From the Departments of Physiology and Medicine and the Cardiovascular Research Laboratories, University of California, School of Medicine, Los Angeles, California 90095-1760
Received for publication, February 16, 2001, and in revised form, March 21, 2001
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ABSTRACT |
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The Na+-Ca2+
exchanger has nine transmembrane segments, with a large cytoplasmic
loop between the fifth and sixth transmembrane segments. The protein
was split within the cytoplasmic loop into two domains consisting of
the first five transmembrane segments and the last four transmembrane
segments, respectively. The two domains were either expressed
individually or coexpressed. Each of the two domains with different
lengths of the cytoplasmic loop was fused to green fluorescent protein.
We show that coexpression of both domains is required for proper
membrane targeting and for expression of functional exchange
activity. Fusion to green fluorescent protein does not alter
biophysical properties of the exchange process. In addition, truncation
of a large portion of the cytoplasmic loop does not alter important
properties of the exchanger such as
Na+-dependent inactivation, activation by
chymotrypsin, or exchanger inhibitory
peptide (XIP) sensitivity.
The Na+-Ca2+ exchanger is a plasma
membrane protein that utilizes the Na+ electrochemical
gradient to exchange three extracellular Na+ ions for one
intracellular Ca2+ ion, thereby maintaining
Ca2+ homeostasis. Although present in many tissues, the
cardiac Na+-Ca2+ exchanger, NCX1, is the form
that has been most extensively characterized. The 938 amino acids that
constitute the protein include 9 transmembrane segments. The first 5 transmembrane segments are separated from the last four by a large
intracellular loop encompassing residues 218-764 (1). Some important
regulatory regions have been identified within the cytoplasmic loop.
The N-terminal portion (amino acids 219 to 238), called the
XIP1 region (2), plays an
important role in Na+-dependent inactivation
(also referred to as I1 regulation) (3), whereas
amino acids 371-508 constitute a high affinity Ca2+
regulatory site (4). Binding of Ca2+ to this site
stimulates transport activity and also removes
Na+-dependent inactivation (3).
The molecular transitions that lead to ion transport are unresolved.
Two highly conserved repeat motifs, referred to as The goal of this study was to identify domains of the cardiac
Na+-Ca2+ exchanger important for activity,
expression, or trafficking of the protein. The
Na+-Ca2+ exchanger protein was conceptualized
as consisting of two domains: a N-terminal domain (N), constituted by
the first five transmembrane segments, and the C-terminal domain (C),
made up of the last four transmembrane segments. We then constructed a
variety of "split" exchangers composed of the N or C domains with
varying lengths of the intracellular loop attached. The N or C domains
were expressed alone or coexpressed. Transport activity was determined
by Na+-gradient-dependent
45Ca2+ uptake or by electrophysiology, and
surface expression was studied by linking GFP (9) to either the N or C domain.
Functional reassembly of protein domains has been demonstrated for both
ion channels and membrane transporters (10-15). This approach has been
useful in defining the roles of domains within a protein. Heterologous
expression of domains from two Ca2+-activated potassium
channels with different Ca2+ sensitivities led to
identification of the region responsible for Ca2+
regulation (15). Coexpression of segments of CFTR in Xenopus oocytes helped define boundaries of the cytoplasmic nucleotide-binding site (10).
We demonstrate that the cardiac Na+-Ca2+
exchanger is also divisible into two domains that can reassemble in the
membrane of Xenopus oocytes. Reassembly of the two domains
gives Na+-Ca2+ exchange activity as judged by
45Ca2+ uptake and by electrophysiology. The
currents generated by the coexpression of the two domains are similar
to those of the wild type (unsplit) exchanger in retaining
Ca2+ regulation, Na+-dependent
inactivation, and sensitivity to XIP and chymotrypsin. GFP fusion
constructs are used to monitor the pattern of expression of split
exchangers in Xenopus oocytes.
Molecular Biology--
Four GFP-exchanger chimeras were
generated. Three fusion proteins were made with the GFP inserted at the
C-terminal end of split exchanger proteins, and one fusion protein was
made with GFP inserted at the N-terminal end of a split exchanger
protein (see Fig. 3). To construct N265-GFP, the vector pEGFPN-1
(CLONTECH, Palo Alto, CA) was digested with
XhoI/BglII, and the exchanger was excised from
pCDNA3.1 with SalI/BamHI. The exchanger
fragment was ligated into the pEGFPN-1 vector, giving the following
amino acid linker region between the exchanger and GFP:
ELKLRILESTVPPAPDPPVAT. To construct N358-GFP, the vector pEGFPN-1 was
digested with PstI/BglII, and the exchanger was
excised from pCDNA3.1 with NsiI/BamHI. The exchanger
fragment was ligated into the pEGFPN-1 vector, giving the following
amino acid linker region between the exchanger and GFP: STVPPAPDPPVAT.
