Isoform-specific regulation of the Na+/Ca2+ exchanger in rat astrocytes and neurons by PKA.
The Na+/Ca2+ exchanger is a major transporter of Ca2+ in neurons and glial cells. The Na+/Ca2+ exchanger gene NCX1 expresses tissue-specific isoforms of the Na+/Ca2+ exchanger, and the isoforms have been examined here quantitatively using primary cultures of astrocytes and neurons. We present a PCR-based quantitative method, quantitative end-labeled reverse transcription-PCR (QERT-PCR), to determine the relative amounts of the NCX1 isoforms present in these cells. Six exons (A, B, C, D, E, and F) are alternatively spliced to produce the known NCX1 isoforms. Three exon B-containing isoforms (BDEF, BDF, and BD) are the predominant transcripts in primary rat cortical astrocytes and in C6 glioma cells. In contrast, exon A-containing isoforms (ADF and AD) are the predominant transcripts in primary rat hippocampal neurons. Functional differences between full-length constructs of NCX1 containing either the astrocyte isoform BD or the neuron isoform AD were examined in a Xenopus oocyte expression system. Although both isoforms function normally, the activity of the AD isoform can be increased 39% by activation of protein kinase A (PKA), whereas that of the BD isoform is not affected. We conclude that specific NCX1 isoforms are expressed in distinct patterns in astrocytes and neurons. Furthermore, the activity of a neuronal (but not glial) isoform of the Na+/Ca2+ exchanger can be altered by the activation of the PKA pathway.
He
S
S
Department of Microbiology and Immunology, University of Maryland Biotechnology Institute, University of Maryland at Baltimore, School of Medicine, Baltimore, Maryland 21201, USA.
Ruknudin
A
A
Bambrick
L L
LL
Lederer
W J
WJ
Schulze
D H
DH
D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000166:cytology / Q000201:enzymology*; D002540:Cerebral Cortex / Q000166:cytology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004789:Enzyme Activation / Q000502:physiology; D005786:Gene Expression Regulation / Q000502:physiology; D005910:Glioma; D008565:Membrane Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000201:enzymology*; D009865:Oocytes / Q000502:physiology; D010766:Phosphorylation; D016133:Polymerase Chain Reaction / Q000379:methods; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D019831:Sodium-Calcium Exchanger / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D014407:Tumor Cells, Cultured / Q000737:chemistry / Q000378:metabolism; D014981:Xenopus
D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; C110927:Slc8a2 protein, rat; D019831:Sodium-Calcium Exchanger; C110374:sodium-calcium exchanger 1; D017868:Cyclic AMP-Dependent Protein Kinases