ⫾ 41.1 pA/pF, n ⫽ 11); 0.1 ␮g
FL-Kv1.5Glyc⫺ cotransfected with 0.1 ␮g
Cav-3 and 2.0 ␮g GFP-⌬SAP (132.4 ⫾ 19.0
pA/pF, n ⫽ 13); and 0.1 ␮g FL-Kv1.5Glyc⫺
cotransfected with 1.0 ␮g SAP97, 0.1 ␮g
Cav-3, and 2.0 ␮g GFP-⌬SAP (170.2 ⫾
29.5 pA/pF, n ⫽ 15; P ⫽ NS) are shown. E:
0.1 ␮g Cav-3 did not inhibit the currents
encoded by 0.1 ␮g FL-Kv1.5Glyc⫺A
(291.7 ⫾ 43.0 vs. 402.8 ⫾ 48.3 pA/pF; P ⫽
0.09). F: GFP1.5C abrogates the Cav-3 inhibition. Cotransfection of 0.1 ␮g FLKv1.5Glyc⫺ with 1.0 ␮g GFP1.5C (293.0 ⫾
65.1 pA/pF, n ⫽ 7) or with 1.0 ␮g GFP1.5C
and 0.1 ␮g Cav-3 (279.4 ⫾ 45.1 pA/pF, n ⫽
10; P ⫽ NS) are shown. Data are means ⫾
SE. Student’s t-test and ANOVA were used
to evaluate the statistical significance. Current density is in pA/pF at ⫹60 mV. The
amount of DNA in all transfections was kept
constant with vector DNA (pcDNA3).
SCAFFOLDING COMPLEX OF CAVEOLIN-3 AND SAP97
AJP-Heart Circ Physiol • VOL
ACKNOWLEDGMENTS
We thank Thomas Michel and Barbara Wible for Myc-tagged Cav-3 and
Kv2.1 cDNAs, respectively. We also thank Giselle Martı́nez Nöel for contributing to the experiment shown in Fig. 1B.
GRANTS
G. Koren is a recipient of grants from the National Heart, Lung, and Blood
Institute.
REFERENCES
1. Babila T, Moscucci A, Wang H, Weaver FE, and Koren G. Assembly
of mammalian voltage-gated potassium channels: evidence for an important role of the first transmembrane segment. Neuron 12: 615– 626, 1994.
2. Bennett E, Urcan MS, Tinkle SS, Koszowski AG, and Levinson SR.
Contribution of sialic acid to the voltage dependence of sodium channel
gating. A possible electrostatic mechanism. J Gen Physiol 109: 327–343,
1997.
3. Brunner M, Guo W, Mitchell GF, Buckett PD, Nerbonne JM, and
Koren G. Characterization of mice with a combined suppression of I(to)
and I(K,slow). Am J Physiol Heart Circ Physiol 281: H1201–H1209,
2001.
4. Butz S, Okamoto M, and Sudhof TC. A tripartite protein complex with
the potential to couple synaptic vesicle exocytosis to cell adhesion in
brain. Cell 94: 773–782, 1998.
5. Craven SE and Bredt DS. PDZ proteins organize synaptic signaling
pathways. Cell 93: 495– 498, 1998.
6. Eldstrom J, Choi WS, Steele DF, and Fedida D. SAP97 increases Kv1.5
currents through an indirect N-terminal mechanism. FEBS Lett 547:
205–211, 2003.
7. Galbiati F, Engelman JA, Volonte D, Zhang XL, Minetti C, Li M, Hou
H Jr, Kneitz B, Edelmann W, and Lisanti MP. Caveolin-3 null mice
show a loss of caveolae, changes in the microdomain distribution of the
dystrophin-glycoprotein complex, and t-tubule abnormalities. J Biol Chem
276: 21425–21433, 2001.
8. Garner CC, Nash J, and Huganir RL. PDZ domains in synapse
assembly and signalling. Trends Cell Biol 10: 274 –280, 2000.
9. Godreau D, Vranckx R, Maguy A, Goyenvalle C, and Hatem SN.
Different isoforms of synapse-associated protein, SAP97, are expressed in
the heart and have distinct effects on the voltage-gated K⫹ channel Kv1.5.
J Biol Chem 278: 47046 – 47052, 2003.
10. Godreau D, Vranckx R, Maguy A, Rucker-Martin C, Goyenvalle C,
Abdelshafy S, Tessier S, Couetil JP, and Hatem SN. Expression,
regulation and role of the MAGUK protein SAP-97 in human atrial
myocardium. Cardiovasc Res 56: 433– 442, 2002.
