From the
Department of Microbiology and Immunology,
School of Medicine, and ¶Institute of Molecular
Cardiology, Medical Biotechnology Center, University of Maryland Biotechnology
Institute, University of Maryland, Baltimore, Maryland 21201
Received for publication, January 23, 2003 , and in revised form, May 7, 2003.
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ABSTRACT |
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INTRODUCTION |
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Early studies suggested that ATP-dependent regulation of NCX1 occurred in squid axons (24, 25) and in cardiac sarcolemmal vesicles (26), but these studies did not distinguish between direct ATP binding and ATP-dependent phosphorylation. Several studies were designed to resolve this problem. Hilgemann and colleagues (11, 12, 27, 28) investigated the question by measuring NCX1 currents in giant excised patches. The work used two preparations, NCX1-expressing Xenopus oocytes (27) or cardiac myocytes expressing native NCX1 (11, 12, 28). These investigations found no functional change in cardiac NCX1 activity following application of PKA or protein kinase C (PKC) catalytic subunits to the intracellular side of the giant patch. Condrescu et al. (19) came to similar conclusions by examining the phosphorylation state of NCX1 in a heterologous expression system (19). Less direct investigations, however, suggested that PKA affected NCX1 function in heart (29, 30). The indirect nature of these studies in support of phosphorylation did not exclude the possibility that the measured changes were due to PKA phosphorylation of other proteins. The first unambiguous demonstration that phosphorylation affected NCX1 was provided by Iwamoto et al. (31). They showed that NCX1 from rat aorta smooth muscle cells is phosphorylated by PKC and is activated in response to growth factors (32). Further studies demonstrated that cardiac NCX1 is regulated by PKC phosphorylation (20). Additionally, Iwamoto et al. (20) provided evidence that the intracellular loop of NCX1 was phosphorylated by PKC and PKA. We were the first to show that PKA-dependent phosphorylation of NCX1 increased NCX1 activity both in Xenopus oocytes expressing cardiac NCX1 and in adult rat ventricular cardiomyocytes (33, 34). To determine how such divergent conclusions could arise, we undertook an investigation of the molecular organization of NCX1 in heart.
Regulation of NCX1 could arise through cytosolic enzymes bathing NCX1 or through local clustering of the regulatory proteins. The variable loss of cytosolic regulatory proteins during experiments on NCX1 phosphorylation could, in principle, account for the divergent findings. Alternatively, if NCX1 was part of a macromolecular complex that included other regulatory proteins, differences in experimental results may reflect the different levels of expression and activity of associated proteins under different experimental conditions. Recently, local signaling complexes have been shown to regulate ion channels similar the L-type Ca+ channel (35) and specific K+ channels (36) and cardiac ryanodine receptors (RyR2) (37). These complexes are composed of kinases, phosphatases, and kinase-anchoring proteins (AKAPs) and regulate activation state, substrate specificity, and subcellular localization. Recently, it was shown that the RyR2 could be more rapidly de-phosphorylated by the phosphatases of the macromolecular cluster than the kinases phosphorylated RyR2 (38). This recent observation and the large number of Ca2+-regulatory proteins associated with macromolecular complexes that regulate function suggested that this hypothesis is particularly appealing for NCX1. The experiments that we present below examine the macromolecular complex hypothesis for NCX1.
Recently, we have shown that phosphorylation of NCX1 is induced by
-adrenergic stimulation in pig heart resulting increased NCX1 exchanger
current (INCX). More importantly, we found that NCX1 is
hyperphosphorylated in pig model of heart failure but
-adrenergic
response is attenuated in heart failure
(39). These findings
underscore the significance of NCX1 phosphorylation in pathological heart, and
we have investigated the regulation of NCX1 phosphorylation in this paper.
Preliminary findings of this work were presented in abstract form previously
(40).
