From the C. H. Best Institute, Banting and Best
Department of Medical Research, University of Toronto, Toronto,
Ontario M5G 1L6, Canada, the ¶ Departments of Physiology and
Medicine and the Cardiovascular Research Laboratories, UCLA School of
Medicine, Los Angeles, California 90095-1760, and the
Departments of Biochemistry & Molecular Biology and Physiology & Biophysics, University of Calgary,
Calgary, Alberta T2N 4N1, Canada
Received for publication, March 14, 2001, and in revised form, April 9, 2001
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ABSTRACT |
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We describe here the identification and
characterization of a novel member of the family of
K+-dependent
Na+/Ca2+ exchangers, NCKX3
(gene SLC24A3). Human NCKX3 encodes a protein of 644 amino
acids that displayed a high level of sequence identity to the other
family members, rod NCKX1 and cone/neuronal NCKX2, in the hydrophobic
regions surrounding the " Plasma membrane Na+/Ca2+ exchangers are an
important component of intracellular Ca2+ homeostasis and
have been extensively studied in various cell systems (1).
Na+/Ca2+ exchangers are encoded by a protein
superfamily present in organisms ranging from bacteria to man (2). All
the members of this family share sequence similarity in two hydrophobic
and internally homologous domains, commonly referred to as The NCX family of exchangers is best exemplified by the mammalian
cardiac Na+/Ca2+ exchanger, NCX1, first cloned
from canine heart (6), which plays a crucial role in the relaxation
process of heart muscle by extruding the Ca2+ that enters
at the beginning of systole. NCX1 is also expressed in a variety of
other tissues (7-9) suggesting an important role in the physiological
processes of different cell types. The tissue-specific expression
pattern of NCX1 has been demonstrated to be under the control of a
multipartite promoter (10-12). Moreover, a complex pattern of
alternative splicing in the large intracellular loop of NCX1 generates
isoforms of the protein, which are also expressed in a highly
tissue-specific way (7-9). Functional studies have revealed complex
regulatory mechanisms of the NCX1 protein, some of which differ between
alternatively spliced isoforms (2, 13). The NCX protein family contains
two other members, products of genes NCX2 and
NCX3, whose expression is restricted largely to brain and
skeletal muscle (9, 14, 15). All three NCX proteins share a high degree
of sequence identity, especially within the transmembrane spanning
domains, and are believed to share the same overall topology, modeled
to have two clusters of hydrophobic membrane-spanning helices separated
by a large hydrophilic, intracellular loop. The hydrophobic domains are
thought to pack together in the membrane, forming the ion translocation pathway (16). Indeed, the three NCX proteins show very similar functional properties when assayed in heterologous expression systems
(17, 18).
The second major group in the Na+/Ca2+
exchanger superfamily is exemplified by the
Na+/Ca2++K+ exchanger from retinal
rod outer segments, NCKX1. NCKX1 was first cloned from bovine retina
(19) and, more recently, from other species including dolphin, rat,
buffalo, and man (20-22). This protein plays a critical role in the
visual transduction process of the mammalian retina (5). In darkness,
the cyclic nucleotide-gated ion channels of the outer segment are
largely open, and both Na+ and Ca2+ ions flow
in. Ca2+ homeostasis must still be maintained under these
conditions of membrane depolarization and reduced sodium gradient, and
NCKX1 is the principal means by which Ca2+ is extruded from
rod outer segments. Such a function could not be achieved by a molecule
operating with the 3:1 Na+:Ca2+ stoichiometry
of NCX1, and indeed it has been demonstrated that NCKX1 couples the
entry of four Na+ ions in exchange for the exit of one
Ca2+ and one K+ ion (23, 24).
Whereas NCX1 proteins from mammalian species are over 90% identical in
their amino acid sequences, NCKX1 orthologs display relatively low
sequence identities of around 60%, largely due to differences in the
two large hydrophilic loops, an extracellular one near the N terminus
and a cytoplasmic one near the center of the molecule. NCKX1 has been
modeled to have a similar arrangement of transmembrane-spanning
segments as NCX1, although actual amino acid sequence similarity is
very limited and restricted only to the two Functional measurements have pointed toward the presence of
K+-dependent Na+/Ca2+
exchangers in tissues other than eye (25, 26). Molecular evidence has
recently confirmed expression of NCKX1 in cells of hematopoietic origin
(27) and identified a second K+-dependent
Na+/Ca2+ exchanger (NCKX2) in brain neurons and
cone photoreceptors (28, 29). Moreover, sequence analysis of the
genomes of model organisms, such as Drosophila and
Caenorhabditis elegans, has revealed several new
hypothetical proteins with similarity to the NCKX family (2, 3,
29).2 Two of these have
recently been characterized functionally (30, 31). In addition,
analysis of NCKX mRNA expression using probes from regions of
sequence conservation under conditions of reduced stringency has
revealed evidence for further, as yet uncharacterized, mammalian
members of the NCKX family
(20).3 These findings lend
support to the idea that the NCKX gene family predates the
evolution of vertebrate vision and that the encoded proteins are an
essential component of intracellular Ca2+ homeostasis in
many different cells and tissues. A more generalized role for NCKX
proteins in current schemes of cellular Ca2+ homeostasis
has yet to be considered, largely because there has been no systematic
analysis of their expression in tissues other than retina (28).
In this study, we have identified, starting from genome project EST
data in three mammalian species, a novel member of the K+-dependent Na+/Ca2+
exchanger gene family, NCKX3. We demonstrate that
transcripts from this gene are prominent in brain and other tissues and
that, when expressed in HEK293 cells, NCKX3 indeed encodes a
K+-dependent Na+/Ca2+ exchanger.
cDNA Manipulation and Sequence Analysis--
Common chemical
reagents were purchased from Fisher, Sigma, or BDH and were of
"molecular biology grade" or better. All molecular procedures
followed standard protocols (32, 33), or those provided by the reagent
suppliers, unless noted otherwise. DNA sequencing was done at local
core facilities of the participating institutions, using either Li-Cor
or Applied Biosystems automated fluorescent sequencing equipment.
Full-length clones were sequenced on both strands. DNA contig assembly
was done with either MacVector (Genetics Computer Group, Inc., Madison,
WI) or Sequencher (Gene Codes, Ann Arbor, MI), and sequence analysis
was done in MacVector or via web interface at the Expasy molecular
biology server. BLAST (34) searches of the sequence data bases
were run at the National Center for Biotechnology Information web site,
at the Sanger Center web site, or at the Ensembl genome server of the
European Bioinformatics Institute.
Identification and Cloning of the Human, Mouse, and Rat Brain
NCKX3 cDNAs--
BLAST searches of the human EST data bases
identified two sequences (R21117 and T06656) with similarity to
Cloning of the Transcription Initiation Region from the Murine
NCKX3 Gene--
A mouse embryonic stem cell genomic library in the
vector Lambda Fix II (Stratagene) was probed with mouse NCKX3 cDNA.
