Split Na+-Ca2+ Exchangers

IMPLICATIONS FOR FUNCTION AND EXPRESSION*

Michela Ottolia, Scott John, Zhiyong Qiu, and Kenneth D. PhilipsonDagger

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha -repeats (5), are important for ion transport (6, 7) and may line the ion translocation pathway (8). Studies indicate that the alpha -repeats face opposite sides of the membrane and may include re-entrant loops analogous to the P loops of ion channels (1, 7).

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.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 -20 °C, and sectioned (approx 8 µm thick).

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.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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).

                              
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Table I
Activity of split exchangers relative to wild type activity
Transport activity was measured as Na+-dependent uptake of 45Ca2+ into oocytes expressing each of the constructs. Activity was normalized to wild type activity. Uptake was performed 3 or 4 times in each case with 6 or more oocytes used each time. The asterisks indicate split exchangers with overlapping residues. Control oocytes were injected either with cRNAs encoding for GFP or Shaker K+ channel. Constructs are shown schematically in Fig. 3. Errors represent S.E.


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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.

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.


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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).

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.


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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.

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. 


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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).

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.


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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 (tau ). 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, tau ), 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).

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. 


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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

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 alpha  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).

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 (>= 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.

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 Delta 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).

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.

    ACKNOWLEDGEMENTS

We thank Dr. R. Olcese for helpful discussions and Dr. Y. Lu and G. Mottino for technical assistance.

    FOOTNOTES

* 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.

Dagger 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.

    ABBREVIATIONS

The abbreviation used is: XIP, exchanger inhibitory peptide, GFP, green fluorescent protein.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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