From the * Departments of Physiology and Medicine and the Cardiovascular Research Laboratories, UCLA School of Medicine, Los Angeles, California 90095-1760; and Department of Physiology, University of Texas, Southwestern Medical Center at Dallas, Dallas, Texas
75235-9040
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ABSTRACT |
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We have cloned the squid neuronal Na+-Ca2+ exchanger, NCX-SQ1, expressed it in Xenopus oocytes,
and characterized its regulatory and ion transport properties in giant excised membrane patches. The squid exchanger shows 58% identity with the canine Na+-Ca2+ exchanger (NCX1.1). Regions determined to be of functional
importance in NCX1 are well conserved. Unique among exchanger sequences to date, NCX-SQ1 has a potential protein kinase C phosphorylation site (threonine 184) between transmembrane segments 3 and 4 and a tyrosine
kinase site in the Ca2+ binding region (tyrosine 462). There is a deletion of 47 amino acids in the large intracellular loop of NCX-SQ1 in comparison with NCX1. Similar to NCX1, expression of NCX-SQ1 in Xenopus oocytes induced cytoplasmic Na+-dependent 45Ca2+ uptake; the uptake was inhibited by injection of Ca2+ chelators. In giant
excised membrane patches, the NCX-SQ1 outward exchange current showed Na+-dependent inactivation, secondary activation by cytoplasmic Ca2+, and activation by chymotrypsin. The NCX-SQ1 exchange current was
strongly stimulated by both ATP and the ATP-thioester, ATPS, in the presence of F
(0.2 mM) and vanadate
(50 µM), and both effects reversed on application of a phosphatidylinositol-4',5'-bisphosphate antibody. NCX1
current was stimulated by ATP, but not by ATP
S. Like NCX1 current, NCX-SQ1 current was strongly stimulated
by phosphatidylinositol-4',5'-bisphosphate liposomes. In contrast to results in squid axon, NCX-SQ1 was not stimulated by phosphoarginine (5-10 mM). After chymotrypsin treatment, both the outward and inward NCX-SQ1 exchange currents were more strongly voltage dependent than NCX1 currents. Ion concentration jump experiments were performed to estimate the relative electrogenicity of Na+ and Ca2+ transport reactions. Outward current transients associated with Na+ extrusion were much smaller for NCX-SQ1 than NCX1, and inward current
transients associated with Ca2+ extrusion were much larger. For NCX-SQ1, charge movements of Ca2+ transport
could be defined in voltage jump experiments with a low cytoplasmic Ca2+ (2 µM) in the presence of high extracellular Ca2+ (4 mM). The rates of charge movements showed "U"-shaped dependence on voltage, and the slopes
of both charge-voltage and rate-voltage relations (1,600 s
1 at 0 mV) indicated an apparent valency of
0.6
charges for the underlying reaction. Evidently, more negative charge moves into the membrane field in NCX-SQ1
than in NCX1 when ions are occluded into binding sites.
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INTRODUCTION |
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Na+-Ca2+ exchangers mediate large Ca2+ fluxes across
the plasma membranes of many cell types and thereby
modulate diverse cell functions (for overview, see Hilgemann et al., 1996). Their major physiological role is to
extrude Ca2+ in exchange for extracellular Na+, which
is subsequently extruded by the ATP-dependent Na+-K+
pump.
Historically, two experimental models were used to
characterize Na+-Ca2+ exchange function: cardiac sarcolemmal vesicles (for review, see Philipson and Reeves,
1989) and perfused squid giant axons (for review, see
DiPolo, 1989
; DiPolo and Beaugé, 1991
). Molecular studies of the cardiac Na+-Ca2+ exchanger (NCX1)
were made possible by its cloning (Nicoll et al., 1990
).
This was followed by the cloning and functional characterization of two other mammalian exchangers (NCX2
and NCX3; Li et al., 1994
; Nicoll et al., 1996a
, 1996b
)
and an NCX-type exchanger from Drosophila (Ruknudin et al., 1997
; Schwarz and Benzer, 1997
). Mutational studies of NCX1 have identified functional domains involved in both regulation and transport (Matsuoka et
al., 1993
, 1995
, 1997
; Levitsky et al., 1994
; Nicoll et al.,
1996a
). Given the extensive nature of Na+-Ca2+ exchange studies in squid giant axons, cloning and molecular characterization of the squid exchanger could
provide many unique insights.
Basic properties of the cardiac and squid exchangers
are clearly similar. These include a 3 Na+ to 1 Ca2+ stoichiometry, regulatory activation of exchanger-mediated Ca2+ influx by cytoplasmic Ca2+ (DiPolo, 1979;
Kimura et al., 1986
), and stimulation by ATP-dependent mechanisms (DiPolo, 1974
; Baker and McNaughton, 1976
; Hilgemann, 1990
). Nevertheless, recent results suggest that there are important differences in the
function and regulation of the different exchangers. (a) The ATP dependence of the squid exchanger appears to reflect its phosphorylation by a protein kinase
(DiPolo and Beaugé, 1994
; DiPolo et al., 1997
), while
the ATP-dependent activation of the cardiac exchanger
appears to reflect the generation of phosphatidylinositol 4'-,5'-bisphosphate (PIP2)1 from phosphatidylinositol (Hilgemann and Ball, 1997
). ATP
S activates the
squid exchanger but not the cardiac exchanger. Cationic agents that bind anionic lipids inhibit the cardiac
exchanger (Hilgemann and Collins, 1992
), but agents
such as pentalysine do not inhibit the squid exchanger
(R. DiPolo and L. Beaugé, 1993). (b) The squid exchanger is regulated by a phosphoarginine-dependent
process that may involve protein kinases unique to invertebrates (DiPolo and Beaugé, 1995
); phosphoarginine is
without effect on the cardiac exchanger (D.W. Hilgemann, unpublished observations). (c) The Ca2+-Ca2+
exchange operation of the squid exchanger (DiPolo et
al., 1985
; DiPolo and Beaugé, 1990
) and of barnacle
muscle (Rasgado-Flores et al., 1996
) appears to be
strongly voltage dependent, while Na+-Na+ exchange
shows almost no voltage dependence in isotope flux
studies. For the cardiac exchanger, on the other hand,
Na+ transport has been shown to be strongly electrogenic (Hilgemann et al., 1991
; Matsuoka and Hilgemann, 1992
; Powell et al., 1993
), while Ca2+-Ca2+ exchange is only weakly voltage dependent (Hilgemann
et al., 1991
; Matsuoka and Hilgemann, 1992
; Powell et
al., 1993
; Niggli and Lederer, 1991
; Kappl and Hartung, 1996
).
To better compare the function and structure of the exchangers, we have now cloned the squid neuronal Na+-Ca2+ exchanger, successfully expressed it in Xenopus oocytes, and studied its function in giant membrane patches. We describe here functional similarities and differences of the two exchangers, which should facilitate the development of structure/function models and the elucidation of exchanger regulatory mechanisms.