A 5-kb ClaI restriction fragment that hybridized to a probe
containing only exon 1 sequences was then subcloned from a positive Cloning of the Full-length NCKX3 cDNA from Human Skeletal
Muscle--
Human EST R21117 was identified as a candidate for a new
NCKX protein by data base searches using the amino acid sequence of the
cardiac Na+/Ca2+ exchanger NCX1 and comparison
to other known K+-dependent and -independent
Na+/Ca2+ exchanger isoforms. The NCKX3 "long
clone" was subsequently isolated from Human Skeletal Muscle cDNA
libraries (5'-Stretch, a generous gift of Dr. Ira Kurtz, and 5'-Stretch
Plus from CLONTECH, Palo Alto, CA) using a
recursive PCR-based screening approach that combined primers from the
vector with primers based initially on the R21117 sequence and
subsequently on sequence from the 5'-end of isolated clones. The Structure and Chromosomal Location of the Human NCKX3
Gene--
BLAST searches of the human high through-put genomic
sequence identified several PAC clones that contained sequence
stretches showing >99% match with portions of human NCKX3 cDNA.
Analysis of these data eventually provided the exon structure of the
NCKX3 gene. Similarly, assignment of the NCKX3
transcript to a Unigene cluster, and to contiguous sequenced stretches
of the draft genome sequence, determined its chromosome location.
Mapping the 5'-End of the Murine NCKX3 Transcript by RNase
Protection Assay--
Mouse brain poly(A)+ mRNA was
isolated from fresh or frozen whole mouse brain using the Trizol
reagent (Life Technologies, Inc.), followed by DNase I digestion and
further isolation with RNeasy Midi columns (Qiagen, Valencia, CA), and
poly(A) Spin columns (New England Biolabs, Beverly, MA). Three
riboprobes were constructed based on murine NCKX3 sequences. Probe 1 was prepared by RT-PCR from mouse brain mRNA, beginning 16 nucleotides upstream of the initiation codon and extending into exon 2 (see Fig. 4), and subcloned between the EcoRI and
BamHI sites of pBluescript II SK ( In Vitro Translation of Human NCKX3 cDNA--
cDNA
clones in the pcDNA3.1(+) vector encoding short (isolated from
brain) and long (isolated from skeletal muscle) versions of human NCKX3
were transcribed and translated in vitro using the TNT-T7
system from Promega (Madison, WI) together with
[35S]methionine (Amersham Pharmacia Biotech), in the
presence of either 0.1% Triton X-100 or canine pancreatic microsomes
with or without 50 µM of the competitive glycosylation
inhibitory peptide, acetyl-Asn-Tyr-Thr-amide. Following an incubation
of 90 min at 30 °C, the products were resolved on an
SDS-polyacrylamide gel (35), dried, and detected by autoradiography
using Biomax MR film (Eastman Kodak Co.).
Tissue-specific Distribution of the NCKX3
Transcript--
Distribution of the human NCKX3 transcript
was studied using commercially available Multiple Tissue Northern and
Multiple Tissue Expression Array filters (CLONTECH)
with 32P-labeled probes corresponding to the entire clone
(Northern blots), or encompassing nucleotides 403-620 of the human
skeletal muscle "long" clone, generated by PCR (dot blot array).
Hybridization and washing of the membrane was carried out under highly
stringent conditions, following the manufacturer's protocol.
Distribution of the NCKX3 transcript in rodent tissues was
studied on Northern blots of total tissue RNA using a
digoxigenin-labeled antisense riboprobe from murine NCKX3 spanning the
region from BglII (356) to EagI (2392).
Hybridization and washing at high stringency were essentially according
to instructions of the manufacturer (Roche Molecular Biochemicals) as
described previously (36). In situ hybridization was
performed on parasagittal sections of mouse brain using base-hydrolyzed
digoxigenin-labeled NCKX3 probes, as described previously (29). The
antisense probe spanned BglII (356) to XbaI
(2193), whereas the sense control probe covered the entire clone
(nucleotides 1-2392).
Analysis of NCKX3 Function by Ca2+ Imaging and
Electrophysiology in HEK293 Cells--
cDNAs encompassing the
predicted protein coding sequence for both human brain ("short")
and skeletal muscle ("long") and mouse brain NCKX3 were cloned into
the expression vector pcDNA3.1(+). HEK293 cells grown on coverslips
were transfected with the NCKX3 constructs or with vector DNA alone
using a standard Ca2+-phosphate precipitation protocol and
analyzed using fluorescent digital imaging as described previously
(29). In brief, transfected cells were loaded with fura-2, mounted in a
perfusion chamber on a microscope stage, and the ratio of fluorescence
observed with excitation at 340 or 380 nm followed with time. Cells
were initially perfused with Na+-containing
K+-free solution (145 mM NaCl, 10 mM D-glucose, 0.1 mM
CaCl2, 10 mM HEPES-trimethylamine (HEPES-TMA),
pH 7.4) for 5 min, followed by alternating changes to solutions in
which the NaCl was substituted with LiCl and various concentrations of KCl.
For analysis of ion currents, carried by the electrogenic movement of
Na+ through NCKX3, in exchange for Ca2+ and
K+, HEK293 cells were co-transfected with the NCKX3
constructs and a cDNA-encoding green fluorescent protein.
Transfected cells were identified under fluorescent illumination and
then analyzed by whole-cell patch clamp with a seal resistance of 5-10
G Na+ Gradient-dependent
45Ca2+ Uptake into Transfected HEK293
Cells--
NCKX3 cDNA from either human skeletal muscle or brain,
canine cardiac NCX1 cDNA in pcDNA3.1(+), or vector alone, were
transfected into HEK293 cells using GenePORTER (Gene Therapy Systems,
San Diego, CA). Forty eight hours following transfection the cells were
harvested, washed twice with washing buffer (140 mM NaCl, 10 mM MOPS, pH 7.4), and loaded with Na+ (140 mM NaCl, 1 mM MgCl2, 10 mM MOPS, pH 7.4, 0.4 mM ouabain, 25 µM nystatin) for 10 min at room temperature. Cells were
washed twice with washing buffer to remove nystatin, and the cell
pellet was resuspended in assay medium as follows: 140 mM
KCl, choline-Cl or NaCl, 10 mM MOPS, pH 7.4, 25 µM 45CaCl2 (5 µCi/ml), 0.4 mM ouabain. After 1 min the reaction was stopped by the
addition of ice-cold 140 mM KCl, 1 mM EGTA, and cells were washed two times with the same solution. Cell pellets were
dissolved in 1 N NaOH at 80 °C for 30 min, and aliquots
of the samples were subjected to scintillation counting and protein assay (Micro BCA Assay, Pierce).
Identification in Human Brain and Skeletal Muscle of a Novel
cDNA with Similarity to K+-dependent
Na+/Ca2+ Exchangers--
Comparison of the
well characterized mammalian cardiac Na+/Ca2+
exchanger, NCX1, to the sequence data bases of simple model organisms has revealed a surprisingly complex family of related sequences (2, 3,
29).2 In a further effort to characterize mammalian
counterparts to these putative molecules, the human EST data base was
screened with a cDNA fragment corresponding to the conserved amino
acid sequence of the
The human skeletal muscle clone (HuNCKX3-L, for "long") extended
254 nucleotides further 5' than the human fetal brain clone (HuNCKX3-S,
for "short"), but otherwise these clones were identical aside from
several, presumably allelic, single nucleotide polymorphisms. The
5'-ends of both clones were found within contiguous genomic DNA
sequence of what appears to be the first exon of the human gene (Fig.