To construct N468-GFP, the vector pEGFPN-1 was digested with
SmaI/BglII, and the exchanger in pCDNA3.1 was digested with StuI, blunt-ended, and then excised with
BamHI. The exchanger fragment was ligated into the pEGFPN-1
vector, giving the following amino acid linker region between the
exchanger and GFP: GDPPVAT. The cDNA for the fusion protein
GFP-532C was constructed by digesting the pEGFPC-1 vector with
XhoI/HindIII and ligating a
XhoI/HindIII exchanger fragment into the vector.
For all oocyte expression analyses, the fusion constructs were
subcloned into the pGEM vector between Xenopus globin 5'-
and 3'-untranslated regions. RNA was synthesized using the mMessage
mMachine T7 RNA kits (Ambion, Austin TX). Xenopus laevis
oocytes were injected with 46 nl of cRNA and kept at 18 °C
for 4-7 days.
Preparation of Oocyte Sections--
Sections of oocytes were
prepared according to Terada et al. (16). Briefly, oocytes
were incubated in 4% paraformaldehyde at 4 °C for 1 h,
immersed in phosphate-buffered saline solution containing 30% sucrose
at 4 °C for 18 h, fixed in Tissue-Tek Optimal Cutting
Temperature compound, frozen overnight at Electrophysiological Experiments--
Giant inside-out patches
were excised from oocytes 4-7 days after cRNA injection. Records were
low pass-filtered at 50 Hz and acquired on line at 4 ms/point.
Experiments were performed at 35 °C at a holding potential of 0 mV.
To excise a membrane patch, the vitelline membrane was manually removed
in a solution of 110 mM KCl, 10 mM HEPES, 2 mM MgCl2, pH 7. After membrane excision, solutions were rapidly changed using a computer-controlled 20-channel solution switcher. Recordings were obtained with the following solutions: pipette solution, 100 mM
N-methylglucamine, 20 mM HEPES, 20 mM tetraethylammonium hydroxide, 8 mM
Ca(OH)2, 0.1 mM niflumic acid, and 0.15 mM ouabain, pH 7 (using methanesulfonic acid); bath
solution, 100 mM CsOH or NaOH, 20 mM
tetraethylammonium hydroxide, 20 mM HEPES, 10 mM EGTA, and 0 or 5.75 mM Ca(OH)2
(0 or 1 µM free Ca2+), pH 7 (using
methanesulfonic acid). Solutions with higher Ca2+ (15 µM) were prepared by adding 10 µM
Ca(OH)2 in the absence of EGTA.
Electrophysiological Characterization of a Split
Na+-Ca2+ Exchanger--
One goal was to split
the cardiac Na+-Ca2+ exchanger into two
segments and restore transport activity by coexpression of the two
domains in Xenopus oocytes. We generated two expression
constructs: one coding for the N domain, consisting of the first five
transmembrane segments and the cytoplasmic loop up to residue 671 (N671), and one coding for the C domain (672C), starting at residue 672 including the remainder of the protein.
Exchange current was measured using excised inside-out giant patches
from oocytes expressing the split exchangers. Alternatively, exchange
activity was monitored as Na+
gradient-dependent 45Ca2+ uptake.
Expression of either the N or the C domain alone produced no detectable
exchange activity, as assayed by either technique (Table
I). However, coexpression of the N + C
domains produced transport activity. Fig.
1 shows representative traces recorded from oocytes expressing the wild type (i.e. intact; Fig.
