The Putative Amino-terminal Signal Peptide of the Cloned Rat Brain Na+-Ca2+ Exchanger Gene (RBE-1) Is Not Mandatory for Functional Expression

Abstract

The rat brain Na(+)-Ca2+ exchanger (RBE) gene, as well as other isoforms of this protein family, can be organized into 12 transmembrane alpha helices, the first of which was proposed by Durkin et al. (14) to constitute a cleavable signal peptide. We have prepared three amino-terminal mutants, in which 21, 26, and 31 amino acids beyond the initiating methionine were deleted. The deletions include the hydrophobic core of the putative signal peptide (N21), the entire putative signal peptide and parts of the putative signal peptidase cleavage site (N26), and the entire putative signal peptide and putative signal peptidase cleavage site (N31). All three mutant clones were transiently expressed in HeLa cells. The average Na+ gradient-dependent Ca2+ transport activity of the mutant exchangers was 108% (N21), 37.2% (N26), and 60.06% (N31) of the wild-type clone. Mutation of the putative cleavage site by an exchange of Ala-32 --> Asp, resulted in a decrease in Na(+)-Ca2+ exchange activity to 7.7%, relative to the wild-type exchanger. Functional reconstitution of the proteins that were expressed in the transfected cells, resulted in transport activities of: 60.1% (N21), 26.75% (N26), 85.36% (N31), and 31% (Ala-32 --> Asp) relative to the wild-type exchanger. Western blot analysis of the protein profile of RBE-1, N21, N26, N31 and Ala-32 --> Asp-transfected HeLa cells was carried out by using an antipeptide antibody directed against a pentadecapeptide segment derived from the large putative cytoplasmic loop of the cloned rat exchanger gene. In the total cell extract and in the plasma membrane-enriched fraction, in addition to a major protein band of about 125 kDa, which corresponds to the molecular mass of the mature fully processed Na(+)-Ca2+ exchanger, an additional protein of about 135 kDa is revealed in the profile of N21- and N26-transfected cells. This band is not detected in the protein profile of RBE-1, N31, or Ala-32 -->Asp. The amino-terminal truncated mutants of the cloned Na(+)-Ca2+ exchanger could be expressed and processed also in a reticulocyte lysate supplemented with dog microsomes. Our results suggest that the putative signal peptide of the cloned Na(+)-Ca2+ exchanger gene does not play a mandatory role in functional expression of the protein in HeLa cells.

Association of Genetic Polymorphisms of Sodium-Calcium Exchanger 1 Gene, NCX1, with Hypertension in a Japanese General Population

“…Arg5 is located within the signal peptide sequence consisting of the first N-terminal 35 amino acids of NCX1, which are removed during biosynthesis (1). We expressed a mutant canine NCX1 with the Arg5Gln substitution in the fibroblastic cell line CCL39, and found that this mutant NCX1 was properly targeted into the plasma membrane and exhibited the normal Na /Ca 2 exchange activity (unpublished observations), consistent with previous reports stating that signal sequence is not essential for functional expression of the NCX1 protein (25,26). On the other hand, Arg703 is located within the large cytoplasmic loop connecting the transmembrane segments 5 and 6, which are not essential for the functional expression of the NCX1 protein (1).…”

Molecular Cloning of a Fourth Member of the Potassium-dependent Sodium-Calcium Exchanger Gene Family, NCKX4

“…Indeed, previous studies on the NCX1 molecule have indicated that, although not essential, M0 does play a role in optimal functional expression (37). Similarly, activity of NCKX3 in HEK293 cells was reduced when a clone lacking the M0 region was expressed.…”

The Transport Activity of the Na+-Ca2+Exchanger NCX1 Expressed in HEK 293 Cells Is Sensitive to Covalent Modification of Intracellular Cysteine Residues by Sulfhydryl Reagents

“…(9). Preparation, testing, and purification of the antibodies was done as described (9,17). Goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, Inc.) was used to detect antigen-antibody complexes using the ECL kit from Amersham Pharmacia Biotech.…”