3). 5'-Rapid amplification of cDNA
ends experiments using human brain mRNA were unable to extend past
the end of HuNCKX3-S. As this sequence is very GC-rich, the difference
in length between brain and skeletal muscle clones could be due to
technical reasons or might indicate that transcription starts at
different sites in these different tissues. Mapping of the potential
transcriptional start was performed using the mouse genomic clone and
mouse brain mRNA (see below), suggesting that the longer clone most
likely corresponds to authentic NCKX3 transcripts.
Structure of the Human NCKX3 Gene and Mapping to Its Chromosomal
Location--
Comparison of the NCKX3 cDNA sequences with the HTGS
data base allowed the intron-exon structure of the gene to be
reconstructed (Figs. 1 and 3). Human NCKX3 consists of 17 exons spanning over 500 kilobase pairs. The first three exons are
spread out over more than half of this distance, followed by two
clusters comprising exons 4-5 and 7-17, which are more conventionally
spaced. Surprisingly, the unusually long first coding exon found as a
conserved feature in NCX1, NCX3, NCKX1, and NCKX2
genes (analyzed by BLAST search; also see Refs. 37 and 38) is not
present in NCKX3. Also, unlike its family members, there is no evidence
for alternative splicing of NCKX3 among available clones
isolated from human cDNA libraries or by RT-PCR analysis of mouse
or rat tissues. However, the last exon of NCKX3, encoding
the C-terminal end of the protein and all of the 3'-untranslated
region, is the longest in the gene, in line with the gene structure of
other members of the Na+/Ca2+ exchanger gene superfamily.
The sequence of the brain and skeletal muscle NCKX3 cDNA clones
overlapped in the 3'-untranslated region with more than 20 different
EST entries, originating from various human tissue libraries. The
3'-end of the transcript was found to correspond in the human genome to
position 30,641 of GenBankTM accession number AL121761, and
the nearest sequence-tagged sites were Em:G25896, Em:T03762, and
Em:L30728. A representative EST entry that contains the 3'-end of this
transcript, including a poly(A) tail (GenBankTM accession
number F10455; data base EST entry 132788), belongs to the UniGene EST
cluster Hs.12321, which has been mapped to the interval
D20S182-D20S106 on chromosome 20p13. This gene has been given the
designation SLC24A3. The genes for the other two members of
the K+-dependent
Na+/Ca2+ exchanger family have also been
mapped, NCKX1 (gene SLC24A1) to chromosome 15q22
(38) and NCKX2 (gene SLC24A2) to chromosome 9q22.1-22.3.
Identification of Rodent NCKX3 cDNA and the Putative Promoter
Region of the Mouse SLC24A3 Gene--
Searches using completed human
NCKX3 cDNA identified a set of mouse ESTs from which primers were
designed to amplify the mouse NCKX3 cDNA by RT-PCR from brain
poly(A)+ mRNA. Similar primers, based on identity
between the human and mouse cDNA sequences, were also used to
isolate the rat brain NCKX3. We were unable to amplify products from
either mouse or rat mRNA using primers upstream of the presumed
initiating methionine residue of human NCKX3, however, and only clones
that extended to include the Arg residue at position 20 of the human
sequence were obtained (Fig. 1). Nevertheless, 5'-rapid amplification
of cDNA ends experiments (data not shown) as well as EST entries (Fig. 4) indicated that the mouse
NCKX3 transcript encodes the same N-terminal protein region
as the human one. The sequences of the resulting deduced proteins from
human, mouse, and rat are >95% identical, as shown in Fig. 1. The
only areas with clustered differences are at the immediate N terminus
and in a repetitive acidic sequence found in the central, presumptive
cytoplasmic, loop.
The mouse NCKX3 cDNA was used as a probe to isolate a genomic
clone, which was found to contain the sequence corresponding to the
first exon of the mouse SLC24A3 gene (Fig. 4). The 5'-end of
the mouse NCKX3 transcript was mapped using probes derived from cDNA and genomic sequence, as shown in Fig. 4. Probes 1 and 2 were both protected to their full lengths by brain mRNA (184 and
383 nucleotides, respectively), whereas the majority of probe 3 was
protected to a point just upstream of the NcoI site that delineates probe 2 (although a small amount of a full-length protected fragment of 496 nucleotides is also visible). These data indicate that
transcription of the mouse NCKX3 gene likely initiates close to position 325 of Fig. 4B, which would produce a transcript
analogous to the HuNCKX3-L clone. The abrupt loss of cross-species
sequence identity close to this site is also consistent with
transcription initiation in this region of the gene. The 5'-flanking
sequence is very GC-rich and contains no obvious TATA box. There are,
however, numerous Sp-1-like sites, CACC box sites, as well as an AP-2
site at 311 and a CArG site at 208, consistent with a ubiquitously expressed gene.
In Vitro Translation of the Cloned NCKX3 cDNAs--
Neither
the human nor mouse NCKX3 transcripts contain in-frame stop
codons upstream of the 5'-most methionine residue. In light of the two
different clones isolated, the presence of several fairly closely
spaced potential initiator AUG codons, and the relatively weak
consensus sequence surrounding each (39), we decided to use in
vitro translation to examine the choice of starting methionine. As
illustrated in Fig. 5, when translated
using a reticulocyte lysate system, HuNCKX3-L produced a single major protein product of about 72 kDa, whereas HuNCKX3-S produced a 64-kDa
protein. It is noteworthy that the HuNCKX3-L clone produced almost no
product of 64 kDa, demonstrating a high degree of preference for the
upstream methionine as the starting point. An NCKX3 protein synthesized
from this site would also contain a hydrophobic region analogous to the
M0 region of other Na+/Ca2+ exchanger family
members. Synthesis of NCKX3 in the presence of canine pancreatic
microsomes resulted in an upward shift in the mobility of the product,
especially evident with the HuNCKX3-L clone, consistent with the
attachment of oligosaccharide chains to the two glycosylation site
motifs present in the extracellular loop between M0 and M1. Synthesis
in the presence of the competitive glycosylation inhibitor,
acetyl-Asn-Tyr-Thr-amide, prevented appearance of the reduced mobility
species characteristic of a glycosylated polypeptide and revealed a
more rapidly migrating polypeptide instead, suggestive of signal
peptide processing and consistent with the assignment of the M0 region.
The HuNCKX3-S protein product of 64 kDa appeared to be too short to
start from the first methionine codon in its cDNA sequence,
suggesting initiation from a more downstream site. Since this product
was glycosylated (albeit weakly), a start site just before the two
potential glycosylation sites is predicted.
Tissue Distribution of NCKX3 Gene Expression--
Northern
blotting data of NCKX3 distribution is shown for RNA from different rat
and mouse tissues and from various human brain regions, in Fig.