1A) and N671 + 672C exchangers (Fig. 1B). Upon
application of bath Na+ (100 mM) to the
cytoplasmic surface of the patch in the presence of 8 mM
Ca2+ in the pipette, an outward exchange current was
recorded. For both the wild type and split exchangers, current peaked
and then decayed to a steady state level. The decay is caused by the
presence of cytoplasmic Na+, which drives some exchangers
into an inactive state (Na+-dependent
inactivation or I1) (3). Current decay was fit with a
single exponential to determine the time constant. Values were 2.9 ± 0.2 s (n = 11) for the wild type and 3.2 ± 0.4 s (n = 15) for the N671 + 672C exchanger
(see Fig. 6). Although the time constant did not change significantly,
the number of split exchangers that enter the inactive state was less
compared with the wild type. A measure of the distribution of active
and inactive state is the fractional activity,
Fact, defined as the ratio of steady state to
initial current. Fact for the wild type
exchanger was 0.26 ± 0.02 (n = 22), whereas for
the N671 + 672C exchanger Fact was 0.60 ± 0.04 (n = 15). Thus, the split exchanger retains
inactivation although the magnitude of inactivation is decreased (see
Fig. 6).
The wild type exchanger is regulated by cytoplasmic Ca2+,
which binds to a high affinity site in a region of the intracellular loop between residues 371 and 508 (4). This binding site is distinct
from the Ca2+ transport site. For the wild type exchanger
(Fig. 1A), increases of internal regulatory Ca2+
evoke an increase of the peak current, whereas removal of internal Ca2+ drives the exchanger into a
Ca2+-dependent inactive state
(I2) (17). The effects of regulatory Ca2+ on the activity of the split exchanger were examined.
Fig. 1B shows traces recorded from two different oocytes,
both expressing split exchanger N671 + 672C, and their response to
change in internal Ca2+ concentrations. In one case,
removal of internal Ca2+ evoked a decay of exchange current
(upper trace), whereas in another case, the level of the
current was insensitive to cytoplasmic Ca2+ (lower
trace). Of 15 experiments, 8 showed an absence of Ca2+
regulation, whereas 7 displayed Ca2+ regulation. Note that
in all cases, transported Ca2+ is always present within the
patch pipette at the extracellular surface of the membrane patch; only
intracellular regulatory Ca2+ is varied.
The ionic regulatory mechanisms controlled by Na+ and
Ca2+ interact. Cytoplasmic Ca2+ alters the rate
of entry and exit from the I1-inactive state and, at high
concentrations (10-30 µM), completely eliminates Na+-dependent inactivation (17). Fig. 1
demonstrates the effect of high concentrations of cytoplasmic
Ca2+ on Na+-dependent inactivation
for the wild type and the split exchangers. Upon application of
Na+ in the presence of 15 µM internal
Ca2+, the wild type exchanger current increased and then
decayed, reaching a steady state that was 87 ± 5%
(n = 15, [Ca2+]i = 15 µM) of the peak current. In contrast, when a high
concentration of internal Ca2+ in the presence of
Na+ was applied to an excised patch expressing split
exchangers (Fig. 1B), the current peaked and then slowly
decayed to a new steady state that was 55 ± 9%
(n = 4, [Ca2+]i = 15 µM) of the peak current.
Brief exposure of the intracellular surface of the giant patch to
chymotrypsin deregulates the Na+-Ca2+ exchanger
(18). Deregulation removes both Na+- and
Ca2+-dependent regulatory properties, leaving
transport properties intact (19, 20). The mechanism and the site of
action of this proteolytic enzyme are not known. Fig.
2A shows the effect of chymotrypsin on the split exchanger N671 + 672C. As with the wild type
exchanger, application of cytoplasmic chymotrypsin removed Na+-dependent inactivation and Ca2+
regulation (not shown) and increased the split exchanger current by
1.66 ± 0.27-fold (n = 4). Thus, activity of the
split exchanger can still be up-regulated by chymotrypsin.
The XIP (exchanger inhibitory
peptide) region of the exchanger is a stretch of basic and
hydrophobic amino acids located at the N-terminal region of the large
cytoplasmic loop (amino acids 219-238). A peptide corresponding to
this region, XIP, is a potent inhibitor of
Na+-Ca2+ exchanger activity (2). The location
of the XIP binding site is unknown, although a previous report
suggested that the XIP binding site is located within the intracellular
loop (21). If the XIP peptide is to bind in the large intracellular
loop located between transmembrane segments 5 and 6, the structure and
conformation of the loop could be important for this interaction. The
trace in Fig. 2B displays the effect of 1 µM internal XIP on the exchange current generated by a
split exchanger. As observed for the wild type exchanger, XIP blocked
~90% of exchange current. Similar results were observed in four
additional experiments. Thus, the split exchanger still retains
sensitivity to XIP.
