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 up-regulated, and the pattern of expression of the splice variants of the former changed. The effects on the NCX1 and NCX3 genes were calcineurin-independent.
Highlights
The Na؉/Ca2؉ exchanger (NCX) and the plasma membrane Ca2؉-ATPase export Ca2؉ from the cytosol to the extracellular space
Antibodies Specific for the Naϩ/Ca2ϩ Exchanger Isoforms— Antibodies specific for the NCX1 and NCX2 isoforms were prepared using peptides encompassing the region subjected to alternative splicing (Fig. 1A) as epitopes, because this region shows a low degree of sequence conservation in the three isoforms: The identity of the peptides was below 44% (Fig. 1B)
The NCX1-specific antiserum recognized the exchanger in dog cardiac sarcolemma or the NCX1 expressed in HeLa cells (Fig. 1C)
Summary
The Na؉/Ca2؉ exchanger (NCX) and the plasma membrane Ca2؉-ATPase export Ca2؉ from the cytosol to the extracellular space. The expression of NCX2 became strongly up-regulated during development, whereas comparatively minor effects were seen for NCX1 This was observed in cultured granule cells induced to mature in physiological concentrations of potassium. Whereas NCX1 is expressed at high levels in heart, and is normally referred to as the “cardiac form” of the protein even if present in other tissues, significant amounts of NCX2 and NCX3 mRNAs have only been detected by Northern blots in brain and skeletal muscles. In particular neurons, contain large amounts of all three basic NCX isoforms and of their splice variants and are good models for study. In this research, their expression was investigated during the development of rat cerebellum and of cultured cerebellar granule neurons. The work has shown that Ca2ϩ and calcineurin are critical to the expression of the exchanger genes, supporting the idea that one of the major differences among the NCX genes is the regulation of their transcription
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