A new Na/Ca exchanger splicing pattern identified in situ leads to a functionally active 70 kDaNH2-terminal protein

doi:10.1054/ceca.2001.0223

Cell Calcium

Volume 30, Issue 3, September 2001, Pages 191-198

https://doi.org/10.1054/ceca.2001.0223 Get rights and content

Abstract

The Na/Ca exchanger (NCX) is an ubiquitous transporter that plays an important role in regulating cellular Ca²⁺balance. On gel electrophoresis, the NCX1 protein migrates as two major bands of 120 and 70kDa. While the120kDa is thought to represent the native protein, the nature of the 70kDa protein remains unclear. In this report, we describe a new NCX1 splicing pattern, identified during the cloning of NCX1 isoforms from human eye. The insertion of a newly identified sequence upstream exons B and D of the NCX1.3 isoform, generates a stop codon in frame with the NCX1 coding sequence, that should lead to a truncated Na/Ca exchanger (that we called NCX1.33) comprising only the N-terminal portion of the exchanger and a shortened intracellular loop. Insulin-secreting cells were stably tran sfected with NCX1.33. Overexpression was assessed at the mRNA and protein level, the truncated exchanger migrating as a70kDa band. Appropriate targeting to the plasma membrane was assessed by microfluorescence and by the increase in Na/Ca exchange activity. The results of the present study constitute a clear piece of evidence indicating that the Na/Ca exchanger 70kDa protein corresponds to the N-terminal portion of the exchanger, and is functionally active.

References (19)

KD Philipson et al.
Purification of the cardiac Na⁺⁻Ca²⁺exchange protein
Biochim Biophys Acta
(1988)
JT Durkin et al.
Purification and amino-terminal sequence of the bovine cardiac sodium-calcium exchanger: evidence for the presence of a signal sequence
Arch Biochem Biophys
(1991)
T Scheller et al.
Cloning of the multipartite promoter of the sodium-calcium exchanger gene NCX1 and characterization of its activity in vascular smooth muscle cells
J Biol Chem
(1998)
A Kraev et al.
The organization of the human gene NCX1 encoding the sodium-calcium exchanger
Genomics
(1996)
X -F Li et al.
A circularized sodium-calcium exchanger exon 2 transcript
J Biol Chem
(1999)
N Gabellini et al.
An alternative splicing site modifies the carboxyl-terminal trans-membrane domains of the Na⁺/Ca²⁺exchanger
J Biol Chem
(1995)
T Iwata et al.
The 70kDa component of the heart sarcolemmal Na⁺/Ca²⁺-exchanger preparation is the C-terminal portion of the protein
Cell Calcium
(1995)
F Van Eylen et al.
Identification, expression pattern and potential activity of Na/Ca exchanger isoforms in rat pancreatic B-cells
Cell Calcium
(1997)
A Herchuelz et al.
A role for Na/Ca exchange in the pancreatic B cell. Studies with thapsigargin and caffeine
Biochem Pharmacol
(1993)

There are more references available in the full text version of this article.

Cited by (27)