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EXPERIMENTAL PROCEDURES |
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Immunoprecipitation and in Vitro Phosphorylation
The cardiac lysate was incubated with NCX polyclonal antibody
1113 (Swant, Bellinzona, Switzerland) overnight, and the
antigen-antibody complex was precipitated by mixing with protein A-Sepharose
beads (Sigma). To identify the full-length mAKAP, immunoprecipitation was done
using monoclonal NCX1 antibody (R3F1, Swant). The complex was washed in buffer
containing 150 mM NaCl, 6 mM EDTA, 50 mM Tris
(pH 7.4), 0.1% Triton X-100, and 0.02% SDS. Phosphorylation was performed by
incubation of the pellet with 1 µg of the catalytic subunit of PKA (PKA-CS
reconstituted in 5 mM dithiothreitol) and 10 µCi of
[32P]ATP (3000 Ci/mmol) in phosphorylation buffer (25 mM
Hepes, 5 mM MgCl2,5mM EGTA, and 0.2% Triton
X-100 (pH 7.4)) for 10 min at 37 °C. In experiments with inhibitor, PKA
inhibitor (Sigma) was included along with PKA catalytic subunit. The reaction
was stopped by washing with 1 ml of RIA buffer (50 mM sodium
phosphate buffer (pH 7.4), 50 mM KF, 75 mM NaCl, 2.5
mM EDTA, 0.01% NaN3, and 25 mM Tris (pH 7.4))
(41). Samples were heated to
75 °C for 3 min in gel loading buffer containing 100 mM
dithiothreitol and analyzed using 8% polyacrylamide gels. The proteins from
the gel were transferred to nitrocellulose membrane (Amersham Biosciences) and
exposed to Kodak X-Omat AR at 80 °C.
Western Blotting and Antibodies
After the immunoprecipitation with NCX1 antibodies and protein A-Sepharose
beads, the protein sample was treated as described above. After
electrophoresis, the proteins were transferred to nitrocellulose membrane and
the primary antibodies were added. The following antibodies were used for
immunoblotting: rabbit polyclonal to NCX1 (1113, Swant); mouse
monoclonals to PKA catalytic subunit (clone 5B), to PKA RI subunit (Clone 18),
to PKC (Clone MC5), and to PP1 (clone 24) (BD Biosciences); rabbit polyclonal
to mAKAP (Upstate Biotechnology); and mouse monoclonal to PP2A (Clone 6F9)
(CRP Inc.). HRP-conjugated appropriate secondary antibodies (Jackson
Immunoresearch Laboratories, West Grove, PA) were added, and the proteins in
the nitrocellulose membranes were identified. The proteins were visualized
using ECL kit (Amersham Biosciences), and the images were obtained using Kodak
Biomax-MR film.
Immunofluorescence
Enzymatically isolated adult rat cardiomyocytes
(33) were fixed in cold
ethanol, permeabilized, and incubated with primary antibodies to NCX1 (mouse,
Swant) and mAKAP (rabbit, Upstate Biotechnology). Another set of
cardiomyocytes was incubated with antibodies to RI (Pan-antibody mouse, Clone
18, BD Biosciences) and NCX1 (rabbit, Swant) followed by appropriate secondary
antisera (Alexa488 and Alexa633, Molecular Probes, Sunnyvale, CA) and imaged
by confocal microscopy (Zeiss LSM 510). Images at each wavelength (488 and 633
nm) were collected separately to ensure that there was no fluorescent channel
bleed-through. The fluorescence intensities in a chosen area of the cell were
analyzed using Metamorph software (Universal Imaging Corporation, Downingtown,
PA).
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RESULTS |
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Protein Kinases and NCX1
To examine the possibility that NCX1 associates with relevant kinases, NCX1
was immunoprecipitated and immunoblotting was performed. The PKA holoenzyme is
a kinase composed of two identical catalytic subunits and two identical
regulatory subunits (43). We
show that the components of the PKA holoenzyme, both the catalytic and
regulatory subunit RI, are recognized in the NCX1 immunoprecipitate by the
antibodies specific for those proteins
(Fig. 2AI). The other
PKA regulatory subunit, RII, was not detected in the macromolecular complex.
This indicates that only RI is associated with NCX1. All of the PKA components
(including RII) were found in the cardiac lysate
(Fig. 2, AII). The
presence of both catalytic and regulatory subunits in the cardiac lysate shows
that PKA components are also found in the soluble components of the cell as
has been reported previously
(44). Using the same
immunoprecipitation strategy, we investigated whether or not PKC is associated
with NCX1. As shown in Fig.
2B, a sharp single band is present on the immunoblot
using a pan-PKC antibody.
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Phosphatases and NCX1
The absence of measured phosphorylation of NCX1 following PKA activation in
some studies could be compatible with our results if there was a difference in
the rate of dephosphorylation in the various studies. Conditions that may lead
to such differences could occur if phosphatases colocalized with NCX1 as has
been shown for RyR2 (38).