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MATERIALS AND METHODS |
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The squid optic lobe cDNA library was kindly provided by Dr. F. Bezanilla (UCLA) and the stellate ganglion cDNA library and squid tissues were a gift from Drs. W. Gilly and J. Rosenthal (Stanford University, Stanford, CA). Vesicles from squid optical lobe were obtained from Dr. L. Beaugé (Cordoba, Argentina).
Cloning Procedures
PCR with degenerate primers was used to amplify a fragment of
NCX-SQ1 from squid optic lobe cDNA. Primers were synthesized and reverse-phase purified by Retrogen (San Diego, CA). The
primer pairs were designed from conserved amino acid sequences of putative transmembrane segments 6 and 9 of NCX1
and NCX2. The forward primer was a 29-mer with 32-fold degeneracy (5'-TTAAGAATTCTGGAA(A/G)GT(C/T) CT(C/G) TT (C/T)
GC(A/C)T-3') and the reverse primer was a 33-mer with 64-fold
degeneracy (5'-TTAAGAATTCCCAG(A/G)AA(C/G)AC(A/G)TT (C/G)AC(A/C)GC(A/G) TTG-3'). The PCR reaction was carried out for 30 cycles (94°C, 30 s; 42°C, 60 s; 72°C, 120 s). The
PCR product was cloned into the pCRII vector using the TA
Cloning Kit (Invitrogen Corp., San Diego, CA). Two identical
clones were identified as squid exchanger based on their sequence similarity to NCX1 and NCX2 and were used as probes to
screen a squid optic lobe ZAPII cDNA library. A partial clone of
1.5 kb was isolated with a nucleotide sequence ~60% identical to
the 3' end of NCX1, but no clones extending further into the
missing 5' end sequence were isolated. An EcoRI fragment (0.5 kb) from the 5' end of the 1.5-kb clone was then used to screen a
squid stellate ganglion
ZAPII cDNA library. Two clones (SG12
and SG14), each containing the complete coding sequence of
the squid exchanger, were isolated.
Expression of NCX-SQ1 in Xenopus Oocytes
Initially, expression level of SG12 was relatively low in Xenopus oocytes. Therefore, we used the same strategy that improved expression of NCX1 by replacing the 3' untranslated region of SG12
with that of the Na+-glucose cotransporter clone, which possesses
a poly(A)+ tail (Matsuoka et al., 1993). Expression of NCX-SQ1
was improved only moderately after modification. Expression was
optimized when SG12 was subcloned from pBluescript SK+ into
the pBST4 vector (provided by Dr. Bezanilla's laboratory) at the
BglII site. The vector contains the 5' and 3' untranslated regions
of Xenopus
globin, which flank the NCX-SQ1 coding region in
the final construct. The pBluescript SK+ or pBST4 vector containing the NCX-SQ1 full-length cDNA was linearized with XhoI
or SacII, and cRNA was synthesized using the T3 or T7 mMessage
mMachine Kit (Ambion Inc., Austin, TX), respectively. Unincorporated nucleotides were removed with Chromaspin-100 columns (Clontech, Palo Alto, CA). Oocytes were prepared as described by Longoni et al. (1988)
. Oocytes were injected with 46 nl
of cRNA or water, and exchange activity was measured 4 d after
injection as Na+ gradient-dependent 45Ca2+ uptake (Longoni et
al., 1988
; Nicoll et al., 1990
) or as exchange current across giant
excised patches (see below).
Northern Blot Analysis
Total RNA from squid optic lobe and stellate ganglion were prepared using the protocol of Chomczynski and Sacchi (1987) as modified by Quednau et al. (1997)
. Poly (A)+ RNA was isolated
from 100 µg of total RNA using the Poly (A)Tract mRNA Isolation System (Promega Corp., Madison, WI). 1 µg poly (A)+ RNA
from each tissue was fractionated on a 1% agarose/6% formaldehyde gel, transferred to Hybond-N membrane (Amersham Corp.,
Arlington Heights, IL), hybridized with an antisense probe (see
below), and washed as described previously (Nicoll et al., 1996).
Final washing was at 0.2× SSC at 42°C.
A 32P-labeled antisense probe was generated by asymmetric
PCR as described by Sturzl and Roth (1990). A plasmid containing the ApaI to SacI cDNA fragment of NCX-SQ1 (nucleotides
[nts] 1987-2592) was linearized by ApaI restriction enzyme digestion downstream from the primer annealing site. Phenol-purified
template (200 ng) was used together with 200 pmol primer in a
100-µl reaction mix containing 50 µCi of [
-32P]dCTP, 5 U Taq
DNA Polymerase (GIBCO BRL, Gaithersburg, MD). The primer
for PCR was a 20-mer (5'-ATACGCACTT CCACTTCACC-3'; nts
2574-2554). PCR was carried out for 35 cycles (94°C, 45 s; 55°C, 1 min; 72°C, 2 min). Unincorporated nucleotides were removed by
two consecutive steps of ammonium acetate precipitation.
Preparation of NCX-SQ1 Fusion Protein and Antibody Production
An expression construct containing the cDNA coding for the
large intracellular loop of NCX-SQ1 was constructed by PCR.
The 5' end of the forward primer (5'-AAGCATGCGGTGTGATTGTCCAATGT-3'; nts 1767-1785) included an introduced SphI
restriction site and the reverse primer (5'-TTCTGCAGAATGGCCTCAATAAACTG-3'; nts 3007-2989) contained an introduced
PstI restriction site at the 3' end. pBluescript SK+ vector containing the NCX-SQ1 full-length cDNA (10 ng) was amplified using
0.2 mM each of dATP, dCTP, dGTP, and dTTP, 1 mM MgCl2, 5% DMSO, 50 pmol of forward and reverse primer, and 2.5 U of Taq DNA polymerase to produce a 1,240-bp fragment (nts 1767-
3007; amino acids 259-665). PCR was carried out over 35 cycles
(94°C, 30 s; 55°C, 1 min; 72°C, 2 min). The PCR product was
cloned into the pCR II vector using the TA Cloning Kit, and the
identity of the clone was confirmed by sequencing. The PCR construct was digested with SphI and PstI and the resulting fragment
cloned into the expression vector pQE (QIAGEN Inc., Chatsworth, CA). The loop fusion protein was expressed and purified
as described by He et al. (1997) and used as antigen for generation of polyclonal antisera in rabbits (HRP Inc.; Denver, PA).