6. The major NCKX3 transcript
was about 4.5 kb in length and was abundant in various brain regions,
as well as several tissues rich in smooth muscle (aorta, intestine, and
lung). Lower levels were found in a variety of other tissues, whereas
kidney and liver were essentially negative. This pattern of size and
distribution clearly distinguishes NCKX3 from the other two
known members of the SLC24 gene family, NCKX1 (6 kb), and NCKX2 (~11 kb), whose expression is almost
exclusively restricted to eye and brain (20). Larger transcripts of
somewhat variable size were evident in several tissues, as well as an
~1.8-kb pair species that was present in all rat tissues but not in
mouse or human. Possibly these species represent cross-reaction of the NCKX3 probe with as yet uncharacterized NCKX family members. The 1.8-kb
band is reminiscent of a similarly sized species corresponding to a
circularized NCX1 transcript of the very large exon 2 (40). Since NCKX3 has no such large exon in the region
corresponding to the probe, a similar scenario cannot be the
explanation, nor is the observed transcript likely due to
cross-reaction with the NCX1 exon 2 product itself, as the
sequences are too divergent, and the tissue-specific expression
patterns quite different (40).
The expression pattern of the human NCKX3 gene was
investigated in more detail using a Multiple Tissue Expression Array
(CLONTECH), comprising a collection of
poly(A)+ RNAs isolated from 61 different samples of adult
human tissue, 7 fetal human tissues, 8 human cancerous cell lines, and
8 negative control RNAs and DNAs. The amount of each mRNA dotted
onto the membrane is normalized to the expression of eight different
housekeeping genes allowing for direct reading of semi-quantitative
levels of expression. Fig. 7 summarizes
all tissues with hybridization signals above background as determined
by densitometry. As shown above with Northern blot analyses, NCKX3
mRNA was expressed in almost all regions of the brain with
thalamus, hippocampus, amygdala, and cerebellum showing the highest
levels. Out of 21 brain regions tested on the array, only three (pons,
spinal cord, and pituitary gland) did not show any detectable signal.
NCKX3 mRNA expression was also relatively strong in aorta, uterus,
and skeletal muscle, and a lower level of expression was observed in
various other tissues. Of note, NCKX3 transcripts could not
be detected in heart, ascending colon, liver, pancreas, genitourinary
tissues (kidney, placenta, prostate, testis, and ovary), the immune
system (spleen, thymus, peripheral blood leukocytes, lymph node, and
bone marrow), the pulmonary tract (trachea and lung), or in
endo/exocrine glands (adrenal gland, thyroid gland, salivary gland, and
mammary gland). All eight cancerous human cell lines on the membrane
tested negative as well. In fetal tissue, NCKX3 mRNA was detected
only in brain and not in heart, kidney, liver, spleen, thymus, or
lung.
Spatial Distribution of the NCKX3 Transcript in Mouse
Brain--
The high level of NCKX3 expression in brain
prompted an examination of regional distribution using in
situ hybridization, as illustrated in Fig.
8. This analysis revealed a unique
neuronal staining pattern. NCKX3 transcripts appeared most
abundant in thalamus, where there was a striking pattern within
discrete nuclei. Expression levels were also high in the hippocampus,
especially in the CA1 neurons. Within the cortex, a clearly laminar
pattern was evident, with higher expression of NCKX3 in
large neurons of layer IV than in other layers. In cerebellum,
NCKX3 appeared to be restricted to stellate cells of the
molecular layer. These data on NCKX3 expression in mouse
brain are consistent with the regional distribution in human brain,
inferred from the dot blot data of Fig. 7 and the Northern blot data of
Fig. 6C.
Analysis of NCKX3 Function--
The ability of the NCKX3 protein
to function as a Na+/Ca2+ exchanger was tested
by transfecting HEK293 cells with various constructs and measuring
Na+-dependent Ca2+ fluxes, as
illustrated in Fig. 9. When fura-2-loaded
cells were perfused with Li+, to reverse the
Na+ gradient across the plasma membrane, a large rise in
intracellular [Ca2+] (evident as an increase in the
340:380 ratio of the dye) was observed only in the presence of
extracellular K+, consistent with the operation of a
K+-dependent Na+/Ca2+
exchanger. Cells transfected with each of the three NCKX3 cDNA clones (HuNCKX3-L, HuNCKX3-S, and MuNCKX3, corresponding to the short
human clone) demonstrated this behavior, whereas the cells transfected
with vector alone showed no significant rise in [Ca2+].
The rate of change and the magnitude of the fura-2 response were
consistently greater for the longer human cDNA clone than for
either shorter one (see Fig. 9B). Thus, while the M0 region of NCKX3 seemed not to be essential for functional expression, as
previously documented for NCX1 (41-43), it appeared as though the
level of expression and/or the efficiency of plasma membrane delivery
was much greater for the complete exchanger molecule than for the
N-terminal truncated one, hence resulting in more robust function.
Whole-cell patch clamp data from HEK293 cells were also consistent with
the K+-dependent
Na+/Ca2+ exchange function of NCKX3, as shown
in Fig. 10. A significant outward
current, carried by Na+ movement, was observed only when
cells transfected with HuNCKX3-L were perfused with medium containing
both K+ and Ca2+ and not with either ion alone
or with control transfected cells. Inclusion of 1 mM
Mg2+ in the perfusate significantly reduced the magnitude
of the currents observed, consistent with competition of
Mg2+ at the external Ca2+ transport site, as
observed with NCKX1 (44). Interestingly, we were unable to observe
currents with either shorter version of NCKX3 (data not shown).
Uptake experiments using 45Ca2+ were also
performed on Na+-loaded HEK293 cells, as illustrated in
Fig. 11. Na+
gradient-driven Ca2+ uptake was observed for cells
transfected with HuNCKX3-L in medium containing K+ but not
choline. In contrast, Ca2+ uptake into NCX1-transfected
cells was much larger and was relatively insensitive to the presence or
absence of K+. These data again confirm that NCKX3
functions as a K+-dependent
Na+/Ca2+ exchanger. As with the
electrophysiological measurements, cells transfected with the HuNCKX3-S
clone did not demonstrate any uptake above control levels. Since
HuNCKX3-S did display function, albeit much less than HuNCKX3-L, when
measured using digital fluorescent Ca2+ imaging, it seems
likely that lack of function in the other assays indicates a level of
expression too low to detect. These observations also suggest that the
digital fluorescent Ca2+-imaging technique is at least an
order of magnitude more sensitive than other assays.
In this study we have described the cloning and characterization
of a cDNA encoding a novel, third, member of the family of K+-dependent Na+/Ca2+
exchangers, NCKX3. Like the other two members, rod NCKX1 and cone/neuronal NCKX2, NCKX3 demonstrated
K+-dependent Na+/Ca2+
exchange activity when measured with the fluorescent Ca2+
dye fura2, with ion currents, or with 45Ca2+
uptake. Although we did not formally determine whether K+
was actually transported together with Na+ and
Ca2+, there was an absolute requirement for K+
in all of the functional assays. That NCKX3 can be measured
electrically, with charge moving in the same direction as
Na+, also places limits on the ionic stoichiometry of
transport. Combined with the high degree of amino acid sequence
similarity to NCKX1, which has been clearly established to transport 4 Na+ in exchange for 1 K+ and 1 Ca2+
(23, 24), it seems likely that NCKX3 has a similar stoichiometry.