Surface Expression of the GFP-tagged Split Exchangers--
To
visualize the distribution of the exchanger N and C domains in
Xenopus oocytes, we linked the GFP (9) to a variety of constructs. In addition, the length of the cytoplasmic portions of the
N and C domains was varied to examine the role of the large cytoplasmic
loop in controlling function and surface expression of the transporter.
Constructs are designated as N or C, referring to the N or C domains,
with a number indicating either the last amino acid residue of the N
domain or the first residue of the C domain. If GFP is fused to a
domain, the amino acid number also indicates the position at which GFP
is linked. cRNA coding for the constructs shown schematically in Fig.
3 was injected into Xenopus
oocytes individually and in combinations. Four to six days after
injection, the localization of the chimeric proteins was examined using
a standard epifluorescence microscope. Fig. 4 shows examples of fluorescence
microscopic images of intact oocytes expressing the indicated
constructs. Whole oocyte views (Fig. 4A) show that
expression of GFP-tagged halves N358, N468, and 532C generated a
diffuse green fluorescence signal that was mainly restricted to the
vegetal pole. A reduced fluorescence from the animal pole is likely due
to a filtering effect of the pigments present only in this region of
the oocytes. N265-GFP failed to produce any fluorescence, and the
signal was comparable with background autofluorescence.
Fig. 4B shows fluorescence images of intact oocytes acquired
at higher magnification to better resolve plasma membrane labeling. Since the pigments, located below the plasma membrane on the animal pole, partially filter cytoplasmic fluorescence, we analyzed this region of the oocytes (except for N265-GFP; see Fig. 4 legend). Only
coexpression of the two domains generates a fluorescent signal in the
plasma membrane of the oocytes. Plasma membrane localization was most
intense when the N domains were coexpressed with a long C domain
fragment (i.e. 272C). Oocytes injected with cRNA encoding GFP alone showed a uniform cytoplasmic fluorescent signal, without plasma membrane staining. This indicates that the fluorescence observed
at the plasma membrane reflects the true localization of GFP-tagged
split exchangers.
To visualize in more detail the distribution of the GFP-tagged split
exchangers, cryosections of oocytes were analyzed. Examples of
fluorescent images obtained from oocyte sections (8-10
µM thick) are shown in Fig.
5. Uninjected oocytes or oocytes
expressing an untagged exchanger were used as negative controls.
Analysis of the cryosections confirmed the membrane localization of
split exchangers only when both domains were coexpressed. As seen with intact oocytes, the intensity of the signal detected at the plasma membrane depended on two factors: the presence of both domains and the
length of the large cytoplasmic loop located between transmembrane segments 5 and 6.
In oocyte sections, it was possible to occasionally observe a weak
plasma membrane fluorescence in oocytes expressing single domains (with
the exception of N265-GFP, which did not generate any signal above
background). This signal was restricted to the animal pole,
whereas coexpression of the N and C domains delivered the protein to
both the animal and vegetal poles. In addition, oocytes expressing the
wild type exchanger with no GFP tag also sometimes exhibited weak
surface autofluorescence (Fig. 5). Thus, the significance of the weak
membrane labeling by individual domains of the exchanger is unclear.
Functional Activity of the GFP-tagged Split Exchangers--
To
determine whether linkage to GFP altered transport properties of split
Na+-Ca2+ exchangers, the activities of fusion
proteins were analyzed by Na+-gradient dependent
45Ca2+ uptake and by the giant patch technique.
Table I shows the activity of GFP-tagged split exchangers relative to
the wild type activity, as determined by Na+
gradient-dependent 45Ca2+ uptake.
In no case did expression of individual domains of the protein generate
significant exchanger activity. Transport activity was reconstituted
only after coinjection of both N and C domains. Exchanger activity
correlated with the intensity of fluorescence in the plasma membrane,
and the highest level of transport was obtained when the full length of
the protein was reconstructed. For example, the GFP-labeled split
exchanger composed of the two halves N265-GFP and 672C showed only
24 ± 7% of the activity of the wild type (not split) exchanger,
whereas the exchanger N265-GFP + 272C restored 52 ± 13% of the
activity of the wild type exchanger. Similar results were obtained when
fragments N365-GFP and N468-GFP were expressed with either 672C or 272C.
