Reverse mode Na+/Ca2+ exchangers trigger the release of Ca2+ from intracellular Ca2+ stores in cultured rat embryonic cortical neurons.

The importance of Na+/Ca2+ exchangers in the regulation of the physiological and pathological functions of the nervous system has been widely recognized. In this study, we used primary cultured E14.5 cortical neurons as a model system to study the possible roles of the reverse mode Na+/Ca2+ exchange activity in neurotransmission. Using RT-PCR, several exchanger isoforms, ncx1, ncx3 and nckx2-4 were found to be expressed in freshly isolated and cultured cortical neurons. Expression of ncx2 was undetectable in freshly isolated neurons but increased with time in culture. Neurons were treated with ouabain to increase the intracellular Na+ concentration and the extracellular Na+ was replaced by N-methyl-D-glucamine to activate reverse mode Na+/Ca2+ exchange. During the maturation of the neurons, the exchange activity shifted from mostly K+-dependent exchange to both K+-dependent and K+-independent exchange. The [Ca2+]i rises were mostly suppressed by ryanodine and thapsigargin treatments, indicating contributions from the intracellular Ca2+ stores. This [Ca2+]i elevation could propagate to the axon terminal and resulted in elevated [Ca2+]i at the postsynaptic neurons based on the fact that the elevation in the postsynaptic neuron was inhibited by 6-cyano-7-nitroquinoxaline-2,3-dione and tetanus toxin. When neurons were stimulated by AMPA to increase the intracellular Na+ concentration, the [Ca2+]i elevations were significantly inhibited by thapsigargin pretreatment and by KB-R7943. These results demonstrate that, in cultured cortical neurons, the influx of Na+ through the ionotropic glutamate receptor activates reverse Na+/Ca2+ exchange, which then triggers the release of Ca2+ from intracellular Ca2+ stores to enhance Ca2+ signaling and neurotransmitter release.

Ca2+ binding protein-1 inhibits Ca2+ currents and exocytosis in bovine chromaffin cells.

Calcium binding protein-1 (CaBP1) is a calmodulin like protein shown to modulate Ca2+ channel activities. Here, we explored the functions of long and short spliced CaBP1 variants (L- and S-CaBP1) in modulating stimulus-secretion coupling in primary cultured bovine chromaffin cells. L- and S-CaBP1 were cloned from rat brain and fused with yellow fluorescent protein at the C-terminal. When expressed in chromaffin cells, wild-type L- and S-CaBP1s could be found in the cytosol, plasma membrane and a perinuclear region; in contrast, the myristoylation-deficient mutants were not found in the membrane. More than 20 and 70% of Na+ and Ca2+ currents, respectively, were inhibited by wild-type isoforms but not myristoylation-deficient mutants. The [Ca2+]( i ) response evoked by high K+ buffer and the exocytosis elicited by membrane depolarizations were inhibited only by wild-type isoforms. Neuronal Ca2+ sensor-1 and CaBP5, both are calmodulin-like proteins, did not affect N(+, Ca2+ currents, and exocytosis. When expressed in cultured cortical neurons, the [Ca2+]( i ) responses elicited by high-K+ depolarization were inhibited by CaBP1 isoforms. In HEK293T cells cotransfected with N-type Ca2+ channel and L-CaBP1, the current was reduced and activation curve was shifted positively. These results demonstrate the importance of CaBP1s in modulating the stimulus-secretion coupling in excitable cells.