Western Blot
Samples from oocytes expressing NCX-SQ1 protein were prepared as follows: 10 oocytes were sonicated in 100 µl of homogenization buffer (0.1 M NaCl, 1% Triton X-100, 20 mM Tris-HCl, pH 7.6) and centrifuged at 12,000 rpm for 10 min. The supernatant was filtered twice through Spin-X filters to remove lipids and yolk protein. Supernatant (5 µl) or squid optic lobe vesicles (20 µg total protein) were electrophoresed on a 7% SDS-polyacrylamide gel. Proteins were transferred onto nitrocellulose membrane and the filter was blocked with 5% (wt/vol) milk in MTBST buffer (140 mM NaCl, 20 mM MOPS/Tris, pH 7.4, 0.05% TWEEN 20). The membrane was incubated with the antiserum raised against the loop fusion protein of NCX-SQ1 or preimmune serum at a dilution of 1:5,000, followed by incubation with goat anti-rabbit IgG conjugated with horseradish peroxidase (1:3,000; Bio-Rad Laboratories, Richmond, CA) in MTBST. The antigen-antibody complexes on the membrane were visualized using 3,3'-diaminobenzidine.
Electrophysiological Analyses: Endogenous Oocyte Conductances
Na+-Ca2+ exchange currents were isolated and studied in giant
excised membrane patches from Xenopus oocytes, as described
previously, using solutions that minimize endogenous currents in
the oocyte membrane (Matsuoka et al., 1993, 1995
). The extracellular solution contained 4 mM Ca-sulfamic acid, 1 mM Mg-
sulfamic acid, 40 mM Na-2-(morpholino)ethanesulfonic acid
(MES), 20 mM Cs-MES, 20 mM tetraethyl ammonium (TEA)-
MES, 40 mM N-methyl-D-glucamine (NMG)-MES, and 20 mM
HEPES, adjusted to pH 7.0 with NMG. The cytoplasmic solution
contained (mM): 10 EGTA, 6 Ca-sulfamic acid, 0.5 Mg-sulfamic acid, 60 Cs-MES, 20 TEA-MES, and either 40 additional Cs-MES
or 40 Na-MES to activate outward exchange current, at pH 7.0 with NMG (pCa 6.5). Gigaohm seals were made in a solution containing (mM): 80 K+-aspartate, 40 KCl, 4 MgCl2, 5 EGTA, and 10 HEPES, at pH 7.0 with NMG. Experiments were performed at
32°C. Concentrated stock solutions of nucleotides were prepared
as Mg2+/TRIS salts, with the Mg2+ concentration adjusted to 75%
of the total nucleotide concentration. In this way, the free Mg2+
concentration (0.3 mM) is not changed on addition of nucleotide. Free Ca2+ and Mg2+ concentrations were calculated with
the binding constants given by Fabiato (1988)
, and for ANP-PNP
(adenosine 5'-(
,
-imido)triphosphate) we assumed the same
Mg binding constant as for ATP. Unless indicated otherwise, the
membrane potential was 0 mV. PIP2 liposomes were prepared by
sonicating 1 mM PIP2 (Boehringer Mannheim, Mannheim, Germany) in distilled water. Reconstituted monoclonal PIP2 antibody (PerSeptive Biosystems, Framingham, MA) was diluted 40-fold into the experimental solution.
Endogenous conductances of the oocyte membrane were found to be activated by ATP and anionic lipids, so more extensive control experiments were needed to test for the influence of contaminating currents. Most importantly, we established conditions such that current changes in patches from uninjected (or water-injected) oocytes amounted to at most a few picoamperes using the same conditions and protocols employed in RESULTS (i.e., at 0 mV).
Since our efforts to understand and minimize endogenous
conductances are relevant to many expression studies with Xenopus oocytes, we describe here the two major oocyte currents activated by ATP and PIP2; Ca2+-activated Cl and voltage-activated
Na+ currents. Activation of Ca2+-activated Cl
current by ATP
(Hilgemann, 1997
) and anionic lipids in oocyte patches (Hilgemann, 1995
) has been described previously. As shown in Fig. 1,
Ca2+-activated Cl
current is stimulated by many polyvalent anions (0.1-2 mM) and by F
when cytoplasmic free Ca2+ is submaximal. Fig. 1 A shows results for phosphate. Activation by polyvalent anions typically takes ~1 min, but the effect decays on removal of anions in only a few seconds. Possibly, these effects reflect chelation of trace polyvalent cations in solutions (or from
the pipette) by these anions (Hilgemann, 1997
). Since the pipette tip can become contaminated with Cl
during seal formation, there is a danger that outward Cl
current can occur during
exchange current measurements. Inclusion of Cl
current blockers in the pipette solution (0.3 mM niflumic acid + 0.3 mM flufenamic acid) effectively blocked the residual Cl
currents, while
results with PIP2 and nucleotides in exchanger-expressing patches were not changed.
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As shown in Fig. 2 A, a Na+ current can be activated in oocyte
patches by depolarization for a few seconds beyond 0 mV. As described in previous two-electrode voltage clamp studies (Baud and Kado, 1984), this current activates and deactivates rapidly in
response to voltage steps after it has first been primed by a long
depolarization. Fig. 2 B shows the steady state current-voltage relation of an oocyte patch with symmetrical Na+-containing solutions (40 mM). We stress that the Na+ conductance was always
negligible at potentials more negative than ~
20 mV. In the absence of ATP and PIP2, the magnitude of the Na+ conductance
was variable and depended on the oocyte batch. Na+ conductance could be induced in the majority of patches by ATP or anionic lipids. Fig. 2 C shows the activation by ATP, reversal after
ATP removal, and suppression by 40 mM aluminum in the presence of 10 mM EGTA (50 ms cumulative voltage pulses). To
avoid contamination of exchange currents with Na+ current, we
made our recordings with 40 mM Na+ on both sides at 0 mV (i.e.,
at the reversal potential of the Na+ current). Also, we monitored
current-voltage relations using 2-ms voltage steps and verified
that the current activated by ATP and PIP2 remained outward in
direction at potentials as negative as
70 mV with 40 mM extracellular and cytoplasmic Na+ concentrations. Thus, the underlying mechanism could not involve Na+ channels.
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RESULTS |
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Cloning the cDNA Coding for the Squid Na+-Ca2+ Exchanger
A fragment of the squid exchanger cDNA was obtained
by PCR amplification of cDNA derived from a squid optic lobe library. Degenerate primers were designed
based on conserved amino acid sequences of proposed
transmembrane segments 6 and 9 of the mammalian exchangers NCX1 and NCX2. An appropriately sized
PCR product (~320 bp) was subcloned. Sequence analysis indicated that the DNA coded for a protein homologous to NCX1. The PCR product was used to screen a
squid optic lobe library and a partial clone of 1.5 kb was
isolated with ~60% identity to NCX1 at the amino acid
level. Longer clones were not found in the optic lobe library. A fragment of the partial 1.5-kb clone was then
used to screen a squid stellate ganglion cDNA library.
Two clones containing the complete coding sequence
of the squid exchanger were isolated. These clones
were identical at the 3' end. The complete nucleotide
and amino acid sequences of the longest clone, SG14,
are shown in Fig. 3. SG14 is 4,096 bp long with an open
reading frame of 2,676 nucleotides encoding for a protein of 892 amino acids, which we refer to as NCX-SQ1.
The most 5' ATG, in the proper reading frame, begins at nucleotide 994 with some features of a Kozak (1989)
consensus initiation site. The 3' end of the cDNA terminates with 16 adenosines preceded by multiple consensus polyadenylation sites.