We investigated the biophysical properties of the GFP-tagged split
exchangers using the giant patch technique. All GFP-tagged split
exchangers were active, displaying an outward current upon application
of cytoplasmic Na+. As with the wild type exchanger,
current peaked and then decayed (Na+-dependent
inactivation) to a steady state level. The extent of Na+-dependent inactivation for the GFP-tagged
split exchangers was measured as fractional activity,
Fact. Fig. 6 shows
recordings from excised patches from oocytes expressing the indicated
constructs and the corresponding Fact and
inactivation time constants. The magnitude of inactivation was
generally decreased with respect to that of the wild type exchanger,
although split exchangers N468-GFP + 272C and N265-GFP + 272C had
Fact values similar to wild type. We also
examined the kinetics of the Na+-dependent
inactivation by fitting the current decay to an exponential. The rate
by which N549 + GFP-532C, N265-GFP + 672C, and N468-GFP + 672C entered
the inactivated state did not significantly deviate from that of the
wild type. In contrast, the split exchangers containing the N358-GFP
domain and N468-GFP + 272C had faster time constants, whereas N265-GFP + 272C entered the inactivated state with a slower rate.
Similar to the untagged split exchanger, the constructs N265-GFP + 272C, N358-GFP + 272C, and N468-GFP + 272C showed regulation by
intracellular Ca2+, whereas splits N549 + GFP-532C,
N358-GFP + 672C, and N265-GFP + 672C lacked
Ca2+-dependent regulation (not shown). The data
indicate that the addition of GFP to the exchanger and the omission or
overlap of large portions of the cytoplasmic loop did not drastically
affect the molecular processes involved in
Na+-dependent inactivation or
Ca2+-dependent regulation.
All the GFP-tagged split exchangers retained chymotrypsin and XIP
sensitivity. As an example, Fig. 7 shows
traces recorded from a patch expressing the N265-GFP + 672C exchanger.
Outward current was activated upon application of 100 mM
internal Na+ in the presence of 1 µM
cytoplasmic Ca2+. After exposure of the internal side of
the patch to chymotrypsin, current increased, and the
Na+-dependent inactivation disappeared
(n = 4). Subsequently, chymotrypsin-activated current
was inhibited by the addition of 1 µM cytoplasmic XIP (n = 2). The result indicates that the sites recognized
by chymotrypsin and XIP are not located between residues 266 and
671.
The mature Na+-Ca2+ exchanger has nine
transmembrane segments. The first five transmembrane segments are
separated from the last four by a large intracellular loop (1). Some
functionally important regions of the exchanger protein have been
elucidated. The In this study, we artificially split the exchanger within the large
cytoplasmic loop and examined the consequences for split exchangers
with respect to function, trafficking, and regulation. The N domain
consisted of the first five transmembrane segments and part of the
intracellular loop, whereas the C domain included a portion of the
intracellular loop and the last four transmembrane segments. Individual
domains were unable to generate any transport activity; activity was
restored only after coexpression of paired N and C domains. Analyses of
sections of oocytes expressing domains of the exchanger labeled with
GFP are consistent with these findings. Oocytes expressing single
halves showed a diffuse fluorescence within the cytoplasm, indicating
that the proteins were translated but were mainly retained in internal
membranes or were largely degraded. An exception is the N265-GFP
fragment, which failed to produce any fluorescence when expressed
alone. Since the domain N358-GFP did express protein, residues
encompassing 265 and 358 could play a role in protein stability.
A weak plasma membrane signal on the animal pole of oocytes expressing
single domains was occasionally observed. Although a similar signal was
sometimes also observed in control oocytes, we cannot completely rule
out the possibility that a small amount of individual halves of the
exchanger could be stably inserted in the plasma membrane.
Nevertheless, when the two domains were coexpressed, markedly different
results were observed. The amount of fluorescence in the plasma
membrane drastically increased in both the animal and the vegetal
poles. The intensity of the signal in the plasma membrane was
correlated to the length of the large cytoplasmic loop: split
exchangers with large loop truncations generated less plasma
membrane fluorescence than split exchangers in which the full length of
the exchanger was reconstituted. These findings suggest a role for the
cytoplasmic loop in modulating the trafficking and stability of the
protein. Our data on the activity of split exchangers, measured as
Na+-dependent
45Ca2+ uptake, support this hypothesis; the
highest levels of activity were measured in oocytes expressing split
exchangers containing the full length of the large intracellular loop
(Table I).
To analyze the consequences of an interruption in the protein backbone
and GFP fusions on secondary regulation of the exchanger, we recorded
current from split exchangers using the giant excised patch technique.