Fig. 3 examines this by
immunoblot analysis with antibodies to PP1 and PP2A as shown in
Fig. 3, A and
B, respectively. We found that the serine/threonine
phosphatases PP1 and PP2A were precipitated along with the NCX1. These
phosphatases were also readily detected in the cardiac lysate.
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AKAP and NCX1
Targeting of kinases and phosphatases to specific proteins has been
described extensively by Scott and co-workers
(45,
46). These studies have
identified a family of "A kinase
anchoring proteins" or AKAPs that serves as
scaffolding proteins for this function. To explore the possibility that an
AKAP may be associated with NCX1, we probed immunoblots with antibodies
against six of the major AKAPs. Fig.
4A and B, shows that mAKAP coprecipitates with
NCX1 while the other AKAPs tested (79, 95, 121, 149, and 220) did not. The
size of mAKAP is 300 kDa and is identified by the specificity of the mAKAP
antibody.2 The cardiac
lysate probed with the same antibody contained a 300-kDa band as well as the
smaller bands (Fig.
4A). Others have also detected the proteolytic fragments
of the 300-kDa mAKAP (47).
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Localization of NCX1, mAKAP, and PKA-RI in Rat Cardiomyocytes
To identify the NCX1 and associated proteins of the macromolecular complex
in situ, we used immunocytochemical techniques to localize these
proteins in rat ventricular cardiomyocytes. NCX1 was identified in the
sarcolemmal membrane and in the Z-lines of cardiomyocytes
(Fig. 5) as already reported
(48,
49). Interestingly, mAKAP is
also localized in the Z-lines and the dual staining of NCX1 and mAKAP
confirmed that both of these proteins are present in the same location in rat
cardiomyocytes (Fig.
5A, overlay). The regulatory subunit of PKA, RI
has been identified in the Z-lines and also in other regions of the cell
(Fig. 5C). The overlay
of the RI and NCX1 clearly shows that both of these proteins are colocalized
in cardiomyocytes (Fig.
5C). Analysis of the fluorescence intensities in selected
regions of the cell indicates that there is clear correspondence between NCX1
and mAKAP (Fig. 5B)
and that there is even better support for the matching of PKA-RI and NCX1
(Fig. 5D). The mAKAP
and PKA-RI are also expressed in places that do not contain NCX1 and that are
also shown in immunofluorescence images.
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DISCUSSION |
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Organization of the Macromolecular Complex
The data presented here and by others elsewhere suggest that the
intracellular loop is the probable site of phosphorylation by PKA, PKC, and
CamKII-dependent phosphorylation. Consequently, the intracellular loop of NCX1
is the most likely region to be associated with mAKAP. Our current thinking is
best summarized by the cartoon shown in
Fig. 6 that draws on our
findings shown in Figs. 1,
2,
3,
4 and the findings of others
(20,
50,
5255).
In addition to the nine transmembrane regions (TM), there are two pore-forming
regions (between TM2 and 3 and between TM7 and 8) and a large intracellular
loop between TM5 and 6. TM2, 3, 7, and 8 are organized to form the
"transport pore" while the other TM regions are thought to form an
outer ring (Fig. 6, right
panel). There are two short TM linker intracellular regions between TM1
and 2 and between TM3 and 4. The intracellular loops and the C terminus remain
as possible sites for anchoring of the macromolecular cluster. We favor the
large intracellular loop because it is the site of measured phosphorylation
(20) and its clearly
established role in NCX1 regulation
(33,
34,
56).
mAKAPThe same large 300-kDa AKAP (mAKAP) that is involved in NCX1 signal modulation also targets the cardiac RyR2. RyR2 and mAKAP are linked to each other through leucine/isoleucine-zipper (LZ) regions (57). In analyzing mammalian NCX1 sequences, 7 putative LZ motifs are conserved in sequence and position within the intracellular loop. It remains to be seen whether these motifs are responsible for mAKAP association with NCX1.
PKAThe catalytic subunits and the regulatory subunits were found associated with NCX1. The attachment of the catalytic subunits is facilitated by the regulatory subunits through the AKAP to NCX1. Whether RI binds to NCX1 through mAKAP or through another AKAP is yet to be investigated. The finding of RI as the regulatory subunit in the NCX1 complex is important. RI subunits are dynamic and developmentally regulated (58). The association of RI with NCX1 complex suggests that the PKA modulation of the NCX1 is more dynamic than previously appreciated and that phosphorylation may be an important signal or regulator during development.