The SLC8 gene family of sodium-calcium exchangers (NCX)-Structure, function, and regulation in health and disease
2013, Molecular Aspects of Medicine
Citation Excerpt :
Rapamycin reduces NCX2 and NCX3 activity but has no effect on their surface expression or the total NCX expression, whereas the regulatory f-loop of NCX might be involved in acquisition of immunosuppressive drug specificity. The β-cells express two specific isoforms of NCX1, NCX1.3, and NCX1.7 (Van Eylen et al., 2001; Herchuelz et al., 2002, 2007). In β-cells, NCX displays a high capacity and contributes to controlling [Ca2+] and insulin release.
The SLC8 gene family encoding Na⁺/Ca²⁺ exchangers (NCX) belongs to the CaCA (Ca²⁺/Cation Antiporter) superfamily. Three mammalian genes (SLC8A1, SLC8A2, and SLC8A3) and their splice variants are expressed in a tissue-specific manner to mediate Ca²⁺-fluxes across the cell-membrane and thus, significantly contribute to regulation of Ca²⁺-dependent events in many cell types. A long-wanted mitochondrial Na⁺/Ca²⁺ exchanger has been recently identified as NCLX protein, representing a gene product of SLC8B1. Distinct NCX isoform/splice variants contribute to excitation–contraction coupling, long-term potentiation of the brain and learning, blood pressure regulation, immune response, neurotransmitter and insulin secretion, mitochondrial bioenergetics, etc. Altered expression and regulation of NCX proteins contribute to distorted Ca²⁺-homeostasis in heart failure, arrhythmia, cerebral ischemia, hypertension, diabetes, renal Ca²⁺ reabsorption, muscle dystrophy, etc. Recently, high-resolution X-ray structures of Ca²⁺-binding regulatory domains of eukaryotic NCX and of full-size prokaryotic NCX have become available and the dynamic properties have been analyzed by advanced biophysical approaches. Molecular silencing/overexpression of NCX in cellular systems and organ-specific KO mouse models provided useful information on the contribution of distinct NCX variants to cellular and systemic functions under various pathophysiological conditions. Selective inhibition or activation of predefined NCX variants in specific diseases might have clinical relevance, although this breakthrough has not yet been realized. A better understanding of the underlying molecular mechanisms as well as the development of in vitro procedures for high-throughput screening of “drug-like” compounds may lead to selective pharmacological targeting of NCX variants.
Intestinal Na+/Ca2+ exchanger protein and gene expression are regulated by 1,25(OH)<inf>2</inf>D<inf>3</inf> in vitamin D-deficient chicks
2011, Archives of Biochemistry and Biophysics
Citation Excerpt :
The 70 kDa is a proteolytic product and the 120 kDa is the full-length protein. Both proteins seem to be functionally active [31]. We did not exclude the presence of other isoforms of NCX protein in chick intestine.
The role of 1,25(OH)₂D₃ on the intestinal NCX activity was studied in vitamin D-deficient chicks (-D) as well as the hormone effect on NCX1 protein and gene expression and the potential molecular mechanisms underlying the responses. Normal, -D and -D chicks treated with cholecalciferol or 1,25(OH)₂D₃ were employed. In some experiments, -D chicks were injected with cycloheximide or with cycloheximide and 1,25(OH)₂D₃ simultaneously. NCX activity was decreased by -D diet, returning to normal values after 50 IU daily of cholecalciferol/10 days or a dose of 1 μg calcitriol/kg of b.w. for 15 h. Cycloheximide blocked NCX activity enhancement produced by 1,25(OH)₂D₃. NCX1 protein and gene expression were diminished by -D diet and enhanced by 1,25(OH)₂D₃. Vitamin D receptor expression was decreased by -D diet, effect that disappeared after 1,25(OH)₂D₃ treatment. Rapid effects of 1,25(OH)₂D₃ on intestinal NCX activity were also demonstrated. The abolition of the rapid effects through addition of Rp-cAMPS and staurosporine suggests that non genomic effects of 1,25(OH)₂D₃ on NCX activity are mediated by activation of PKA and PKC pathways. In conclusion, 1,25(OH)₂D₃ enhances the intestinal NCX activity in -D chicks through genomic and non genomic mechanisms.
Molecular cloning and characterization of two novel truncated isoforms of human Na+/Ca2+ exchanger 3, expressed in fetal brain
2005, Gene
The human gene encoding the Na⁺/Ca²⁺ exchanger family member 3 (NCX3) undergoes extensive alternative splicing, with four variants previously identified. In this study, we report two novel alternative transcripts encoding two N-terminally truncated NCX3 proteins specifically expressed in human fetal brain. The identified transcripts, designated NCX3-tN.1 and NCX3-tN.2, are approximately 2.8 kb and 2.9 kb, respectively. The open reading frames (ORFs) are predicted to encode separately a 284 and a 298 amino acid (aa) polypeptide. Sequence analysis and bioinformatics reveal that NCX3-tN.1 and NCX3-tN.2 are the result of alternative splicing of the NCX3 gene. They have their own potential start codons and unique 5′ untranslated regions (UTRs) that are different from those of the known NCX3 variants. The variants include a part of intron 2 of the original gene organization as their first exon (exon “a”) at the 5′ end of the novel transcripts. NCX3-tN.2 consists of six exons including exon “a” and exons 4, 6, 7, 8 and 9 of NCX3, while NCX3-tN.1 lacks exon 4, but is otherwise similar to NCX3-tN.2. Expression studies show that both variants can be translated into protein and NCX3-tN.1 seems more efficiently translated. Based on their structural features, NCX3-tN.1 and NCX3-tN.2 proteins are potentially involved in regulation of Na⁺/Ca²⁺ homeostasis.
Ca2+ Signaling by TRPC3 Involves Na+ Entry and Local Coupling to the Na+/Ca2+ Exchanger
2004, Journal of Biological Chemistry
Citation Excerpt :
