Two functional lsoforms of the rat brain Nat-Ca'+ exchanger were isolated from a 1ZAP hippocampus cDNA library. The open reading frame of clone RBE-1 codes for a protein 935 amino acids long, and that of clone RBE-2 codes for a protein 958 amino acids long. Expression in HeLa cells of Na' gradient dependent Ca" transport activity was determined following transfection of the cells with either RBE-1 or RBE-2. Both clones expressed proteins that exchange Na' with Ca" m an electrogemc manner and none of them exhibited a dependency of the antiport on K', since they transported Ca'+ in an Na+ gradient dependent manner in external choline chloride as well.

The C terminus of the rat brain Na exchanger and from C10 indicated that resistance to the digestion was acquired after 1 and 5 h of chase, respectively. C29 did not acquire detectable resistance to endoglycosidase H digestion even after 10 h of chase. Taken together, these results suggest that the "cellular quality control machinery" can tolerate the structural change introduced by truncation of the C terminus up to Ser-893 albeit with reduced rate of ER3 Golgi transfer and reduced surface expression of the truncated protein. Further truncation of C-terminal amino acids leads to retention of the truncated protein in the ER, no transfer to the Golgi, and no surface expression.

The effect of 1,25‐dihydroxyvitamin D3 (1,25‐(OH)2D3) on the expression of macrophage colony‐stimulating factor (M‐CSF), leukemia inhibitory factor (LIF), and tumor necrosis factor‐α (TNF‐α) genes in primary rat astrocytes and C6 glioma cells was examined. The results show that the hormone differentially regulates the cytokine mRNA in the two cell types. 1,25‐(OH)2D3 augments M‐CSF and LIF mRNA in C6 glioma cells, while lipopolysaccharide (LPS) has minimal effects. When LPS and 1,25‐(OH)2D3 are used in combination, a strong synergistic effect upon the induction of M‐CSF and LIF genes is observed. No TNF‐α transcript has been detected in C6 glioma cells under any stimulus conditions used. In contrast, 1,25‐(OH)2D3 has no pronounced effect on M‐CSF, LIF, and TNF‐α transcripts in primary astrocytes when used as a sole stimulus, while treatment with LPS strongly enhances the levels of the three cytokines. However, when 1,25‐(OH)2D3 is used in combination with LPS, a partial reduction in LPS‐induced levels of M‐CSF and TNF‐α mRNA is observed. The overall results indicate that genes coding for some inflammatory cytokines obey distinct regulatory mechanisms in C6 cells and in primary astrocytes. They also suggest that 1,25‐(OH)2D3, by altering the response of astrocytes to an inflammatory stimulus, could participate in the regulation of the CNS immune response. © 1996 Wiley‐Liss, Inc.

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.