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The amino acid sequence of the squid stellate ganglion Na+-Ca2+ exchanger, aligned with the sequence
of the canine cardiac Na+-Ca2+ exchanger (NCX1.1 in
the terminology of Quednau et al., 1997) is shown in
Fig. 4. Like NCX1, NCX-SQ1 has 12 hydrophobic segments that could form transmembrane segments. In
NCX1, the first hydrophobic segment at the NH2 terminus is a cleaved leader peptide and is removed from the
protein during biosynthesis in the endoplasmic reticulum (Durkin et al., 1991
; Hryshko et al., 1993
). NCX-SQ1 has a potential signal peptidase-recognition site
(Von Heijne, 1983
) after alanine 26, and we predict
that a signal peptide is removed from this protein as
well. Thus, the mature protein is modeled to have 11 transmembrane segments with a large intracellular loop separating transmembrane segments 5 and 6. The
NH2 terminus would be extracellular and the COOH
terminus would be intracellular. NCX-SQ1 has nine potential sites for N-linked glycosylation. If the glycosylation pattern is similar to that of NCX1 (Hryshko et al.,
1993
), only the first site (asparagine 31) would be glycosylated. Overall, the mature NCX-SQ1 and NCX1
proteins are 58% identical, ignoring the gap in NCX-SQ1 in the intracellular loop region. More sequence
analysis will be presented below.
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The transcript size of NCX-SQ1 was determined by Northern blot analysis. Poly (A)+ RNA from squid optical lobe and stellate ganglion hybridized with an NCX-SQ1 probe at 9.6 kb (Fig. 5 A). The signal was substantially stronger in stellate ganglion than in optical lobe. The NCX-SQ1 mRNA is larger than that for NCX1 (7 kb), NCX2 (5 kb), or NCX3 (6 kb) (Nicoll et al., 1996). The NCX-SQ1 transcript apparently has a long 5' untranslated region since the clone ends with a poly (A)+ tail. Extensive untranslated regions also occur for the squid Na+ channel that has a transcript >12 kb (Rosenthal et al., 1993), which is larger than that of the mammalian Na+ channels.
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A His-tagged fusion protein encompassing amino acids 259-665 of NCX-SQ1 was expressed in Escherichia
coli. After affinity purification, the fusion protein was
used for polyclonal antibody production in rabbit. Expression of NCX-SQ1 in membrane vesicles from squid
optic lobe was detected by immunoblot (Fig. 5 B, lane 1). The antibody recognizes a major band at ~120 kD
and two bands at lower molecular weights. NCX1 produces a similar pattern on immunoblots (Nicoll et al.,
1990). Preimmune serum from the same rabbit failed
to produce any signal (not shown). Expression of exchanger protein in Xenopus oocytes injected with NCX-SQ1 cRNA was also examined. A protein band of ~100
kD was detected in a membrane fraction from cRNA-
injected oocytes, whereas no signal was detected in control water-injected oocytes (Fig. 5 B, lanes 2 and 3). The
small difference in apparent molecular weight between the native squid exchanger and NCX-SQ1 expressed in
oocytes is possibly due to a difference in glycosylation.
Functional Expression of NCX-SQ1 in Xenopus Oocytes
cRNA encoding NCX-SQ1 was synthesized from linearized plasmids and injected into Xenopus oocytes. Expression was optimized when the 5'- and 3'-untranslated regions of Xenopus globin flanked the NCX-SQ1
coding region. Expression of Na+-Ca2+ exchange activity was assessed by measuring 45Ca2+ fluxes into intact
oocytes and by measuring exchanger currents using the
giant excised patch technique. An example of Na+ gradient-dependent 45Ca2+ uptake into Na+-loaded Xenopus oocytes is shown in Fig. 6. The first columns show
that NCX-SQ1 RNA induces a substantial uptake of
45Ca2+ in the presence of an outwardly directed Na+
gradient (K+0). This uptake is abolished in the absence
of the Na+ gradient (Na+0). We have validated the use
of this approach to measure Na+-Ca2+ exchange activity in previous studies (Longoni et al., 1988
; Nicoll et al.,
1990
). No Ca2+ uptake was observed in control water-
injected oocytes.
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We used the 45Ca2+ uptake assay to determine
whether NCX-SQ1 exhibited secondary Ca2+ regulation
by intracellular Ca2+. The presence of micromolar levels of [Ca2+]i is required in squid giant axons to activate
Na+i-dependent Ca2+ uptake (Baker and McNaughton,
1976; DiPolo and Beaugé, 1986
). Oocytes expressing
NCX1-SQ1 were injected with EGTA before the Ca2+
uptake assay. Chelation of internal Ca2+ blocked most
Ca2+ uptake (Fig. 6), which is consistent with previous
studies of squid axons and NCX1 (Baker and McNaughton, 1976
; DiPolo and Beaugé, 1986
; Kimura et
al., 1986
; Hilgemann et al., 1992a
, 1992b).
Outward Exchange Current of the Squid (NCX-SQ1) Na+-Ca2+ Exchanger
Fig. 7 shows basic properties of outward Na+-Ca2+ exchange current in patches from oocytes expressing the
NCX-SQ1 exchanger. In brief, no obvious property of
the current is different from NCX1 exchange current.
On application of cytoplasmic Na+, the current activates in the solution switch time and then shows partial
inactivation over several seconds (see Fig. 7, first record, in the presence of 1 µM cytoplasmic free Ca2+). When
cytoplasmic Ca2+ is removed, the current magnitude
decreases, and inactivation on application of Na+ is
subsequently faster. Exchange current remains substantial (second record). With 5 µM free cytoplasmic Ca2+
(third record), or higher concentrations (not shown),
the current did not increase further and ran down with
time. Current run-down prevented us from determining Ca2+-current relations in more detail. Application
of -chymotrypsin (1 mg/ml) activated the exchange
current over 1 min. The final current magnitude was
typically more than twice the peak current magnitude
obtained on applying Na+. After chymotrypsin, the exchange current was insensitive to changes of cytoplasmic free Ca2+ from 0 to 5 µM.