11. Hagiwara Y, Sasaoka T, Araishi K, Imamura M, Yorifuji H, Nonaka
I, Ozawa E, and Kikuchi T. Caveolin-3 deficiency causes muscle
degeneration in mice. Hum Mol Genet 9: 3047–3054, 2000.
12. Khanna R, Myers MP, Laine M, and Papazian DM. Glycosylation
increases potassium channel stability and surface expression in mammalian cells. J Biol Chem 276: 34028 –34034, 2001.
13. Lee S, Fan S, Makarova O, Straight S, and Margolis B. A novel and
conserved protein-protein interaction domain of mammalian Lin-2/CASK
binds and recruits SAP97 to the lateral surface of epithelia. Mol Cell Biol
22: 1778 –1791, 2002.
14. Leonoudakis D, Mailliard W, Wingerd K, Clegg D, and Vandenberg
C. Inward rectifier potassium channel Kir2.2 is associated with synapseassociated protein SAP97. J Cell Sci 114: 987–998, 2001.
15. Martens JR, Sakamoto N, Sullivan SA, Grobaski TD, and Tamkun
MM. Isoform-specific localization of voltage-gated K⫹ channels to distinct lipid raft populations. Targeting of Kv15 to caveolae. J Biol Chem
276: 8409 – 8414, 2001.
16. McNally EM, de Sa Moreira E, Duggan DJ, Bonnemann CG, Lisanti
MP, Lidov HG, Vainzof M, Passos-Bueno MR, Hoffman EP, Zatz M,
and Kunkel LM. Caveolin-3 in muscular dystrophy. Hum Mol Genet 7:
871– 877, 1998.
17. Minetti C, Sotgia F, Bruno C, Scartezzini P, Broda P, Bado M, Masetti
E, Mazzocco M, Egeo A, Donati MA, Volonte D, Galbiati F, Cordone
G, Bricarelli FD, Lisanti MP, and Zara F. Mutations in the caveolin-3
gene cause autosomal dominant limb-girdle muscular dystrophy. Nat
Genet 18: 365–368, 1998.
18. Murata M, Buckett PD, Zhou J, Brunner M, Folco E, and Koren G.
SAP97 interacts with Kv1.5 in heterologous expression systems. Am J
Physiol Heart Circ Physiol 281: H2575–H2584, 2001.
287 • AUGUST 2004 •
www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.3 on August 3, 2017
of Cav-3 to the ␤-turn may be mutually regulated. Furthermore, the occurrence of multiple potential phosphorylation
sites in this segment raises the possibility that the interaction
of SAP97 and Cav-3 is dynamic.
In this study and in our previous work (18) we have
demonstrated that the T-D-L sequence at the COOH terminus
of Kv1.5 is essential for its interaction with SAP97. These
results differ from those recently reported by Eldstrom et al. (6)
and presumably reflect differences in the experimental systems
used (COS-7 vs. human embryonic kidney cells, FLAG-tagged
rKv1.5 vs. T7-tagged hKv1.5) that are likely related to the
presence of distinct sets of endogenous PDZ proteins and
SAP97 isoforms in different cell types. Interestingly, Eldstrom
et al. show that the NH2 terminus of Kv1.5, which we show to
interact with Cav-3, is necessary for the regulation of channel
function by SAP97 and suggest that both Kv1.5 and SAP97 are
in a complex with ␣-actinin. Those results and ours highlight
the importance of the formation of complexes involving multiple protein interactions for channel regulation.
Our observation that Cav-3 selectively inhibits the currents
elicited by glycosylation-deficient Kv1.5 channels is puzzling.
N-linked glycosylation is a ubiquitous modification of potassium channels. Previous studies suggested that this cotranslational modification may control folding, trafficking, and stability of these channels (2). However, the exact role may vary
among different channels. For example, glycosylation reflects
surface expression of HERG (23, 30), alters the voltage dependence of activation of Kv1.1 and KvLQT1/minK, and
modulates the open probability of ROMK1 (20, 31). We and
other investigators have shown that N-linked glycosylation is
not essential for surface expression of Kv1.1 and Shaker
channels in Xenopus oocytes (1). However, recently Khanna et
al. (12) showed that unglycosylated Shaker mutants have a
faster turnover rate and reduced surface expression due to rapid
degradation by the proteasome pathway (12). We are currently
investigating the mechanism underlying the selective downregulation of FL-Kv1.5Glyc⫺ by Cav-3. Our working hypothesis is that the Cav-3/endogenous MAGUK complex plays a
role in quality control of channel assembly, mediating the
selective retention of FL-Kv1.5Glyc⫺ in the secretory pathway
and targeting it for degradation. Alternatively, the complex
may promote the selective internalization of FL-Kv1.5Glyc⫺
from the plasma membrane. However, we cannot rule out the
possibility that the high level of surface expression of wildtype Kv1.5 may somehow mask an eventual regulation of this
channel by Cav-3 as well, an effect that could be unmasked in
the case of the glycosylation-deficient mutant because it has a
lower level of surface expression.