PKCPKC was found to be associated with NCX1, but we do not
know how PKC is attached to the NCX1. The PKC could be linked via mAKAP, but
in other systems such as the neuromuscular junction
(59), distinct AKAPs link PKA
and PKC to their targets. PKC in the neuromuscular junction is
associated with AKAP250 (also known as gravin) to the postsynaptic membrane.
Interestingly, gravin is also reported to be associated with PKA in
erythroleukemia cells (60)
providing a precedent for the possibility that gravin may bind to both PKA and
PKC, but this requires further investigation.
Protein Phosphatases, PP1 and PP2AWe also identified the
phosphatases, PP1 and PP2A associated with NCX1. It has been demonstrated in
the RyR2 macromolecular complex that these proteins are linked to RyR2 through
independent linker proteins. PP1 binds RyR2 through targeting protein,
spinophilin, and PP2A binds RyR2 through the targeting protein, PR130. These
interactions with RyR2 occur through the LZ regions
(57). As noted above, we have
identified several LZ regions in the intracellular loop of NCX1 and these
regions could be responsible for PP1 and PP2A association with NCX1. Another
possibility is that PP1 alone could directly bind NCX1. Others have reported
that the PP1 binds directly to ion transporters through specific sequence
motifs in the target protein
(6163).
We have identified at least four possible PP1 binding regions in the rat NCX1.
We doubt the involvement of three of these possibilities based on either their
location in the protein or lack of conservation across species. However, there
is one region that is conserved by sequence and position in NCX1 of mammalian
species and NCX of Drosophila and squid
(Fig. 7). This region
(asterisk in Fig. 6,
left) is an -helical region that was initially modeled to be a
transmembrane segment. More recently, re-examination of the topology now
places this region at the end of the intracellular loop
(52,
53).
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Regulators and NCX1
Table I shows that there are
many common features shared by NCX1 and other macromolecular complexes. The
theme of macromolecular complexes is increasingly appreciated by recent work
investigating how signaling can be targeted locally while many of the critical
elements are also seen widely within the cell. NCX1 itself has important
cell-wide (i.e. "global") influence in overall
Ca2+ homeostasis but also has a role in
Ca2+ signaling that is more "local" in
nature.
Interaction with IonsThe macromolecular complex of NCX1 is associated with the intracellular components of NCX1 and probably with the large intracellular loop found between transmembrane (TM) segments 5 and 6. It is this part of NCX1 that has both known binding sites for PP1 and a possible association site with mAKAP (Figs. 6 and 7). The intracellular loop also contains a regulatory Ca2+ binding site, Na+ association site, sites for H+ regulation, and a "XIP" binding site. Although definite view on the spatial organization of the intracellular loop will await a structural investigation of this part of the protein, the preliminary data to date suggest that these regions of association in the intracellular loop interact.
Regulation by PhosphorylationEarly experiments suggested that ATP played an important regulatory role in NCX1 function (26, 64, 65). Possible roles for ATP were "direct" regulation or through phosphorylation of NCX1. The role of phosphorylation, however, has been uncertain until now. For example, experiments using the giant patch method have found no effect of phosphorylation on NCX1 function (11, 12). In contrast, other studies using both intact cells (20, 66) and biochemical approaches (20, 33) came to the opposite conclusion. Here we provide an explanation of these paradoxical observations. The presence of colocalized kinases and phosphatases make it possible for rapid dephosphorylation through phosphatase activity so that physiological phosphorylation may be absent or not found even when it is present and important in vivo (38, 39). In contrast, if kinases were to dominate the local process, physiological phosphorylation would be apparent.
Interaction with KinasesIn this report, we show that the macromolecular complex of NCX1 contains the kinase, PKA holoenzyme consisting of two PKA catalytic subunits and two identical PKA regulatory subunits composed of RI (Figs. 2 and 6). Importantly, only the regulatory subunit RI is found in the complex, whereas both RI and RII are present in the heart cell. Immunofluorescence studies clearly reveal the presence of RI in the Z-lines of rat ventricular cardiomyocytes (Fig. 5). This is the first demonstration of the AKAP targeting of RI in heart. All previous reports have found RII associated with AKAPs in heart (36, 37, 57).