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Stimulation of NCX1 Exchange Current by ATP and Reversal of Stimulation by Anti-PIP2 Antibody
ATP strongly activates exchange current in excised cardiac membrane patches and vanadate is without effect
on the stimulatory effect or its reversal after removal of
ATP (Collins et al., 1992). However, in our initial studies of the cardiac exchanger (NCX1) in oocyte patches,
no effects of ATP were observed (Matsuoka et al.,
1993
). We now report that ATP indeed can be effective
when the phosphatase inhibitors, F
and vanadate, are
included in the cytoplasmic solution. The concentrations of F
and vanadate are selected to maintain free
Mg2+ in the range of 0.1-0.3 mM, and vanadate alone
was usually effective. Fig. 8 describes typical results with
0.1 mM F
and 50 µM vanadate (0.5 mM total Mg2+ in
nucleotide-free solution). The outward current activated by Na+ shows the usual inactivation when 40 mM
cytoplasmic Na+ is applied. As shown in Fig. 8 A, 2 mM
of the nonhydrolyzable ATP analogue, AMP-PNP, was
without effect, while 2 mM ATP increased the current
to a magnitude somewhat greater than the peak current on initial application of Na+. On removal of ATP,
the current returned partially toward baseline over 2 min. The thio-analogue of ATP, ATP
S (2 mM), typically had no effect, or only a small stimulatory effect
(Fig. 8 B). After removal of ATP
S, application of ATP
stimulated the exchange current, as usual. As mentioned in the INTRODUCTION, the stimulatory effect of
ATP in cardiac membrane appears to reflect the generation of PIP2 from phosphatidylinositol. PIP2 also activates some potassium channels (Hilgemann and Ball,
1997
) and, with inward rectifier potassium channels,
application of an antibody to PIP2 reverses the ATP effects within 1 min (Huang et al., 1998
). As shown in the
latter part of the record in Fig. 8 B, the same antibody
to PIP2 (AB) reverses the stimulatory effect of ATP on
the NCX1 exchanger; the antibody was without effect
when exchange currents were stimulated by other anionic lipids (e.g., cardiolipin).
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Stimulation of Squid (NCX-SQ1) Na+-Ca2+ Exchange by ATP and Its Reversal by Anti-PIP2 Antibody
Fig. 9 shows typical effects of ATP on the outward NCX-SQ1 exchange currents. In Fig. 9 A, the current was
first activated by cytoplasmic Na+, and it then inactivated by ~85%. With application of 2 mM ATP, the
current increased to a magnitude higher than the peak
current on initial application of Na+. Notably, this result was often obtained in the absence of F and vanadate; stimulation by ATP was observed only rarely with NCX1 in this condition. After removal of ATP, the current declines about half-way toward baseline over 1 min
before it declines more slowly. After allowing current to
return nearly to the control level (not shown), Na+ was
removed and reapplied in the presence of 0.2 mM F
and 50 µM vanadate. The current increased to the
same extent as previously upon addition of ATP, but on
removal of ATP the current remained stable; presumably, the maximal currents in these records reflect a
"ceiling" for simulatory effects. Reversal rates are ~10×
slower than without the phosphatase inhibitors. Fig. 9 B
demonstrates that the PIP2 antibody is also effective in
reversing the stimulatory effects of ATP on the squid
exchanger. F
and vanadate are present throughout
the experiment. After application of the antibody, current returned nearly to baseline in ~2 min in the presence of antibody.
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Stimulation of the Squid (NCX-SQ1) Na+-Ca2+ Exchange
Current by ATPS and Its Reversal by PIP2 Antibody
The NCX-SQ1 Na+-Ca2+ exchange current, in contrast
to NCX1 current, was usually strongly stimulated by applying ATPS (four of six results). Typical results are
shown in Fig. 10. In the presence of F
and vanadate,
application of a nonhydrolyzable ATP derivative, AMP-PNP (2 mM) was nearly without effect (Fig. 10 A); the
small inhibition of exchange current may be due to a
small increase of free Mg2+. Thereafter, application of
2 mM ATP
S stimulates the exchange current roughly
to the extent observed with ATP in NCX-SQ1-expressing patches. The stimulatory effect reverses within a few
minutes on removal of the ATP
S. As shown in Fig. 10
B, PIP2 antibody can reverse the effect of ATP
S over
the course of 90 s, suggesting a possible involvement of
PIP2 in the effects of ATP
S as well as those of ATP.
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Stimulation of the Squid (NCX-SQ1) Na+-Ca2+ Exchange Current by PIP2
Fig. 11 A shows the typical stimulatory effect of PIP2 liposomes on NCX-SQ1 exchange current. Exchange current was activated by application of cytoplasmic Na+, and it was allowed to run-down for 3 min to a very small magnitude. Then, 50 µM PIP2 was applied and current increased over several minutes, comparable to the effect of chymotrypsin (see Fig. 7). To check for conflicting ionic currents, current-voltage relations were acquired at several times. The records 1, 2, and 3, indicated in Fig. 1 A, were subtracted so as to define the current activated by PIP2 (±PIP2; Fig. 11 B, 2-1) and the current switched off by removing Na+ at the end of the recording period (±Na+; Fig. 11 B, 2-3). The two current-voltage relations are very similar, indicating that PIP2-activated outward current is also activated by cytoplasmic Na+, and the shapes of current-voltage relations are typical for outward exchange current. We note also that similar results were obtained in patches from HEK cells in which NCX-SQ1 was expressed by transient transfection, although the maximum exchange currents were only ~15 pA (not shown).
|
Fig. 11 C shows the inactivation time courses observed on application of cytoplasmic Na+, before and
after applying PIP2 in another NCX-SQ1-expressing patch. Single exponential functions were fitted to the
inactivation phases, and they are plotted as dotted
lines. The time constant of inactivation increases from
3.9 s in control records to 8.2 s after application of
PIP2. This is similar to the slowing of inactivation observed with cardiac exchange current when ATP is applied (Hilgemann et al., 1992b), indicating that similar
molecular mechanisms might be involved.
Lack of Effect of Phosphoarginine on Outward Squid (NCX-SQ1) Na+-Ca2+ Exchange Current in Oocyte Patches
As noted in the INTRODUCTION, the high-energy compound phosphoarginine stimulates the Na+-Ca2+ exchange process in squid axons, probably by a mechanism that involves phosphorylation (DiPolo and Beaugé,
1995). As shown in Fig. 12, phosphoarginine (5 mM)
was without effect on the outward exchange current in
oocyte patches (four observations, applying phosphoarginine with Mg2+). In the same patch, 50 µM PIP2
was highly effective. As with all nucleotides, other phosphates, and citrate, large stimulatory effects were observed in patches when phosphoarginine was applied without added Mg2+ (not shown). The probable explanation for our results in patches is that all of these anions chelate Mg2+ and thereby relieve an inhibition of
exchange current by cytoplasmic Mg2+.
|
Voltage Dependence of Chymotrypsin-deregulated Outward NCX-SQ1 Exchange Currents
To compare the voltage dependencies of NCX-SQ1 exchange current with those of cardiac exchange current,
we examined both the outward and inward exchange
currents under "zero trans" conditions (i.e., with Na+
and no Ca2+ on one membrane side, and Ca2+ but no
Na+ on the other side). This was carried out in chymotrypsin-treated patches, so that regulatory mechanisms
were absent. We allowed chloride and sodium currents
to "run down" for a few minutes in Mg2+-containing solution before application of -chymotrypsin (1 mg/ml for 30 s). Current changes were negligible in patches
from uninjected oocytes, using the same conditions
and protocols.