All active split exchangers showed
Na+-dependent inactivation. The
Na+-dependent inactivation or
I1, describes entry of the exchanger into an
inactive state in the presence of high concentrations of cytoplasmic
Na+. This process is associated with a region of the
protein located at the N-terminal side of the large cytoplasmic loop
(endogenous XIP region, amino acids 219-238) (23). Interaction of the
endogenous XIP region with another region of the exchanger may produce
the inactive state. Our data indicate that
Na+-dependent inactivation is not significantly
affected by 1) partitioning of the protein into two domains, 2) large
deletions of the cytoplasmic loop, or 3) fusion of GFP to the loop.
Even the split exchanger N265-GFP + 672C retains
Na+-dependent inactivation, although in this
case the GFP is in close proximity (within 30 residues) to the XIP region.
All split exchangers, including N265-GFP + 672C, with more than 400 loop residues deleted, were blocked by the synthetic peptide with the
same sequence as the endogenous XIP region (XIP peptide) (2). This
result indicates that the site with which the endogenous XIP region
interacts is not located between amino acids 265 and 672. Hale et
al. (21) have previously identified the segment between residues
445 and 455 of the exchanger as a XIP binding region. Although this
segment may be able to interact with XIP, our data indicate that this
cannot be the primary site responsible for XIP inhibition. In addition,
in an early report, Matsuoka et al. (24) report that a
mutant exchanger with a large deletion (amino acids 240-679) in the
intracellular loop was insensitive to inhibition by XIP peptide.
Subsequently, we have been unable to reproduce this
finding,2 consistent with the
data presented here. Our data do not rule out the possible involvement
of the C-terminal region of the cytoplasmic loop (amino acids 672-764)
in the binding of XIP peptide. These residues include a highly
conserved region previously thought to be a transmembrane segment
(former TMS6 (1)). More mutagenesis and biophysical studies are needed
to elucidate the events that lead to
Na+-dependent inactivation and XIP inhibition.
Cytoplasmic Ca2+ regulates Na+-Ca2+
exchange activity. Regulatory Ca2+ binds to a high affinity
binding site, located in the cytoplasmic loop. Increasing intracellular
Ca2+ activates transport activity, whereas removal of
cytoplasmic Ca2+ drives the exchanger into an inactive
state referred to as Ca2+-dependent
inactivation or I2 (17, 25). In testing the
Ca2+ sensitivity of split exchangers, we found that the
GFP-tagged split exchangers N265-GFP + 272C, N358-GFP + 272C,
N468- GFP + 272C, and the split exchanger N671 + 672C were activated
by cytoplasmic Ca2+. However, a complete loss of
Ca2+ regulation was sometimes observed (Fig. 1). Possible
explanations are that the split exchangers are more susceptible to
proteases, causing the loss of Ca2+ regulation, or that
alternative conformations exist with split exchangers. The split
exchangers N265-GFP + 672C, N368-GFP + 672C, and N549 + GFP-532C lacked
secondary Ca2+ regulation. Of particular interest is the
lack of Ca2+ regulation in the N549 + GFP-532C construct,
since it still retains the Ca2+ binding site (residues
371-508 (4)). The presence of GFP or the location at which the protein
was split could possibly interfere with the binding of Ca2+
or obstruct conformational changes leading to exchanger activation by
Ca2+. In the wild type exchanger, regulatory
Ca2+ not only activates exchanger current but also
modulates Na+-dependent inactivation. At high
internal Ca2+ concentrations ( Exposing the cytoplasmic side of the wild type exchanger to
chymotrypsin eliminates both Ca2+ and Na+
regulation (18), leaving the exchanger in an activated state. The
site(s) at which proteolysis induces activation are not known. It is
assumed that the chymotrypsin site is located in the large cytoplasmic
loop, although there is no evidence to validate this hypothesis. Our
data indicate that a GFP-tagged split exchanger with a large portion of
the loop deleted (amino acids 266-671) is deregulated after exposure
to chymotrypsin. Two considerations arise. First, chymotrypsin can
still activate the exchanger with a split loop; second, if the
proteolytic site responsible for activation is to be found in the
cytoplasmic loop, it is not located between residues 266 and 671. Previous investigations showed activation of the deletion mutant
Two studies report that expression of truncated forms of the exchanger,
comparable with N domains characterized here, induced Na+-Ca2+ exchange activity in transfected cells
(29, 30). Our results are inconsistent with these reports. We were
never able to observe exchange activity with either the N or C domains
expressed individually. Likewise, the N or C domains did not induce
activity when expressed in human embryonic kidney cells instead of
oocytes.3 In addition, the
fluorescent signal in the plasma membrane of oocytes expressing
individual GFP-tagged domains was marginally, if at all, above
background. Possibly different experimental conditions or different
levels of expression of the single domains could account for the discrepancy.