The macromolecular complex also contains PKC as shown in Fig. 3. Both cloned cardiac NCX1 expressed in CCL39 cells and the neonatal rat cardiac NCX1 showed an increased NCX1 activity when treated with phorbol ester that activates PKC (20, 31). In such experiments, phosphorylation was measured (32). In contrast, studies employing giant patches to measure NCX1 activity did not show any effect of PKC activation (11, 12). The presence of phosphatases in the macromolecular complex may account for these different conclusions.
Interaction with PhosphatasesThe NCX1 macromolecular complex contains two phosphatases, PP1 and PP2A. The PP1 can be associated with NCX1 through mAKAP or directly to NCX1 (see above). The phosphatase PP2A can be associated with mAKAP through the adapter PR130 (57) or through gravin (59). Similar to PP1, PP2A is also serine-threonine phosphatase that is widely found and tightly regulated. The presence of two phosphatases and two kinases in the NCX1 macromolecular complex provides for both specific and subtle control of function.
Macromolecular Complexes in Other Systems, Local Control, and Targeting of the NCX1Similar to the NCX1 complex described in this report, macromolecular complexes have been reported in heart for L-type Ca2+ channels, RyR2 (the SR calcium release channel), and KCNQ1-KCNK1 K+ channels (3537). These complexes and NCX1 complex share the common feature in which the protein kinases and the phosphatases are tied to the central protein. By forcing the colocalization of regulatory enzymes, the central protein can be modulated more quickly and efficiently. The main protein can be organized with respect to other proteins within the cell through association with cytoskeletal proteins. Evidence suggests that NCX1 binds the adapter protein, ankyrin (50, 67), to guide the location of NCX1 within the cell. Ankyrin is thought to interact with spectrin and spectrin with actin. It is not known how ankyrin binds to NCX1. It is also possible that other adapter proteins may also be involved in local positioning of NCX1. It does appear that ankyrin-B is critical to T-tubular targeting of NCX1 (67), and this has important functional consequences. There is also the possibility that AKAPs can directly bind to the cytoskeleton as was demonstrated for ezrin (68).
Colocalization of NCX1 with mAKAP and PKA-RINCX1 has been
identified in the sarcolemma and in Z-line/T-tubules by us and others
(48,
49,
67). The mAKAP has been
localized in the sarcoplasmic reticulum, in the perinuclear region, and in
Z-line/T-tubular regions in cells
(37,
47,
69). We have identified mAKAP
in the Z-lines/T-tubules of adult rat ventricular cardiomyocytes, and mAKAP
colocalized with NCX1 in these cells. These findings are significant because
the L-type voltage-gated Ca2+ channels are
also localized in the T-tubules
(70). Similar to the
modulation of Ca2+ channels by the -adrenergic
stimulation through PKA activation, NCX1 also has been shown to be influenced
(39). The PKA subunit, RI, is
found in the Z-lines/T-tubules and is colocalized with NCX1. These
immunocytochemical results support the biochemical findings that NCX1 exists
as a macromolecular complex in cardiomyocytes.
Significance of the NCX1 Macromolecular Complex
The central role of Ca2+ in EC coupling and cardiac
contractility elevates the importance of tight regulation of NCX1, the primary
Ca2+ extrusion mechanism in heart. The important local
signaling functions carried out by NCX1 also demand careful but flexible and
rapid control of its function. The NCX1 macromolecular complex is the first
such complex reported for a transporter protein other than a channel. As noted
above, virtually all of the proteins with regulatory macromolecular complex
involving AKAPs influence intracellular [Ca2+] like
NCX1.
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FOOTNOTES |
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To whom correspondence may be addressed: Dept. of Microbiology and Immunology,
University of Maryland, 655 W. Baltimore St., Baltimore, MD 21201. Tel.:
410-706-5180; Fax: 410-706-2129.
1 The abbreviations used are: PKA, protein kinase A; PKC, protein kinase C;
RyR, ryanodine receptors. mAKAP, muscle protein kinase A-anchoring protein;
AKAP, A-kinase-anchoring proteins; TM, transmembrane; LZ,
leucine/isoleucine-zipper.
2 J. Scott, personal communication.
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ACKNOWLEDGMENTS |
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REFERENCES |
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