Immunoblotting experiments with HEK293 lysates revealed a pattern of proteins that is typically observed in NCX expressing cells, including a band at 70 kDa and ∼120 kDa. Consistently, the immunoreactivity retained in GST pull-down experiments with a TRPC3 fragment as bait displayed a molecular mass of ∼120 and 70 kDa corresponding to native full-length and truncated forms of NCX1 described in other cell types (16, 26, 27). Moreover, we obtained convincing evidence for a Na+-dependent Ca2+ transport system in HEK293 cells.
TRPC3 has been suggested as a key component of phospholipase C-dependent Ca²⁺ signaling. Here we investigated the role of TRPC3-mediated Na⁺ entry as a determinant of plasmalemmal Na⁺/Ca²⁺ exchange. Ca²⁺ signals generated by TRPC3 overexpression in HEK293 cells were found to be dependent on extracellular Na⁺, in that carbachol-stimulated Ca²⁺ entry into TRPC3 expressing cells was significantly suppressed when extracellular Na⁺ was reduced to 5 mm. Moreover, KB-R9743 (5 μm) an inhibitor of the Na⁺/Ca²⁺ exchanger (NCX) strongly suppressed TRPC3-mediated Ca²⁺ entry but not TRPC3-mediated Na⁺ currents. NCX1 immunoreactivity was detectable in HEK293 as well as in TRPC3-overexpressing HEK293 cells, and reduction of extracellular Na⁺ after Na⁺ loading with monensin resulted in significant rises in intracellular free Ca²⁺ (Ca²⁺_i) of HEK293 cells. Similar rises in Ca²⁺_i were recorded in TRPC3-overexpressing cells upon the reduction of extracellular Na⁺ subsequent to stimulation with carbachol. These increases in Ca²⁺_i were associated with outward membrane currents at positive potentials and inhibited by KB-R7943 (5 μm), chelation of extracellular Ca²⁺, or dominant negative suppression of TRPC3 channel function. This suggests that Ca²⁺ entry into TRPC3-expressing cells involves reversed mode Na⁺/Ca²⁺ exchange. Cell fractionation experiments demonstrated co-localization of TRPC3 and NCX1 in low density membrane fractions, and co-immunoprecipitation experiments provided evidence for association of TRPC3 and NCX1. Glutathione S-transferase pull-down experiments revealed that NCX1 interacts with the cytosolic C terminus of TRPC3. We suggest functional and physical interaction of nonselective TRPC cation channels with NCX proteins as a novel principle of TRPC-mediated Ca²⁺ signaling.
Molecular Cloning of a Sixth Member of the K+-dependent Na +/Ca2+ Exchanger Gene Family, NCKX6
2004, Journal of Biological Chemistry
Citation Excerpt :
Studies from our laboratory have demonstrated an abundant and unusual circular transcript of the NCX1 gene that encodes a truncated protein, containing only the first α-repeat, which is nevertheless functional when expressed in HEK-293 cells (49). Other groups have also reported similar truncated, functional NCX1 proteins (50-52). The mechanism for how such single α-repeat proteins including NCKX6 act as exchangers is not clear but may involve oligomeric association of exchanger proteins (34, 53).
Bioinformatic and molecular cloning tools were used to identify and isolate cDNA clones from mouse and human tissues that encode the sixth member of the K⁺-dependent Na⁺/Ca²⁺ exchanger family, NCKX6. The mouse NCKX6 protein is 585 amino acids long and shares about 62% sequence similarity with previously identified exchangers in the α-repeat regions but has little primary sequence similarity outside these regions. NCKX6 transcripts of 4 kb are abundantly expressed in all tissues examined and are thus more broadly distributed than previously described NC(K)X family members. Two alternatively spliced products of this novel gene were identified that encode proteins of different length. The short isoform differs from the full-length isoform at the C-terminal hydrophobic domain as a result of a shift in the reading frame caused by the deletion of two exons. Both NCKX6 isoforms were expressed in HEK-293 cells. Functional analysis by digital imaging of fura-2 loaded transfected HEK-293 cells demonstrated that the short isoform exhibited K⁺-dependent Na⁺/Ca²⁺ exchange activity whereas the full-length isoform did not. The latter was retained within the endoplasmic reticulum, whereas the short isoform was present at the plasma membrane in transfected cells. Immunofluorescence studies examining NCKX6 expression in native tissue using an NCKX6-specific antibody showed intense labeling of the cardiac sarcolemmal membrane. The discovery of NCKX6 therefore reveals a novel member of the Na⁺/Ca²⁺ exchanger superfamily whose ubiquitous expression in all tissues suggests an important role for K⁺-dependent Na⁺/Ca²⁺ exchange in maintaining cellular Ca²⁺ homeostasis in diverse tissues and cell types.
The human SLC8A3 gene and the tissue-specific Na+/Ca2+ exchanger 3 isoforms
2002, Gene
We have identified the human gene for member 3 of Solute Carrier family 8 (SLC8A3) by bioinformatic analysis of human genomic sequences. The gene is located on chromosome 14q24.2, and spans a region of about 150 kb. The full-length DNA complementary to RNA encoding the Na⁺/Ca²⁺ exchanger isoform 3 (NCX3), amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) from the human neuroblastoma SH-SY5Y RNA, includes seven exons and encodes a protein of about 100 kDa. RT-PCR analysis was performed in different tissues to determine the exon composition in the region encoding the large intracellular loop of the protein. The region underwent modifications by alternative tissue-specific splicing. NCX3.2, including exon 4 but not exon 5, was found in human brain and in the neuroblastoma cell line. In human skeletal muscle two additional isoforms were identified: NCX3.3, including exons 4 and 5, and a truncated isoform (NCX3.4) produced by the skipping of both exons 3 and 4. The skipping causes a frame shift downstream of the exon 2 sequence. The new coding sequence of 25 amino acids terminates with a stop codon in exon 6. The NCX3.4 isoform (68 kDa) is truncated in the C-terminal portion of the domain first found in Drosophila Na⁺/Ca²⁺ exchanger domain (Calxβ) and lacks the C-terminal hydrophobic segments.