In summary, we describe the functional reconstitution of the exchanger
after partition into two domains. The two domains were properly
delivered to the membrane when coexpressed, and both domains were
required for activity. Exchanger activity was maintained after fusion
of GFP to individual domains. The fluorescent fusion proteins may be
useful monitors of conformational changes in future studies.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-repeats (5), are
important for ion transport (6, 7) and may line the ion translocation
pathway (8). Studies indicate that the
-repeats face opposite sides
of the membrane and may include re-entrant loops analogous to the P
loops of ion channels (1, 7).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 °C, and sectioned
(
8 µm thick).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Activity of split exchangers relative to wild type activity
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Fig. 1.
Giant patch recordings from
Xenopus oocytes expressing wild type and split
exchangers. Shown are representative outward currents from
patches of oocytes expressing the wild type exchanger (A)
and an exchanger split between residues 671 and 672 (N671 + 672C)
(B). Currents were elicited by the rapid application into
the bath (intracellular surface) of 100 mM Na+
in the presence of 8 mM Ca2+ in the pipette
(extracellular surface). Cytoplasmic Ca2+ concentrations
are indicated. Activity of the split exchanger was detected only when
the two fragments were coexpressed. As for the wild type exchanger,
outward current of the split exchanger displayed
Na+-dependent inactivation and Ca2+
regulation (upper traces, panel B). However, in
about half the cases, the exchange current of the split protein lacked
Ca2+ regulation (lower traces, panel
B). Currents shown in panel B were measured from two
different oocytes from the same batch. Exchanger currents were recorded
at 0 mV.
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Fig. 2.
Chymotrypsin and XIP sensitivity of split
exchanger N671 + 672C. Shown is the outward
Na+-Ca2+ exchange current from split exchanger
N671 + 672C in a giant inside-out patch. Exchanger activity was
measured in the presence of 1 µM cytoplasmic
Ca2+. Perfusion with chymotrypsin (1 mg/ml) at the
intracellular surface augmented exchanger activity (A).
Split exchanger N671 + 672C also retained XIP sensitivity. Application
of 1 µM synthetic XIP peptide to the internal side of the
patch inhibited exchange current (B).
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Fig. 3.
Topology of the different N- and C-terminal
domains. Schematic representation of the split
Na+-Ca2+ exchanger domains. The intact
exchanger, shown schematically at top, has nine transmembrane segments
(cylinders). The N terminus is extracellular (EXT), whereas
the C terminus is cytoplasmic (INT). The N-domains consist
of the first five transmembrane segments of the protein and different
lengths of the large cytoplasmic loop. They are indicated with the
letter N followed by a number, which designates the last residue of the
peptide, and for N468-GFP, N358-GFP, and N265-GFP, the position to
which GFP was linked. The same nomenclature has been used for the C
domains, composed of the last four transmembrane segments, and
different portions of the cytoplasmic loop. In this case,
(a.a.) the number preceding the letter C indicates the first
amino acid of the peptide, and for GFP-532C, the position to which GFP
was linked.
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Fig. 4.
Surface expression of the GFP-tagged N
and C domains. 4-5 days after cRNA injection, the pattern of
expression was examined by fluorescence microscopy. Panel A
shows whole oocytes expressing the indicated constructs. All oocytes
were from the same batch, and pictures were taken at the same exposure
(10 × 0.6 magnification). Panel B shows fluorescent
images acquired at 20 × 0.6 magnification. Oocytes were from the
same batch, except for the N358-GFP series. All images in panel
B show the animal pole with the exception of N265-GFP, which would
have appeared completely black.
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Fig. 5.
Fluorescent images of oocyte sections
expressing GFP-tagged split exchangers. Images are from thin
sections of oocytes at 20 × 0.6 magnification. All images were
taken at the same exposure. Oocytes expressing the full-length
exchanger (wild type) or uninjected oocytes (control) occasionally
displayed a weak fluorescent signal in the plasma membrane. A green
fluorescence in the plasma membrane clearly distinguishable from the
background was observed only when both fragments of the protein were
coexpressed. Expression of single domains generated a diffuse
fluorescence within the oocytes (not shown).