In summary, our results show that a scaffolding complex
containing Cav-3 and SAP97 can recruit Kv1.5 by means of
multiple protein-protein interactions. Furthermore, we show
that this scaffolding complex regulates the expression of currents encoded by a glycosylation-deficient mutant of Kv1.5. In
excitable cells, the targeting of ion channels to appropriate
membrane subdomains and their organization in multiprotein
complexes are critical for cell excitation. Our results represent
a first step towards the elucidation of the assembly of potassium channel-containing complexes (channelosomes) in the
heart.
H689
H690
SCAFFOLDING COMPLEX OF CAVEOLIN-3 AND SAP97
AJP-Heart Circ Physiol • VOL
26. Tiffany AM, Manganas LN, Kim E, Hsueh YP, Sheng M, and Trimmer JS. PSD-95 and SAP97 exhibit distinct mechanisms for regulating
K(⫹) channel surface expression and clustering. J Cell Biol 148: 147–158,
2000.
27. Woodman SE, Park DS, Cohen AW, Cheung MW, Chandra M,
Shirani J, Tang B, Jelicks LA, Kitsis RN, Christ GJ, Factor SM,
Tanowitz HB, and Lisanti MP. Caveolin-3 knock-out mice develop a
progressive cardiomyopathy and show hyperactivation of the p42/44
MAPK cascade. J Biol Chem 277: 38988 –38997, 2002.
28. Wu H, Reissner C, Kuhlendahl S, Coblentz B, Reuver S, Kindler S,
Gundelfinger ED, and Garner CC. Intramolecular interactions regulate
SAP97 binding to GKAP. EMBO J 19: 5740 –5751, 2000.
29. Zhou J, Jeron A, London B, Han X, and Koren G. Characterization of
a slowly inactivating outward current in adult mouse ventricular myocytes.
Circ Res 83: 806 – 814, 1998.
30. Zhou Z, Gong Q, Epstein ML, and January CT. HERG channel
dysfunction in human long QT syndrome. Intracellular transport and
functional defects. J Biol Chem 273: 21061–21066, 1998.
31. Zhu J, Watanabe I, Gomez B, and Thornhill WB. Determinants
involved in Kv1 potassium channel folding in the endoplasmic reticulum,
glycosylation in the Golgi, and cell surface expression. J Biol Chem 276:
39419 –39427, 2001.
287 • AUGUST 2004 •
www.ajpheart.org
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.3 on August 3, 2017
19. Okamoto T, Schlegel A, Scherer PE, and Lisanti MP. Caveolins, a
family of scaffolding proteins for organizing “preassembled signaling
complexes” at the plasma membrane. J Biol Chem 273: 5419 –5422, 1998.
20. Pabon A, Chan KW, Sui JL, Wu X, Logothetis DE, and Thornhill
WB. Glycosylation of GIRK1 at Asn119 and ROMK1 at Asn117 has
different consequences in potassium channel function. J Biol Chem 275:
30677–30682, 2000.
21. Parton RG, Way M, Zorzi N, and Stang E. Caveolin-3 associates with
developing T-tubules during muscle differentiation. J Cell Biol 136:
137–154, 1997.
22. Pawson T and Scott JD. Signaling through scaffold, anchoring, and
adaptor proteins. Science 278: 2075–2080, 1997.
23. Petrecca K, Atanasiu R, Akhavan A, and Shrier A. N-linked glycosylation sites determine HERG channel surface membrane expression.
J Physiol 515: 41– 48, 1999.
24. Romiti E, Meacci E, Tanzi G, Becciolini L, Mitsutake S, Farnararo M,
Ito M, and Bruni P. Localization of neutral ceramidase in caveolinenriched light membranes of murine endothelial cells. FEBS Lett 506:
163–168, 2001.
25. Rybin VO, Xu X, Lisanti MP, and Steinberg SF. Differential targeting
of beta-adrenergic receptor subtypes and adenylyl cyclase to cardiomyocyte caveolae. A mechanism to functionally regulate the cAMP signaling
pathway. J Biol Chem 275: 41447– 41457, 2000.