Fig. 13 A shows current-voltage relations for outward
NCX-SQ1 exchange current with 4 mM extracellular
Ca2+ and no extracellular Na+. With the Ca2+-free/10
mM EGTA cytoplasmic solution employed in these experiments, Ca2+-activated Cl conductance is zero, and
the same solutions (with 20 mM Cl
) could be used
as in previous measurements with cardiac membrane patches (Hilgemann et al., 1992a
). Cytoplasmic Na+
was varied from 5 to 90 mM, substituting it for Cs+, and
baseline current-voltage relations in the absence of
Na+ were subtracted. The current-voltage relations are
similar in shape and they can be scaled well to each
other (not illustrated). Fig. 13 B shows the Na+ dependence of current at
60 and +60 mV; the K50 (half-maximal concentration) for Na+ is 27 mM at +60 mV
and 24 mM at
60 mV, and the Hill slopes are 1.2 and
1.7, respectively. For the cardiac exchanger, by contrast, current-voltage relations become less steep with
high cytoplasmic Na+, and the K50 for Na+ decreases
somewhat at positive potentials (Matsuoka and Hilgemann, 1992
).
|
Fig. 14 compares inward exchange current-voltage
relations for NCX-SQ1 and NCX1, expressed in the
same batch of oocytes and using the same Cl-free solutions. The cytoplasmic solution was the same as described in MATERIALS AND METHODS without Na+. The
pipette solution contained 120 mM Na+ (see Fig. 14,
legend, for complete composition). Fig. 14, A and B,
shows results for NCX1 and NCX-SQ1, respectively,
with 0.2, 2, 10, and 300 µM free cytoplasmic Ca2+. The
current-voltage relations of the NCX-SQ1 exchanger
are substantially steeper than those for the NCX1 exchanger and have a more "exponential" form. Those
for NCX1 are nearly linear over the entire voltage range. For NCX-SQ1, current doubles in ~30 mV in
the steepest region of the current-voltage relations.
This is close to the slope expected for a single charge
movement across the entire electrical field. Fig. 14, C
and D, shows the Ca2+ dependencies of the inward current at
150 and
30 mV. Both data sets are well-
described by Hill equations with slopes of 1, and there
are small shifts of the K50's to higher concentrations at more negative potentials; 2.5-4.2 µM for NCX1, and
3.5-7.2 µM for NCX-SQ1.
|
Na+ and Ca2+ Concentration Jump-induced Charge Movements of the NCX-SQ1 Exchanger
As mentioned in the INTRODUCTION, Ca2+ flux studies
in squid axons suggest that Ca2+ translocation is substantially more voltage dependent than Na+ translocation (DiPolo et al., 1989; DiPolo and Beaugé, 1990
) and similar results are described for the barnacle Na+-
Ca2+ exchanger (Rasgado-Flores et al., 1996
). To test
directly whether Ca2+ translocation is electrogenic in
NCX-SQ1, ion concentration jump experiments were
designed to isolate the possible charge movements of
outward Ca2+ and Na+ translocation. To do so, the outward translocation of ions is initiated by moving the
patch pipette tip through the interface between two solution streams in 1 ms, whereby one stream contains no
substrate and the other contains a high substrate concentration. A computer-controlled Piezzo-type manipulator is used to move the patch clamp head stage together with the patch pipette (Hilgemann and Lu,
1998
). The speed of the substrate concentration change occurring at the membrane surface in these experiments (~150-ms time constant) is determined by diffusion from the pipette orifice to the membrane surface
(20-70 µm). We point out, however, that the speed of
current activation, observed upon applying a substrate, can be much faster. That is because binding sites can
be saturated quickly, compared with average diffusion
times, if substrate concentrations are high with respect
to binding site affinity. This is the case for experiments
with Na+ jumps, but it appears not to be the case for
Ca2+ jumps.
Fig. 15 illustrates the major experimental results. All
patches were chymotrypsin treated and it is noted that
experiments were successful only in oocyte batches with
high exchanger expression (approximately one batch
out of six). The protocols, based on the predicted function of an alternating-access exchange model, were the
same as used previously to monitor "half-cycles" of ion transport (Hilgemann et al., 1991): in the presence of
substrate on the extracellular side and no substrate on
the cytoplasmic side, the exchanger binding sites will
orient to the cytoplasmic side and will be free of substrate. When a high concentration of substrate is applied to the cytoplasmic side, substrate will bind and
the binding sites will reorient and open to the extracellular side. Substrate will be released, and binding sites
will remain in the extracellular orientation, on average,
if the extracellular substrate concentration is relatively
low. Thus, charge movement observed during this protocol should reflect the electrogenicity of ion transport
for the substrate added.
|
Fig. 15, A and B, shows results for jumping cytoplasmic Na+ from 0 to 40 mM in the presence of 20 mM extracellular Na+ (no Ca2+ on either membrane side).
Results for NCX1 are the same as described previously
(Hilgemann et al., 1991). An outward current transient, ~150 ms in duration with a peak of ~3 pA, is observed on application of Na+, and a slower inward current transient with a peak of ~1 pA is observed on removing Na+. The areas defined by the current transients
correspond to ~300,000 elementary charges, which in
turn corresponds to ~300 charges/µm2 with a 10-pF
patch. Using the same protocols in patches expressing the squid exchanger, current transients were either absent or very small (five observations; Fig. 15 B). That
these results reflect a real difference in exchanger function is supported by the observation that inward currents activated by applying cytoplasmic Ca2+ were of
similar magnitude in the NCX1 and NCX-SQ1 patches
(Fig. 15, C and D). We point out that the current activated by 5 µM Ca2+ corresponds to ~10% of the current activated in patches from the same oocyte batches
when 150 mM Na+ was included in the pipette.
Fig. 15, E and F, shows the equivalent results for
jumps of cytoplasmic Ca2+ from 0 to 5 µM in the presence of 50 µM extracellular Ca2+. As described previously (Hilgemann et al., 1991), charge movements are
small or absent for this protocol with NCX1. For NCX-SQ1, however, inward current transients are obtained
on applying Ca2+ and outward current transients on removing Ca2+. These charge movements may correspond to a movement of negative charge when Ca2+ is
moved by the exchanger. The magnitudes of charge
movements in these experiments were roughly similar
to those for Na+ jumps with NCX1 (Fig. 15 A). Fig. 15,
G and H, shows the time courses with which outward
exchange current was turned on and off with cytoplasmic Na+ jumps.