Previous studies with the cardiac Na+/Ca2+
exchanger, NCX1, had implicated the so-called Hydropathy analysis as well as our in vitro translation
experiments support a proposed transmembrane topology for NCKX3 that is
largely similar to those proposed previously for other family members
(Fig. 1). The NCKX3 protein begins with a functionally dispensable
region that may encode a putative "signal" peptide, in a manner
analogous to that reported for NCX1 (41-43). The presence of this
region in the expressed protein significantly improved the level of
functional expression of NCKX3 (Fig. 9). Thus, whereas all cDNA
constructs demonstrated K+-dependent
Na+/Ca2+ exchange activity when assayed using
fura-2 fluorescent digital imaging, only the HuNCKX3-L clone expressed
activity above background in either electrophysiological or
45Ca2+ uptake assays. Since in vitro
transcription and translation experiments (Fig. 5) did not reveal a
significant difference in the efficiency of protein expression, the
longer NCKX3 protein species containing the N-terminal hydrophobic M0
sequence may be targeted or delivered to the plasma membrane more
efficiently than the protein lacking this sequence.
The processed NCKX3 protein consists of a short, glycosylated,
extracellular loop at the N terminus followed by a cluster of five
hydrophobic, putative transmembrane, segments, a long hydrophilic loop,
and finally, a second cluster of hydrophobic regions. The long central
loop, which is presumed to be cytoplasmic based on a comparison to
NCKX1 and NCX1, contains consensus sequences for several protein
kinases. Recent studies using cysteine-scanning mutagenesis of NCX1
have revealed that the C-terminal hydrophobic region of that molecule
is composed of four helical transmembrane segments and a pore-like
re-entrant loop structure that extends into the membrane (45, 47, 48).
Hydropathy analysis of NCKX3 suggests a somewhat different topology,
with five transmembrane spans separating the central cytoplasmic loop
from the protein C terminus, which would reside on the extracellular
side of the membrane. The validity of such a model, and the notion that
the K+-dependent
Na+/Ca2+ exchangers (NCKX) may have a topology
different from those of the K+-independent (NCX) family,
will need to be tested.
The exon boundaries for human NCKX3 are indicated in Fig. 1, and their
location in genomic sequence on chromosome 20 is shown in Fig. 3. The
following two features of the NCKX3 gene are striking: (i)
the distance over which the exons, especially the first three, are
spaced; (ii) the arrangement of exons in comparison to the genomic
structure of other Na+/Ca2+ exchanger genes.
The NCX1 (SLC8A1), NCX3
(SLC8A3), NCKX1 (SLC24A1), and
NCKX2 (SLC24A2) genes all share an unusually
large second exon that extends from just before the initiating
methionine codon into the central cytoplasmic loop (analyzed by BLAST
search; also see Refs. 37 and 38). In contrast, the corresponding
sequence of NCKX3 is split into 10 separate exons. Furthermore, the
arrangement of exons encoding the C-terminal hydrophobic domain is
specific for NCKX3 and differentiates it from either the arrangement
conserved between NCKX1 and NCKX2 or the distinct arrangement conserved between NCX1 and NCX3. These differences suggest that, despite the
sequence similarity within the The pattern of expression of NCKX3 transcripts in different
tissues was examined in rat and mouse by Northern blot and in human by
Multiple Tissue Expression Array dot blot analyses (Fig. 6). From these
data it is clear that NCKX3 was most abundantly expressed in various
brain regions. Regional distribution was examined in more detail in
parasagittal sections of mouse brain using in situ
hybridization (Fig. 8), which revealed a very specific pattern. The
highest levels of NCKX3 mRNA were found in neurons arranged in
distinct nuclei of the thalamus, followed by the pyramidal CA1 neurons
of the hippocampus, and large neurons of cortical layer IV. This
pattern is quite different from published reports of NCX1 and NCKX2
distribution in rat brain (29, 49). Transcripts for both of these
exchangers were also abundant in cortex but lacked the laminar pattern
evident for NCKX3. All three exchangers were present in hippocampal
pyramidal cells, but only NCKX3 was richest in CA1 neurons. In the
thalamus, each exchanger had a characteristic regional distribution.
NCKX3 and NCKX2 expression largely overlapped in the cerebellar
molecular layer. In contrast, NCKX3 was essentially absent from the
striatum, where NCKX2 was very abundant, and from the septal nuclei,
where NCX1 was highly expressed.
A unique feature of NCKX3 expression in comparison to other
K+-dependent Na+/Ca2+
exchangers was the presence of transcripts in many tissues other than
brain, although at lower levels. The precise pattern appeared to vary
between human and rodent, although part of this may have been due to
the manner in which RNA loading was normalized (to a fixed quantity of
total RNA in the case of rodent and to a varying amount of
poly(A)+ mRNA, normalized to several control
transcripts, in the case of the Multiple Tissue Expression Array), or
due to the difference in the probes used. The tissues that had the next
most abundant level of NCKX3 mRNA, after brain, were those
generally rich in smooth muscle, such as aorta, uterus, and intestine.
Many other tissues expressed lower levels, but only liver and kidney
consistently appeared to be essentially negative for NCKX3. This rather
ubiquitous pattern of expression matches more closely that of NCX1 than
it does any of the other family members, which are thought to have quite restricted tissue expression patterns, NCKX1 in eye, NCKX2 in
brain neurons (including eye), and NCX2 and NCX3 in brain and skeletal
muscle. The selective presence of NCKX3 in specific unique cell types
of the vascular wall (smooth muscle, endothelial cells, or innervating
neurons) has yet to be examined. To our knowledge, however, there have
been no reports of K+-dependent
Na+/Ca2+ exchange activity in these tissues or
cell types.
At present the physiological consequences of the differential
tissue-specific expression patterns for different
Na+/Ca2+ exchanger family members are unclear.
The situation in brain, where at least four different
Na+/Ca2+ exchanger gene products (NCX1, NCX2,
NCKX2, and NCKX3)3 are present at high levels, is
particularly intriguing. It seems likely that unique
Ca2+-handling requirements of specific tissues or cell
types, combined with kinetic, thermodynamic, or regulatory differences
in the function of these exchangers, will provide important clues. The difference in transport stoichiometry between NCKX and NCX families provides an opportunity for NCKX-type exchangers to maintain
Ca2+ homeostasis in environments where the Na+
gradient and/or the membrane potential are lower than normal. Although
this argument was first used to justify the expression of NCKX1 in rod
photoreceptors, there may be situations in other neurons where similar
conditions exist. Another possible role for different exchanger gene
products might be in the transport of Mg2+ instead of
Ca2+ (50). We have not investigated whether NCKX3 might
transport Mg2+. It is noteworthy, however, that 1 mM Mg2+ significantly inhibits NCKX3 activity
(Fig. 10), although under these conditions it had no significant effect
on the amplitude of either NCX1 or NCKX2
currents.4 These observations
suggest that Mg2+ interacts more selectively with the
transport site of NCXK3 than it does with the sites of other family members.
The eventual answer to the question of unique physiological roles for
the different Na+/Ca2+ exchanger family members
will require the development of selective pharmacological blockers
and/or of recombinant gene knock-out animals. The recent developments
of an agent relatively selective for inhibition of NCX-type
Na+/Ca2+ exchangers, KB-R7943 (51), and mice
lacking NCX1 (52), will help in this effort, but additional selective
molecular tools are eagerly anticipated. Most notably, planning complex
gene knock-out experiments will be greatly facilitated by the
completion of the mouse genome sequence (due in 2002) which will set a
unique stage for genetic dissection of mammalian Ca2+ homeostasis.
