Role of Na⁺/Ca²⁺exchanger in Ca²⁺homeostasis in rat suprachiasmatic nucleus neurons

Abstract

Wang YC, Chen YS, Cheng RC, Huang RC. Role of Na ϩ /Ca 2ϩ exchanger in Ca 2ϩ homeostasis in rat suprachiasmatic nucleus neurons. J Neurophysiol 113: 2114 -2126, 2015. First published January 7, 2015 doi:10.1152/jn.00404.2014 is critical to the central clock of the suprachiasmatic nucleus (SCN). However, the role of Na ϩ /Ca 2ϩ exchanger (NCX) in intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i ) homeostasis in the SCN is unknown. Here we show that NCX is an important mechanism for somatic Ca 2ϩ clearance in SCN neurons. In control conditions Na ϩ -free solution lowered [Ca 2ϩ ] i by inhibiting TTX-sensitive as well as nimodipine-sensitive Ca 2ϩ influx. With use of the Na ϩ ionophore monensin to raise intracellular Na ϩ concentration ([Na ϩ ] i ), Na ϩ -free solution provoked rapid Ca 2ϩ uptake via reverse NCX. The peak amplitude of 0 Na ϩ -induced [Ca 2ϩ ] i increase was larger during the day than at night, with no difference between dorsal and ventral SCN neurons. Ca 2ϩ extrusion via forward NCX was studied by determining the effect of Na ϩ removal on Ca 2ϩ clearance after high-K ϩ -induced Ca 2ϩ loads. The clearance of Ca 2ϩ proceeded with two exponential decay phases, with the fast decay having total signal amplitude of ϳ85% and a time constant of ϳ7 s. Na ϩ -free solution slowed the fast decay rate threefold, whereas mitochondrial protonophore prolonged mostly the slow decay. In contrast, blockade of plasmalemmal and sarco(endo)plasmic reticulum Ca 2ϩ pumps had little effect on the kinetics of Ca 2ϩ clearance. RT-PCR indicated the expression of NCX1 and NCX2 mRNAs. Immunohistochemical staining showed the presence of NCX1 immunoreactivity in the whole SCN but restricted distribution of NCX2 immunoreactivity in the ventrolateral SCN. Together our results demonstrate an important role of NCX, most likely NCX1, as well as mitochondrial Ca 2ϩ uptake in clearing somatic Ca 2ϩ after depolarization-induced Ca 2ϩ influx in SCN neurons. Ca 2ϩ homeostasis; Ca 2ϩ imaging; circadian rhythm; Na ϩ /Ca 2ϩ exchanger; suprachiasmatic nucleus

Na^+/Ca^(2+) Exchanger 2 in the Circadian Clock of the Rat Suprachiasmatic Nucleus: Colocalization with Neuropeptides and Daily Profiles of Gene Expression and Protein Levels

“…4B). The results confirms our previous observation of constancy in the NCX2 immunoreactivity intensity between two time points at day (ZT 8) and at night time (ZT 14) (44). Taken together, the results indicate a lack of daily variation in the NCX2 gene expression and protein levels.…”

“…The NCX plays an important role in the regulation of intracellular Ca 2+ homeostasis by being able to extrude or uptake Ca 2+ . The SCN expresses NCX1 and NCX2, with NCX1 distributed in the whole SCN but NCX2 restricted to the retinorecipient ventral SCN (44). We have previously used ratiometric Ca 2+ imaging to demonstrate that NCX1 is responsible for rapid clearance of depolarization-induced Ca 2+ influx in the SCN neurons (44).…”

“…The demonstration of presynaptic localization of NCX2/VIP and NCX2/GRP suggests a possible involvement of NCX2 in the regulation of the release of VIP and GRP, most likely via way of clearing or uptaking Ca 2+ at presynaptic release sites. We recently reported an important role of NCX1 in rapidly clearing depolarization-induced [Ca 2+ ] i increase in the soma of SCN neurons, as the inhibition of NCX slows the clearance of high K + -induced [Ca 2+ ] i increase (44). It is expected that NCX2 would play a similar role in clearing Ca 2+ at presynaptic sites to regulate the release of VIP, GRP or VIP/GRP.…”

“…The proteins (20 μg) were electrophoresed on a 7.5% acrylamide gel and then electrotransferred to a polyvinylidene difluoride (PVDF) membrane (GE Healthcare Biosciences, Piscataway, NJ, USA). Membranes were blocked for 1 h at room temperature with 5% non-fat milk in Tris-buffered saline containing 0.1% Tween 20 (TBST), and then incubated overnight at 4°C with a primary antibody against NCX2 (goat anti-NCX2, directed against a peptide sequence mapping within an extracellular domain of NCX2 of human origin; SC-33528; Santa Cruz, CA, USA) at 1:5,000 dilution (11,44). After washing with TBST, membranes were processed with a horseradish peroxidase-conjugated anti-goat secondary antibody (1:5,000), and the protein bands were visualized using chemiluminescence (ECL reagent; GE Healthcare Biosciences).…”

“…In vivo microdialysis experiments have demonstrated a Ca 2+ -dependent circadian release of AVP, VIP and GRP from the hamster SCN (13,14). To investigate the regulation of Ca 2+ homeostasis in the SCN neurons, we recently showed that the SCN expressed NCX1 and NCX2, with NCX1 distributed in the whole SCN, but NCX2 distribution was restricted to the ventral SCN, a region corresponding to the VIP-and GRP-positive area that receives major afferent inputs (44). Importantly, NCX, most likely NCX1, was also shown to play an important role in the rapid clearance of depolarization-induced Ca 2+ influx in neurons from both dorsal and ventral SCN (44) However, the role of NCX2 in the SCN remains unknown.…”