Calcineurin Controls the Transcription of Na+/Ca2+ Exchanger Isoforms in Developing Cerebellar Neurons

Abstract

The Na ؉ /Ca 2؉ exchanger (NCX) and the plasma membrane Ca 2؉ -ATPase export Ca 2؉ from the cytosol to the extracellular space. Three NCX genes (NCX1, NCX2, and NCX3), encoding proteins with very similar properties, are expressed at different levels in tissues. Essentially, no information is available on the mechanisms that regulate their expression. Specific antibodies have been prepared and used to explore the expression of NCX1 and NCX2 in rat cerebellum. The expression of NCX2 became strongly up-regulated during development, whereas comparatively minor effects were seen for NCX1. This was also observed in cultured granule cells induced to mature in physiological concentrations of potassium. By contrast, higher K ؉ concentrations, which induce partial depolarization of the plasma membrane and promote the influx of Ca 2؉ , caused the complete disappearance of NCX2. Reverse transcription-polymerase chain reaction analysis showed that the process occurred at the transcriptional level and depended on the activation of the Ca 2؉ calmodulin-dependent protein phosphatase, calcineurin. The NCX1 and NCX3 genes were also affected by the depolarizing treatment: the transcription of the latter became upregulated, and the pattern of expression of the splice variants of the former changed. The effects on the NCX1 and NCX3 genes were calcineurin-independent.Hormonal and electrical stimuli promote the penetration of Ca 2ϩ into cells to activate cellular responses. Ca 2ϩ must then be continuously extruded, because its uncontrolled increase in the cytosol would lead to cell death. Two systems, a pump (1) and a Na ϩ /Ca 2ϩ exchanger (2), eject Ca 2ϩ . The latter system, which is particularly active in heart and neurons, uses the Na ϩ gradient generated by the Na ϩ /K ϩ -ATPase to remove Ca 2ϩ from the cytosol; under normal conditions 3 Na ϩ ions are exchanged for 1 Ca 2ϩ . The cDNA of exchanger type 1 (NCX1) 1 has been cloned from mammalian (3-8), amphibian (9), and invertebrate (10, 11) tissues. A comparison of the sequences shows a high level of conservation. The mature NCX1 is a glycosylated protein (12) of 970 amino acids, the first 32 of which are post-translationally cleaved off (13-15). The original membrane topography model based on hydropathy analysis predicted 11 transmembrane domains, separated by small loops and by a large intracellular loop (Ͼ500 amino acids) between transmembrane domains 5 and 6. A more recent model based on cysteine accessibility studies has revised the number of predicted transmembrane domains down to 9 (16), eliminating 2 from the C-terminal half of the exchanger. Although the large intracellular loop is not strictly necessary for the activity of the exchanger, it contains important regulatory elements (17-19). Its C-terminal portion is subjected to alternative splicing (20), which also occurs at the 5Ј-untranslated region of the gene (21-23). Numerous splicing variants have been described for NCX1. The amount of the major variant present in neurons (the AD isoform) is altered by protein kinase...

The Expression of the PDZ Protein MALS-1/Velis Is Regulated by Calcium and Calcineurin in Cerebellar Granule Cells

“…Furthermore, these treatments decreased the relative MALS-1 expression compared with nondepolarized control cells. This finding supports earlier data showing that Ca 2ϩ -and calcineurin-dependent gene expression is active to a small degree also in basal culture conditions and can be blocked by L-type Ca 2ϩ channel blockers and calcineurin inhibitors (17,18).…”

“…This might suggest that the pathway involved in Homer 1a regulation is developmentally regulated and is not present in the early stages of the culture. As a further control to strengthen the reliability of our model, we performed a RT-PCR with specific primers for NCX2, the Na ϩ /Ca 2ϩ exchanger isoform that is selectively controlled by calcineurin (18). As expected, cells grown in 25 mM KCl expressed lower levels of this isoform (Fig.…”

Inhibitory Interaction of the Plasma Membrane Na+/Ca2+ Exchangers with the 14-3-3 Proteins

“…The cells were grown in the presence of physiological concentrations of KCl (5.3 mM) to prevent NCX2 degradation (10). The 14-3-3⑀ protein was found to be associated with the NCX2 protein of the granules (Fig.…”

Expression and function of Na+/Ca2+ exchangers 1 and 3 in human macrophages and monocytes

“…Finally, in monocytes and BHK-NCX1 a large immunoreactive area of high molecular weight (between 140 and 200 kDa) was detected. This could be the internally locked variant of NCX1 that migrates with abnormal electrophoretic mobility [31,33]. Alternatively, this immunoreactivity might be due to glycosylation of the full-length NCX1 that contributes to increase its molecular weight.…”