-repeat" sequences thought to form the
ion-binding pocket for transport. Outside of these regions NCKX3 showed
no significant identity to other known proteins. As anticipated from
this sequence similarity, NCKX3 displayed
K+-dependent Na+/Ca2+
exchanger activity when assayed in heterologous expression systems, using digital imaging of fura-2 fluorescence, electrophysiology, or
radioactive 45Ca2+ uptake. The N-terminal
region of NCKX3, although not essential for expression, increased
functional activity at least 10-fold and may represent a cleavable
signal sequence. NCKX3 transcripts were most abundant in
brain, with highest levels found in selected thalamic nuclei, in
hippocampal CA1 neurons, and in layer IV of the cerebral cortex. Many
other tissues also expressed NCKX3 at lower levels, especially aorta,
uterus, and intestine, which are rich in smooth muscle. The discovery
of NCKX3 thus expands the K+-dependent
Na+/Ca2+ exchanger family and suggests this
class of transporter has a more widespread role in cellular
Ca2+ handling than previously appreciated.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-repeats
(3). Two groups within the Na+/Ca2+ exchanger
superfamily have been characterized so far in considerable detail and
consist of structurally and functionally distinct proteins. Na+/Ca2+ exchangers
(NCX)1 are thought to
catalyze the extrusion of one intracellular Ca2+ ion in
exchange for three extracellular Na+ ions (Ref. 1 but see
Ref. 4). Na+/Ca2+ + K+ exchangers
(NCKX), on the other hand, are thought to transport one intracellular
Ca2+ and one K+ ion in exchange for four
extracellular Na+ ions (5).
-repeats, as mentioned
above. The cloning of NCKX1 from rat eye (20) also revealed the
presence of alternatively spliced isoforms in this species, which
differ by the arrangement of four exons at the N terminus of the large
intracellular loop. Interestingly, the equivalent region of bovine
NCKX1, but apparently not the alternatively spliced region in rat
NCKX1, is responsible for producing a functionally silent protein when
expressed in heterologous systems (20, 21).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-repeats conserved among members of the
Na+/Ca2+ exchanger superfamily (3).
Corresponding cDNA clones were purchased from Research Genetics
(Huntsville, AL), and their inserts sequenced completely. The sequence
information was used to design primer pairs for PCR amplification of
cDNA prepared from human total brain RNA
(CLONTECH, Palo Alto, CA). These products were subsequently used to screen a number of cDNA libraries from human brain (CLONTECH; generous gifts of K. Petrukhin and
B. Soares). These screenings eventually yielded three overlapping
clones, from which a composite transcript was constructed
(GenBankTM accession number AF169257). The completed
transcript was used to re-screen EST data bases of the genome projects,
which identified a set of mouse brain ESTs with a high degree of
sequence identity to the human NCKX3 coding sequence. Primers based on
the mouse EST sequences were used to amplify the NCKX3
transcript from mouse brain poly(A)+ mRNA by RT-PCR
(using Expand Reverse Transcriptase followed by the Expand Long
Template PCR System, both from Roche Molecular Biochemicals). The
roughly 2.5-kilobase pair (kb) product was subcloned into the mammalian
expression vector, pcDNA3.1(+) (Invitrogen, Carlsbad, CA). Six
clones with the correct orientation were pooled, and the sequence from
a large scale DNA preparation was determined. This sequence has been
submitted to the GenBankTM under accession number AF314821.
Rat NCKX3 was amplified by RT-PCR from brain RNA using High Fidelity
Taq polymerase (Roche Molecular Biochemicals), and primers
were chosen from regions of nucleotide identity between human and mouse
NCKX3 cDNAs, lying near the N terminus and just downstream from the
C terminus of the deduced coding region. The amplification product was
subcloned into pBluescript II SK(
) (Stratagene, La Jolla, CA) and
sequenced. The consensus sequence from several independent clones has
been deposited in GenBankTM with accession number
AY009158.
clone into pBluescript II SK(
). The plasmid subclone was mapped
further by restriction enzyme digestion and Southern blotting, and the fragment containing exon 1 was sequenced and deposited in the GenBankTM with accession number AF314822.
DNA of the positive full-length clone was digested with KpnI
and SacI, and the fragment containing the full-length
cDNA was subcloned into pBluescript II SK(
). Subsequently, the
remaining
DNA was removed by a partial EcoRI digestion
that released the entire NCKX3 cDNA insert, which was subcloned
into pBluescript II SK(
). This clone was sequenced entirely, and the
data were deposited in GenBankTM with accession number
AF288087.
). Digestion by
XbaI and transcription from the T7 polymerase promoter
produced a 261-nucleotide-long probe that was expected, when fully
protected by NCKX3 mRNA, to result in a fragment 184 nucleotides in
length. Probes 2 and 3 were both prepared from a BglI to
FseI fragment derived from the murine NCKX3 genomic clone
(see Fig. 3) inserted in the SmaI site of pBluescript II SK
(
). Digestion with NcoI (cutting within the insert) or
XbaI, followed by transcription from the T7 polymerase
promoter, yielded probe 2 (456 nucleotides) and probe 3 (590 nucleotides). Full-length protection of these probes is expected to
result in fragments of 383 and 496 nucleotides, respectively.
Radiolabeled riboprobes were prepared using the MAXIscript T7 kit from
Ambion (Austin, TX), in the presence of [
-32P]UTP
(PerkinElmer Life Sciences), and were gel-purified prior to use.
RNase protection assays were performed using the Hybspeed RPA kit
(Ambion), according to the directions of the manufacturer.
. The pipette solution contained 120 mM NaCl, 5 mM KCl, 1 µM
[Ca2+]free (buffered with 0.5 mM
EGTA), 20 mM tetraethylammonium-Cl, 2 mM
MgCl2, 1 mM ATP, 8 mM
D-glucose, and 10 mM HEPES-TMA, pH 7.2. Cells
were held at 0 mV, and outward currents were elicited by perfusion
switches from a solution composed of 145 mM NaCl, 0.5 mM EGTA, 1 mM MgCl2, 10 mM D-glucose, and 10 mM HEPES-TMA, pH 7.4, to ones in which the NaCl and EGTA were substituted by combinations of LiCl, KCl, EGTA, and CaCl2 as follows: 145 mM LiCl and 0.5 mM EGTA, 145 mM
LiCl, and 1 mM CaCl2, 105 mM LiCl, 40 mM KCl, and 0.5 mM EGTA, or 105 mM LiCl, 40 mM KCl, and 1 mM CaCl2. MgCl2 was omitted from the bath solution
in some experiments. Data were obtained using an Axopatch 200B
amplifier and analyzed with pCLAMP software (both from Axon
Instruments, Foster City, CA).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-repeat regions of the
Na+/Ca2+ exchanger (3). Initially, two entries
were identified independently and simultaneously in the Toronto and Los
Angeles laboratories (GenBankTM accession Numbers R21117
and T06656) that led to isolation of the corresponding cDNA clones
from human fetal brain and skeletal muscle libraries, respectively.
Comparison of the deduced amino acid sequence of this novel gene (Figs.
