Differential regulation of nimodipine-sensitive and -insensitive Ca2+ influx by the Na+/Ca2+ exchanger and mitochondria in the rat suprachiasmatic nucleus neurons

Abstract

Background: Transmembrane Ca²⁺ influx is critical for molecular rhythmicity, metabolic activity, and neuropeptide release in the central clock of the suprachiasmatic nucleus (SCN). We previously reported that both the Na⁺/Ca²⁺ exchanger (NCX) and mitochondria play a role in regulating intracellular Ca²⁺ homeostasis in the rat SCN neurons. Here we present evidence to show differential regulation by NCX and mitochondria of nimodipine-sensitive and -insensitive Ca²⁺ influx.

Methods: Ratiometric Ca²⁺ imaging was used to measure change in [Ca²⁺]_i and patch clamp recordings to study spontaneous firing, membrane potential, and voltage-dependent Ca²⁺ channels in neurons from reduced SCN slice preparations. Immunofluorescent staining was used to determine the distribution pattern of CaV1.2 and CaV1.3 and their colocalization with NCX1.

Results: Ratiometric Ca²⁺ imaging indicates that nimodipine (2 μM) blocked most of 20 (mM) K⁺-induced, but less so of 50 K⁺-induced, Ca²⁺ rise. The nimodipine-sensitive 50 K⁺-induced Ca²⁺ transient rose more rapidly but decayed similarly with the nimodipine-insensitive component, suggesting both components were extruded by NCX. Immunofluorescent stains showed the expression of both CaV1.2 and CaV1.3 and their colocalization with NCX1, whereas functional studies suggest that CaV1.2 mediated most of the nimodipine-sensitive Ca²⁺ rise but had insignificant effect on spontaneous firing. After normalization relative to the Ca²⁺-free solution, nimodipine reduced ~ 65% of basal Ca²⁺ influx, and TTX lowered it by ~ 35%, leaving ~ 25% basal Ca²⁺ influx in the combined presence of TTX and nimodipine. With the mitochondrial uncoupler carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) to inhibit mitochondrial Ca²⁺ uptake, 20 K⁺-induced Ca²⁺ transients became larger and slower, both in the absence and presence of nimodipine. FCCP markedly enhanced nimodipine-insensitive, but not nimodipine-sensitive, Ca²⁺ transients, suggesting that mitochondria preferentially buffer nimodipine-insensitive Ca²⁺ influx. Results from using CaV2 channel blockers further indicate that FCCP enhanced Ca²⁺ transients mediated by N-, P/Q-, and the blocker cocktail-insensitive Ca²⁺ channels.

Conclusions: The differential regulation of transmembrane Ca²⁺ influx by NCX and mitochondria suggests that Ca²⁺ entry via different sources may be regulated differently to play different roles in SCN physiology.

Keywords: Action potential; Ca2+ channels; Ca2+ imaging; Mitochondria; Na+/Ca2+ exchanger; Suprachiasmatic nucleus.