Na(+)/Ca(2+) exchanger isoforms in nervous tissue and isoform-specific regulation by PKA

Abstract

In many tissues including nervous tissue, cytoplasmic Ca2+ concentration is regulated by the plasma membrane Na+/Ca{2+ exchanger. I have measured Na+/Ca2+ exchanger activity in brain-derived primary astrocyte cultures by using Na+-dependent45Ca2+ influx. Multiple Na+/Ca2+ exchanger genes have been identified in the rat brain tissue. I used RT-PCR cloning and RNase protection to show that NCX1 mRNA was the main transcript expressed in primary astrocyte and neuron cultures compared to NCX2. The NCX1 gene has previously been demonstrated to undergo alternative splicing of the primary transcript in the carboxyl end of the intracellular loop of the Na+/Ca2+ exchanger protein to produce multiple tissue-specific isoforms. This alternative splicing is generated by six exons present in the gene (A, B, C, D, E and F), among which exons A and B are mutually exclusive in their expression and the other four are "cassette" type exons. Using RT-PCR cloning, I demonstrated the presence of four NCX1 isoforms (BDEF, BDF, BDE and BD) in the cultured astrocytes and two NCX1 isoforms (ADF and AD) in the cultured neurons. To quantitate the relative mRNA amounts of these NCX1 isoforms, I developed a new method, quantitative end-labeled RT-PCR (QERT-PCR). Using this method, I have demonstrated that three exon B-containing isoforms (BDEF, BDF and BD) are predominant in the primary astrocyte culture and each represents about 23% of the total QERT-PCR product. Interestingly, QERT-PCR analysis of the C6 astrocytoma cell line has shown a similar pattern for the predominant isoforms as in the primary astrocyte culture. In the primary neuron culture, QERT-PCR results demonstrate that two exon A-containing isoforms (ADF and AD) are the predominant representing 57% and 37% of the NCX1 transcripts, respectively. While alternative splicing of NCX1 would predict that up to 32 different Na+/Ca2+ exchanger mRNAs may be produced by various combination of exons A to F, by using QERT-PCR, I have demonstrated there are a total of only 16 isoforms including the 3 predominant ones in the primary astrocyte culture. Each of these isoforms contain either exon A or B and all have exon D in the sequence. To determine the functional significance underlying the different isoforms in these cells, a representative astrocytic isoform (BD) and neuronal isoform (AD) have been subcloned into the full-length NCX1 cDNA. When RNA from these constructs were expressed in Xenopus oocytes, both isoforms showed the Na+/Ca2+ exchanger activity. Importantly, the activity of the AD isoform increased by 31-54% after activating the protein kinase A (PKA) pathway whereas the activity of the BD isoform did not change. Function of chimeric constructs containing parts of exon A and B sequences have been examined, demonstrating that the carboxyl terminal 20 amino acids of exon A were important to enable PKA to increase the Na+/Ca2+ exchanger activity. Additionally, experiments with exon D deletion construct showed that this exon was also necessary for the PKA-dependent modulation of the Na+/Ca2+ exchanger activity.