1 and 2)
revealed a significant level of similarity to the two known members of
the K+-dependent
Na+/Ca2+ exchanger family as follows: NCKX1,
first identified in retinal rod outer segments (19), and NCKX2,
originally identified in rat brain and then in chicken and human
retinal cone and ganglion cells (28, 29). Thus, the novel gene
presented here encodes the third member of the
K+-dependent Na+/Ca2+
exchanger gene family and will be referred to as NCKX3 (gene SLC24A3).
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Fig. 1.
Comparison of the NCKX3 protein from human,
mouse, and rat. A, the deduced amino acid sequences of
human, mouse, and rat NCKX3 clones have been aligned using the ClustalW
algorithm in MacVector. Similar or identical amino acids are
boxed, with identities highlighted in bold with a
dark gray background. Dashes indicate gaps introduced to
maximize the alignment. The positions corresponding to exon boundaries
(see Fig. 3) are marked above the sequences with
inverted carets. Hydrophobic, putative transmembrane
spanning, regions identified by hydropathy analysis (Predict Protein
Server and TopPred II) are highlighted and labeled
M0 to M10. Consensus sites for oligosaccharide
addition (CHO) and for phosphorylation via protein kinase A
(PKA) or protein kinase C (PKC) are also
indicated. The DNA sequences used to deduce the proteins shown have
been deposited in GenBankTM with accession numbers AF169257
and AF288087 (human cDNA), AF314821 and AF314822 (mouse cDNA
and partial genomic sequences, respectively), and AY009158 (rat
cDNA). B, hydropathy profile (generated using DNA
Strider with a window of 19 amino acids) for human NCKX3 indicating the
location of the 11 proposed transmembrane regions (M0 to
M10).
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Fig. 2.
Comparison between human
K-dependent Na+/Ca2+ exchanger
paralogs. Alignment of the deduced amino acid sequences from the
hydrophobic regions of human NCKX3 (hNCKX3) with human
retinal rod NCKX1 (hNCKX1; GenBankTM accession
number AF062922), human cone NCKX2 (hNCKX2;
GenBankTM accession number AF097366), human cardiac NCX1
(hNCX1; GenBankTM accession number M91368), and
C. elegans ncx-4 (cNCKX; GenBankTM
accession number AF005701). A, the region including putative
transmembrane spanning regions M1 to M5 (human NCKX3 amino acids
91-249; see Fig. 1) is aligned. B, the region including
putative transmembrane spanning regions M6 to M10 (human NCKX3 amino
acids 477-644; see Fig. 1) is aligned. The conserved -repeat
regions identified by Schwarz and Benzer (3) are indicated with a
gray bar above the alignment. This figure was generated
using the ClustalW web server of the European Institute of
Bioinformatics and enhanced with the Boxshade 3.21 server at the Swiss
EMBO site.
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Fig. 3.
Genomic organization of human NCKX3
(SLC24A3). Exons were identified by BLAST search
of human NCKX3 cDNA against the Human Genome draft sequence of
overlapping BAC clones, shown as thick lines with
accompanying GenBankTM accession numbers at the
top of the figure. This region corresponds to part of
sequence contig NT_019671 located on chromosome 20 at p11. The position
of each exon within the Gene is indicated by numbered
vertical bars below the BAC clones (exons and short
introns not drawn to scale). The size in base pairs and arrangement of
the Exons within the mRNA is illustrated,
with the NCKX3 coding region shown as a filled bar and the
untranslated regions as open boxes. The leading and trailing
sequence of each intron is listed at the bottom of the
figure. The sequence preceding exon 1 corresponds to genomic sequence
found upstream of the HuNCKX3-L clone and may not represent true
flanking sequence, as the transcriptional starting point has not been
mapped in human tissues.
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Fig. 4.
RNase protection analysis of the 5'-end of
the mouse NCKX3 transcript. mRNA from mouse
brain was hybridized with one of three different probes and subjected
to RNase protection analysis. A, protected fragments were
resolved on 5% polyacrylamide/urea gels. Lane labels are as follows:
B1, B2, and B3, 1 µg of mouse brain
poly(A)+ mRNA hybridized to Probes 1, 2, and
3, respectively; t1, tRNA control hybridization
for Probe 1; P, untreated probes (Probe 1 in gel on
left, Probes 2 and 3 combined in gel on right).
The size of the intact probes, as well as the size of the protected
fragments observed are indicated in nucleotides (nt).
B, sequence from the mouse NCKX3 gene including
exon 1 (GenBankTM accession number AF314822), joined to
cDNA sequence for the first part of exon 2 (GenBankTM
accession number AF314821). The position of the intron that separates
these two sequences is indicated. The cDNA sequence overlapped the
genomic sequence from nucleotide 691 to the end of exon 1. Three mouse
EST clones also overlap with this sequence (AI843091, AI850536, and
BE981094), all ending with the NotI site at nucleotide 577. The sequence from the 5'-end of the human NCKX3-L cDNA is shown
above the mouse sequence. Periods indicate
identities; dashes indicate gaps, and inverted
carets indicate insertions, introduced to maximize the alignment.
The extent of the full-length protected fragments is indicated
below the sequence, with a black bar for Probe 1 (nucleotides 621-804) and with a gray bar for Probe 2 (nucleotides 345-727; the internal NcoI site forming the
5'-end of this probe is indicated with a vertical line) and
Probe 3 (nucleotides 232-727).
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Fig. 5.
In vitro translation of cloned
NCKX3. cDNA clones encoding short and long versions of human
NCKX3 (HuNCKX3-S and HuNCKX3-L, respectively) were transcribed and
translated in vitro, either in the presence of 0.1% Triton
X-100 (Triton), canine pancreatic microsomes
(µsomes), or microsomes and 50 µM
competitive glycosylation inhibitory peptide, acetyl-Asn-Tyr-Thr-amide
(µs+NYT). A, autoradiograph of
SDS-polyacrylamide gel. Filled circles indicate size of
polypeptide produced without modification (i.e. in the
presence of Triton alone). Open circles indicate position of
polypeptide modified by glycosylation. The triangle
indicates a HuNCKX3-L polypeptide that may represent a product of
signal peptidase cleavage. B, amino acid sequence for the
N-terminal region of HuNCKX3, indicating the anticipated size for
polypeptides initiating at each methionine residue. The positions of
the hydrophobic transmembrane spans M0 and M1 are indicated as are
putative sites of signal peptide cleavage (SPase?) and
glycosylation (CHO).
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Fig. 6.
Northern blot analysis of NCKX3
tissue expression. Samples (10 µg of total RNA from rat
(A), or mouse (B), or 2 µg of
poly(A)+ mRNA from human brain regions (C))
isolated from the indicated tissues were separated on
formaldehyde-agarose gels, transferred to nylon membranes, and
hybridized at high stringency with probes from either mouse NCKX3
(A and B) or human NCKX3 (C).
Br, brain; CC, cerebral cortex; Cb,
cerebellum; BS, brainstem (includes pons and medulla);
MB, midbrain (includes thalamus and hypothalamus);
M, medulla; SC, spinal cord; Co,
cortex, occipital lobe; Cf, cortex, frontal lobe;
Ct, cortex temporal lobe; P, putamen;
Sk, skeletal muscle (diaphragm in A, leg in
B); Ht, heart; Ao, thoracic aorta;
St, stomach; SI, small intestine; LI,
large intestine; Lu, lung; Li, liver;
Th, thymus; Sp, spleen; LN, lymph
node; Ad, adrenal; Kc, kidney cortex;
Te, testis.