Voltage Jump-induced Charge Movements of Ca2+ Transport of the NCX-SQ1 Exchanger
For the cardiac exchanger (NCX1), charge movements
of Ca2+ transport have been isolated in voltage-jump
experiments (Hilgemann, 1996). The charge movements were of small magnitude and showed weak voltage dependence with rates of ~5,000 s
1 at 0 mV. Results for NCX-SQ1, after chymotrypsin treatment, are shown in Fig. 16. The rationale of the experiment is
that most of the voltage dependence of Ca2+ transport
comes about during occlusion of Ca2+ from the cytoplasmic side. Therefore, a high concentration of Ca2+,
4 mM, is included on the extracellular side. Without cytoplasmic Ca2+, all exchangers should orient to the cytoplasmic side with empty binding sites. Voltage pulses
are applied first in the absence of cytoplasmic Ca2+,
and then in the presence of 2 µM cytoplasmic Ca2+ to
activate the ion occlusion reaction. Charge rather than
current was recorded, and the holding potential was
40 mV. Records presented in Fig. 16 A are a subtraction of records with cytoplasmic Ca2+ from records
without Ca2+, whereby 16 records were acquired in alternating order with and without cytoplasmic Ca2+, and
results were averaged. The charge movements show
small fast components that appear as charge jumps on
changing voltage, and they show slower components
that saturate progressively as larger voltage pulses are
applied to +160 and
200 mV. Fig. 16 B shows the voltage dependence of the charge movements, fitted to a
Boltzmann relation {1/[1 + expq*(Em+E50)/26.5]}, which
gives an equivalent charge (q) of 0.46 underlying the charge movement. This is twice the value obtained for
NCX1 (Hilgemann, 1996
). Fig. 16 C shows the voltage
dependence of the rates of the charge movements, obtained by fitting the slow components of Fig. 16 A to single exponential functions. The rates have a "U-shaped" dependence on voltage, as expected for a simple reaction with voltage dependence of both the forward and
reverse rates. The rates can be well-described by the
sum of two exponentials, Kf · eq*Em/55 + Kb · e
q*Em/55,
where Kf and Kb are the forward and backward rates at
0 mV. The fit gives an equivalent charge of 0.59. The
overall rate at 0 mV is ~1,600 s
1, which is about threefold lower than rates obtained in equivalent experiments with NCX1 (Hilgemann, 1996
).
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DISCUSSION |
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The Squid Na+-Ca2+ Exchanger, NCX-SQ1
We have cloned and expressed the squid neuronal
Na+-Ca2+ exchanger, NCX-SQ1. The squid exchanger
is a member of the family of NCX-type exchangers, including three mammalian exchangers, NCX1 (Nicoll et
al., 1990), NCX2 (Li et al., 1994
), and NCX3 (Nicoll et
al., 1996), as well as the Drosophila Na+-Ca2+ exchanger
(Schwarz and Benzer, 1997
; Ruknudin et al., 1997
). NCX-SQ1 is 58% identical at the amino acid level to
the canine cardiac Na+-Ca2+ exchanger, NCX1, and
has similar identities (51-64%) to the other NCXs.
There are several features of note. Regions determined
to be of functional importance in previous studies of
NCX1 are well conserved. For example, we have identified specific acidic residues within the binding site for
regulatory Ca2+ (Fig. 4, boxed area) that are important
for Ca2+ binding (Levitsky et al., 1994
; Matsuoka et al.,
1995
). These triple aspartate (DDD) motifs (Fig. 4,
bold) are perfectly conserved in all NCX exchangers.
Likewise, we have described the endogenous XIP region of NCX1 and have proposed that the XIP region is
involved in Na+-dependent inactivation (Li et al., 1991
;
Matsuoka et al., 1997
). A homologous region (Fig. 4,
shaded area) is also present in NCX-SQ1 and is conserved among the exchangers.
The predicted topology of NCX-SQ1 is similar to that
of the other exchangers with 11 transmembrane segments and a large intracellular loop. Sequence conservation among the exchangers is highest in the proposed
transmembrane segments consistent with a catalytic role
of the hydrophobic domains in ion translocation. We
have described that exchanger function is especially
sensitive to mutations within portions of transmembrane
segments 2, 3, 8, and 9 (Nicoll et al., 1996). These regions are known as the repeats (
-1 and
-2). Significantly, the
repeats are highly conserved in species as
divergent as squid and dog, consistent with a proposed
role in ion transport. Proposed transmembrane segment 11 is the least well conserved transmembrane domain among the NCX exchangers. Perhaps the COOH
terminus of the protein has a lesser role in exchange
function. As noted previously (Tsuruya et al., 1994
), the NH2 terminus of the exchanger, which represents a
signal peptide region, is poorly conserved among NCX
proteins.
The squid Na+-Ca2+ exchanger has previously been
reported to be stimulated by phosphorylation reactions
and by phosphoarginine (DiPolo and Beaugé, 1994; DiPolo et al., 1997
) and we analyzed the NCX-SQ1 sequence for potential phosphorylation sites. Potential sites
for phosphorylation by protein kinases A and C, Ca2+/
calmodulin-dependent kinase, and tyrosine kinases are
shown in Fig. 4. The PKC site (threonine 184) between
transmembrane segments 3 and 4 and the tyrosine kinase site in the Ca2+ binding region (tyrosine 462) are
unique among the exchangers. Phosphorylation at either site might be expected to have functional effects.
As shown in Fig. 4, there is a deletion of 47 amino acids in the large intracellular loop of NCX-SQ1 in comparison with NCX1. This region of NCX1 displays extensive alternative splicing (Nakasaki et al., 1993; Kofuji
et al., 1994
; Lee et al., 1994
; Quednau et al., 1997
). Six
small exons are used in different combinations in a tissue-specific manner. The first two of these exons (exons A and B) are mutually exclusive. The NCX1 splice
variant in Fig. 4 is NCX1.1 (Quednau et al., 1997
) using
exons A and C-F. Of the six small exons, NCX-SQ1 apparently uses only exon A. The homologous exons A of
NCX-SQ1 and NCX1 are shaded in Fig. 4. We performed reverse transcriptase-PCR using optic lobe and
stellate ganglion RNA to detect other splice variants of
NCX-SQ1. Nine clones were sequenced, but no other
splicing isoforms were detected.
Regulation of Na+-Ca2+ exchange by intracellular
Ca2+ was first described in the squid axon (Baker and
McNaughton, 1976; DiPolo, 1979
). Micromolar levels
of free intracellular Ca2+ are required to activate function. That is, in addition to transporting Ca2+, the exchanger is separately regulated by Ca2+. This secondary
effect of intracellular Ca2+ has been analyzed at the molecular level for the cloned cardiac exchanger NCX1
(Levitsky et al., 1994
; Matsuoka et al., 1995
). Using
45Ca2+ fluxes, we find that Ca2+ regulation is apparently
intrinsic to the NCX-SQ1 exchanger protein. Chelation
of intracellular Ca2+ with EGTA prevents Na+i-dependent Ca2+ uptake (Fig. 6). This is not surprising since,
as noted above, the regulatory Ca2+ binding site of
NCX1 is conserved in the sequence of NCX-SQ1.