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Fig. 6.
GFP-tagged split exchangers retain
Na+-dependent inactivation. Recordings
from giant excised patches expressing the indicated construct and
corresponding fractional activity (Fact) and
inactivation time constant values ( ). Traces were recorded at 0 mV
upon application and removal of 100 mM internal
Na+ in the presence of 1 µM internal
Ca2+. Fractional activity is calculated as the ratio of
steady state current to peak current. The decay of the current was fit
to a single exponential. Constructs N358-GFP + 672C, N358-GFP + 272C,
N468-GFP + 272C, and N265-GFP + 272C show a significantly different
time constant of inactivation as compared with wild type
(WT). Values for n are 22 (wild type
Fact), 11 (wild type,
), 15 (N671 + 672C), 7 (N549 + GFP-532C), 2 (N468-GFP + 672C), 2 (N468-GFP + 272C), 6 (N358-GFP + 672C), 6 (N358-GFP + 272C), 10 (N265-GFP + 672C), and 12 (N265-GFP + 272C).
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Fig. 7.
Effects of XIP and chymotrypsin on a
GFP-linked split exchanger. Shown are giant patch recordings from
Xenopus oocytes injected with N265-GFP + 672C. Coexpression
of these two domains leads to an exchanger lacking cytoplasmic residues
266-671. Outward currents, upon application and removal of 100 mM internal Na+, were recorded in the presence
of 1 µM internal Ca2+. Traces show activation
of exchange current by chymotrypsin (Chym.) treatment (1 mg/ml) and inhibition by 1 µM XIP.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
repeats (including transmembrane segments 2, 3, and
7) are important for ion transport, whereas the large intracellular
loop confers secondary regulation by Na+ and
Ca2+ ions. Na+ drives a fraction of the
exchangers into an inactive state
(Na+-dependent inactivation), whereas
regulatory Ca2+ stimulates activity. These two processes
are not independent since high cytoplasmic Ca2+ removes
Na+-dependent inactivation (3).
Binding of regulatory Ca2+ to the loop induces a major
change in conformation of the protein (4, 22).
10 µM),
Na+-dependent inactivation is largely
eliminated (17). In contrast, the Na+-dependent
inactivation of N671 + 672C is essentially insensitive to regulatory
Ca2+, and substantial inactivation is observed even at 15 µM internal Ca2+ (Fig. 1). Apparently the
conformational changes induced by Ca2+ binding that
modulate Na+-dependent inactivation are absent
in a split exchanger.
240-679 by chymotrypsin (24), and in preliminary experiments, we
observed chymotrypsin sensitivity in an exchanger with residues
223-265 deleted. These results further constrain the location of the
cleavage site. Chymotrypsin treatment of the exchanger protein shifts
the apparent molecular mass from 120 to 70 kDa (26). Interestingly,
after chymotrypsin treatment, 50 kDa of protein is thus unaccounted
for. Apparently, in one portion of the protein, chymotrypsin cleaves
the exchanger at multiple sites, and the fragments are too small to be
detected in the gel system. In two studies, the N terminus of the
70-kDa proteolytic fragment has been identified to coincide with the N
terminus of the full-length protein (27) or to begin within the
intracellular loop in the 257-269-residue region (28).
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ACKNOWLEDGEMENTS |
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We thank Dr. R. Olcese for helpful discussions and Dr. Y. Lu and G. Mottino for technical assistance.
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FOOTNOTES |
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* This research was supported by Fellowships from the American Heart Association, Western States Affiliate (to M. O. and Z. Q.), a grant-in-aid from the American Heart Association, Western States (to S. J.), and by National Institutes of Health Grants HL49101 and HL48509 (NHLBI) (to K. D. P.).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.
To whom correspondence should be addressed: Cardiovascular
Research Laboratory, MRL 3-645, UCLA School of Medicine, Los Angeles, CA 90095-1760. Tel.: 310-825-7679; Fax: 310-206-5777; E-mail: kphilipson@mednet.ucla.edu
Published, JBC Papers in Press, March 27, 2001, DOI 10.1074/jbc.M101489200
2 S. Matsuoka, and K. D. Philipson, unpublished observation.
3 Z. Qiu, and K. D. Philipson, unpublished observation.
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ABBREVIATIONS |
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The abbreviation used is: XIP, exchanger inhibitory peptide, GFP, green fluorescent protein.
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REFERENCES |
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