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Fig. 7.
Analysis of NCKX3 expression in
human tissues by RNA dot blot array. A multitissue expression
array with poly(A)+ RNA from 76 human tissues and 8 control
RNAs and DNAs was hybridized and washed at high stringency with a
cDNA probe derived from the human NCKX3 clone isolated from
skeletal muscle. The amount of mRNA from each tissue dotted on the
membrane was normalized to eight different housekeeping genes. After 2 weeks of exposure dots were scanned with a densitometer to give the
relative level of expression. Only those tissues giving signals above
the background of the control samples, which included a sample of human
genomic DNA, are shown in the graph. P.G., paracentral
gyrus.
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Fig. 8.
Distribution of NCKX3
transcripts in mouse brain. In situ hybridization
using digoxigenin-labeled NCKX3 probes was performed on parasagittal
sections of mouse brain. Antisense probe was used for sections on the
left of A and on the top of
B-E, whereas sense control probe was used for sections on
the right and bottom, respectively.
B-E are higher magnification views of various regions from
A. B, parietal cortex; C, hippocampus;
D, thalamus; E, posterior cerebellum. The six
cortical layers are indicated in B; the regions
corresponding to nuclei of the CA1, CA3, and
dentate gyrus (DG) neurons of the hippocampus are marked in
C; the molecular (M), granular (G),
and Purkinje cell (P) layers of cerebellum are indicated in
E.
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Fig. 9.
Functional analysis of NCKX3 using
Ca2+ imaging. HEK293 cells grown on coverslips were
transfected with cDNA constructs encoding murine (Mu)
NCKX3 (short version; A), human (Hu) NCKX3 (short
(S) and long (L) versions, B), or
vector alone (Neg Ctl). The cells were then loaded with
fura-2, mounted in a perfusion chamber on a microscope stage, and
observed by fluorescent digital imaging. An initial perfusion of about
5 min with sodium-containing potassium-free solution was followed by
alternating changes to lithium-containing solutions supplemented with
various concentrations (in mM) of potassium as indicated.
Values (mean ± S.E. for the indicated number of cells) represent
the ratio of background-subtracted fluorescence when excited at 340 or
380 nm. This figure is representative of 11 independent experiments for
MuNCKX3, 7 for HuNCKX3-S, and 4 for HuNCKX3-L.
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Fig. 10.
Functional analysis of NCKX3 using
electrophysiology. HEK293 cells were transfected with cDNA
constructs encoding the long version of human NCKX3
(HuNCKX3-L), or vector alone (Control), together
with a cDNA encoding green fluorescent protein. Transfected cells
were identified under fluorescent illumination and then analyzed by
whole-cell patch clamp. A, representative current traces
from cells transfected with HuNCKX3-L or vector control. Cells were
held at 0 mV, and currents were elicited by perfusion switches between
solutions containing LiCl plus 0.5 mM EGTA (Li),
LiCl plus 1 mM CaCl2 (Ca), LiCl/EGTA
plus 40 mM KCl (K), or LiCl plus both KCl and
CaCl2 (Ca & K). Currents corresponding to NCKX3
were observed in greater than 90% of the HuNCKX3-L-transfected cells
but in none of the control cells. B, average current values,
Li/EGTA-background subtracted and normalized to cell capacitance, are
plotted ± S.E. for the indicated number of cells measured under
each perfusion condition, either with or without 1 mM
MgCl2 in the perfusate.
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Fig. 11.
Functional analysis of NCKX3 using
45Ca2+ uptake. HEK293 cells were
transfected with cDNA constructs encoding the human NCKX3 short
(HuNCKX3-S) and long (HuNCKX3-L) versions as well
as NCX1 (positive control) or with vector alone (negative
control; pcDNA). Forty eight hours post-transfection the
cells were loaded with sodium, and 45Ca2+
uptake was measured in the presence of either NaCl, KCl, or choline
chloride (ChCl) for 1 min. The data presented are the
averages ± S.E. of the means for 3 (HuNCKX3-L, NCX1, and
pcDNA), 2 (HuNCKX3-S uptake in NaCl and KCl), and 1 (HuNCKX3-S
uptake in ChCl) independent experiments, each conducted in
triplicate.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-repeat regions of the
molecule in forming the binding pocket for ion translocation (45, 46). Functional studies on a deletion mutant of bovine NCKX1 and a C. elegans paralog (cNCKX) have suggested similar regions also specify the transport sites needed for
K+-dependent Na+/Ca2+
exchangers (30). A comparison of the sequences between NCKX3 and these
molecules (Fig. 2) shows the high sequence similarity anticipated
within the
-repeats. NCKX3 is, however, significantly more divergent
in these regions than the NCKX1/NCKX2/cNCKX trio. Intriguingly, these
positions of divergence often correspond to identities or similarities
with NCX1. Nevertheless, NCKX3 is clearly K+-dependent, in contrast to NCX1, and thus
this new sequence information places further limits on the amino acids
likely to contribute to the ionic specificity of the
K+-dependent class of
Na+/Ca2+ exchangers.
-repeat regions, NCKX3 must have its
origins in a very ancient gene duplication event and subsequent
divergence from other NCKX and NCX family members.
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ACKNOWLEDGEMENTS |
---|
We thank David Andrews (McMaster University) and Reinhart Reithmeier (University of Toronto) for their generous gifts of canine pancreatic microsomes and acetyl-Asn-Tyr-Thr-amide, respectively, and Roland Auer (University of Calgary) for assistance in interpretation of neuronal expression patterns. A. K. thanks David H. MacLennan (University of Toronto) for support, helpful discussions, and the use of his laboratory for some of these studies.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants HL4901 and HL48509 (to K. D. P.) and by the Canadian Institutes of Health Research Grant FRN15035 (to J. L.).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF169257, AF288087, AF314821, AF314822, and AY009158.
§ Both authors contributed equally to this work.
** Senior Scholar of the Alberta Heritage Foundation for Medical Research and an Investigator of the Canadian Institutes of Health Research. To whom correspondence should be addressed: University of Calgary Health Sciences Center, 3330 Hospital Dr. NW, Calgary, Alberta T2N 4N1, Canada. Tel.: 403-220-2893; Fax: 403-283-4841; E-mail: jlytton@ucalgary.ca.
Published, JBC Papers in Press, April 9, 2001, DOI 10.1074/jbc.M102314200
2 A. Kraev, unpublished observations.
3 Analysis of the completed sequence of the human genome (53, 54) reveals another putative NCKX family member on chromosome 14. This gene was given the designation NCKX4 (SLC24A4), although it has not yet been characterized functionally.
4 H. Dong and J. Lytton, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are: NCX, Na+/Ca2+ exchanger; EST, expressed sequence tags; kb, kilobase pair; MOPS, 3-(N-morpholino)propane-sulfonic acid; NCKX, Na+/Ca2+ + K+ exchanger; PCR, polymerase chain reaction; RT-PCR, reverse transcription-coupled polymerase chain reaction.
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