View all citing articles on Scopus

^f1: Correspondence to: A. Herchuelz, Laboratoire de Pharmacodynamie et de Thérapeutique, Université Libre de Bruxelles, Faculté de Médecine, Route de Lennik, 808 – Bâtiment GE, B-1070 Bruxelles, Belgium. Tel.: +3225556201; Fax: +3225556370; e-mail:[email protected]

View full text

Article preview

Cell Calcium

Abstract

References (19)

Purification of the cardiac Na⁺⁻Ca²⁺exchange protein

Biochim Biophys Acta

Purification and amino-terminal sequence of the bovine cardiac sodium-calcium exchanger: evidence for the presence of a signal sequence

Arch Biochem Biophys

Cloning of the multipartite promoter of the sodium-calcium exchanger gene NCX1 and characterization of its activity in vascular smooth muscle cells

J Biol Chem

The organization of the human gene NCX1 encoding the sodium-calcium exchanger

Genomics

A circularized sodium-calcium exchanger exon 2 transcript

J Biol Chem

An alternative splicing site modifies the carboxyl-terminal trans-membrane domains of the Na⁺/Ca²⁺exchanger

J Biol Chem

The 70kDa component of the heart sarcolemmal Na⁺/Ca²⁺-exchanger preparation is the C-terminal portion of the protein

Cell Calcium

Identification, expression pattern and potential activity of Na/Ca exchanger isoforms in rat pancreatic B-cells

Cell Calcium

A role for Na/Ca exchange in the pancreatic B cell. Studies with thapsigargin and caffeine

Biochem Pharmacol

Cited by (27)

The SLC8 gene family of sodium-calcium exchangers (NCX)-Structure, function, and regulation in health and disease

Intestinal Na<sup>+</sup>/Ca<sup>2+</sup> exchanger protein and gene expression are regulated by 1,25(OH)<inf>2</inf>D<inf>3</inf> in vitamin D-deficient chicks

Molecular cloning and characterization of two novel truncated isoforms of human Na<sup>+</sup>/Ca<sup>2+</sup> exchanger 3, expressed in fetal brain

Ca<sup>2+</sup> Signaling by TRPC3 Involves Na<sup>+</sup> Entry and Local Coupling to the Na<sup>+</sup>/Ca<sup>2+</sup> Exchanger

Molecular Cloning of a Sixth Member of the K<sup>+</sup>-dependent Na <sup>+</sup>/Ca<sup>2+</sup> Exchanger Gene Family, NCKX6

The human SLC8A3 gene and the tissue-specific Na<sup>+</sup>/Ca<sup>2+</sup> exchanger 3 isoforms

Regular ArticlesA new Na/Ca exchanger splicing pattern identified in situ leads to a functionally active 70 kDaNH2-terminal protein

Abstract

Biochim Biophys Acta

Arch Biochem Biophys

J Biol Chem

Genomics

J Biol Chem

J Biol Chem

Cell Calcium

Cell Calcium

Biochem Pharmacol

Regular Articles
A new Na/Ca exchanger splicing pattern identified in situ leads to a functionally active 70 kDaNH₂-terminal protein