Regulatory Mechanisms Acting on NCX-SQ1 in Oocyte Giant Patches
Given the sequence similarities to NCX1, it is not surprising that many of the regulatory properties of NCX-SQ1 are similar to those of NCX1. This includes the
properties of Na+-dependent inactivation, secondary
activation by cytoplasmic Ca2+, and deregulation by
chymotrypsin (Fig. 7). Furthermore, similarities include stimulation by ATP and PIP2, and reversal of the
stimulatory effects of ATP by a PIP2 antibody (Figs. 8
and 9). However, there are also evident differences between NCX-SQ1 and NCX1. In the absence of phosphatase inhibitors, NCX-SQ1 exchange current is usually
stimulated by ATP, which is not the case with NCX1. Either the squid exchanger has higher affinity for PIP2 or it
is modulated by an additional ATP-dependent reaction. Consistent with this possibility, the thioester of
ATP, ATPS, stimulates NCX-SQ1 in oocyte patches
but not NCX1 current (Figs. 8 and 10). An attractive speculation is that we are observing phosphorylation of
NCX-SQ1 at one of its consensus phosphorylation sites.
We cannot, however, completely eliminate a possibility
that effects of the ATP
S preparations employed reflect
contaminating ATP and an increased sensitivity of the
squid to PIP2.
The reversal of ATP effects on Na+-Ca2+ exchange
current by a PIP2 antibody is reported here for the first
time. The fact that the PIP2 antibody can reverse stimulatory effects of ATPS on the squid exchanger indicates that PIP2 is involved. Perhaps phosphorylation increases the affinity of NCX-SQ1 for PIP2. Precedents for
such a mechanism come from recent work with inward
rectifier potassium channels. With GIRK-type channels,
activation by G-protein
subunits is accompanied by
an increase of affinity for PIP2 (Huang et al., 1998
),
and, with ROMK-type channels, activation by cAMP- dependent protein kinase is accompanied by an increase
of the apparent affinity for PIP2 (Dr. C.L. Huang, personal communication). Since ATP increases the apparent affinity for Ca2+ at exchanger regulatory sites (DiPolo and Beaugé, 1987
; Collins et al., 1992
), a primary
effect of phosphorylation on Ca2+ affinity is also a possibility. In contrast to our results, it is reported that the
PIP2 antibody is without effect on Na+-Ca2+ exchange
in squid giant axons, either with or without ATP (DiPolo and Beaugé, 1998
). Either the antibody does not
reach the membrane in axons or the exchanger is regulated in a fundamentally different way in the squid
axon environment. The fact that phosphoarginine does
not stimulate the cloned squid exchanger presumably
reflects the absence of appropriate phosphoarginine-
dependent kinases in oocyte giant membrane patches.
Voltage Dependence of NCX-SQ1 in Oocyte Giant Patches
The ion dependencies of the NCX-SQ1 exchange currents, determined here for the cytoplasmic side (Figs.
13 and 14), are consistent with those determined in dialyzed squid axons (DiPolo, 1989) and are only slightly
different from results for the NCX1 exchanger. As described in Figs. 13 and 14, the voltage dependencies of
outward and inward exchange currents of NCX-SQ1
are stronger than for NCX1, and the isolation of
charge movements of Ca2+ transport for the squid exchanger (Figs. 15 and 16) verifies that electrogenic reactions are indeed different in the squid exchanger. Na+ transport is relatively less electrogenic, while Ca2+
transport is more electrogenic (Figs. 15 and 16). An important observation, which allows interpretation of the
current-voltage relations of the squid exchanger, is
that Na+-Na+ exchange by the squid exchanger is substantially greater than Na+-Ca+ or Ca2+-Ca2+ exchange
(DiPolo et al., 1989
; DiPolo and Beaugé, 1990
; Dr. L. Beaugé, personal communication). Thus, Ca2+ transport may in general be rate limiting and the voltage dependence of Ca2+ transport will determine the overall
voltage dependence of transport current. Consistent
with this interpretation, the rates of Ca2+-dependent
charge movements determined for the squid exchanger at 0 mV and 33°C are substantially less (1,700 s
1) than
those determined for the NCX1 exchanger (5,000 s
1;
Hilgemann et al., 1991
; Hilgemann, 1996
).
Our interpretation is tempered to some extent because an alternating access model, or "consecutive" exchange mechanism, has not been rigorously verified
for the squid exchanger. In a consecutive mechanism,
the apparent affinity of one transported ion should increase as the concentration of the countertransported
ion is decreased. For the squid exchanger, it has been
reported that the Na+ dependence of Ca2+ efflux does
not change as the cytoplasmic Ca2+ concentration is reduced (DiPolo, 1989). Thus, further work is required
on transport properties of the squid exchanger; the application of techniques to photo-release Ca2+ in giant
patches within microseconds (Kappl and Hartung,
1996
), rather than 100 ms, will allow much better resolution of these issues.
The differences in the charge-moving reactions of NCX1 and NCX-SQ1 may provide an important key to elucidating the physical basis of exchanger electrogenicity. Ion occlusion reactions may result from the movement of charged residues anywhere in the exchanger protein, but only residues that enter or leave the membrane electrical field together with ions will generate charge movements. Evidently, more than two negative charges must move into the membrane electrical field when Ca2+ is occluded by the squid exchanger from the cytoplasmic side. Studies of chimeras of the two exchangers, and ultimately point mutation studies, should be able to define the involvement of specific groups and provide an understanding of the conformational changes underlying ion transport.
In summary, the squid Na+-Ca2+ exchanger, NCX-SQ1, has been cloned and expressed in Xenopus oocytes, and its function has been characterized electrophysiologically in giant membrane patches. The differences between the sequence and functional properties of NCX-SQ1 and the mammalian NCX1 now provide a new basis to elucidate both regulatory and transport properties of Na+-Ca2+ exchange. NCX-SQ1 is strongly activated by the anionic phospholipid, PIP2, and the presence of phosphorylation sites, not present in NCX1, may correlate with stimulation of the squid exchanger by thioester derivatives of ATP. The molecular basis of differences in the voltage dependence of cardiac and squid exchangers can now be pursued by the combined methods of molecular biology and electrophysiology.
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FOOTNOTES |
---|
Address correspondence to K.D. Philipson, Cardiovascular Research Laboratories, MRL 3-645, UCLA School of Medicine, Los Angeles, CA 90095-1760. Fax: 310-206-5777; E-mail: kenneth{at}cvrl.ucla.edu. D.W. Hilgemann, Department of Physiology, UTSW Medical Center, Rm. K4.103, 5323 Harry Hines Blvd., Dallas, TX 75235-9040. Fax: 214-648-8685; E-mail: hilgeman{at}utsw.swmed.edu
Received for publication 4 December 1997 and accepted in revised form 20 March 1998.
Z. He's current address is Clinical Research Department, DuPont Hospital for Children, Wilmington, DE 19899.We thank Siyi Feng and Chin-Chih Lu for expert technical help.
This work was supported by the National Institutes of Health (HL-5132302 to D.W. Hilgemann and HL-48509 and HL-49101 to K.D. Philipson).
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Abbreviations used in this paper |
---|
MES, 2-(morpholino)ethanesulfonic acid; NMG, N-methyl-D-glucamine; nts, nucleotides; PIP2, phosphatidylinositol-4',-5'-bisphosphate.
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