Circadian regulation and function of voltage-dependent calcium channels in the suprachiasmatic nucleus

Abstract

Individual neurons within the suprachiasmatic nuclei (SCNs) are capable of functioning as autonomous clocks and generating circadian rhythms in the expression of genes that form the molecular clockworks. Limited information is available on how these molecular oscillations in individual clock cells are coordinated to provide for the ensemble rhythmicity that is normally observed from the entire SCN. Because calcium influx via voltage-dependent calcium channels (VDCCs) has been implicated in the regulation of gene expression and synchronization of rhythmicity across the population of SCN clock cells, we first examined the rat SCN and an immortalized line of SCN cells (SCN2.2) for expression and circadian regulation of different VDCC alpha1 subunits. The rat SCN and SCN2.2 cells exhibited mRNA expression for all major types of VDCC alpha1 subunits. Relative levels of VDCC expression in the rat SCN and SCN2.2 cells were greatest for L-type channels, moderate for P/Q- and T-type channels, and minimal for R- and N-type channels. Interestingly, both rat SCN and SCN2.2 cells showed rhythmic expression of P/Q- and T-type channels. VDCC involvement in the regulation of molecular rhythmicity in SCN2.2 cells was then examined using the nonselective antagonist, cadmium. The oscillatory patterns of rPer2 and rBmal1 expression were abolished in cadmium-treated SCN2.2 cells without affecting cellular morphology and viability. These findings raise the possibility that the circadian regulation of VDCC activity may play an important role in maintaining rhythmic clock gene expression across an ensemble of SCN oscillators.

Figure 1.

Differential expression of VDCC α1 subunits in the rat SCN (A; n = 5) and SCN2.2 (B; n = 3) cells. Bars denote determinations of mRNA abundance for L-type (α1C), P/Q-type (α1A), T-type (α1G), R-type (α1E), and N-type (α1B) calcium channels by triplicate real-time PCR analyses of RNA samples.

Figure 2.

Circadian regulation of VDCC expression in vivo and in vitro. Temporal patterns of P/Q- and T-type calcium channel expression in the rat SCN and cerebellum (A; n = 5) and in SCN2.2 and GH3 cells (B; n = 3). Symbols denote the ratios of P/Q-type (α1A) or T-type (α1G) subunit/CypA mRNA signal. For GH3 cells, P/Q-type calcium channel expression was plotted relative to the secondary ordinant (B). Significant differences between peak mRNA levels for P/Q- and T-type calcium channels and those observed during preceding or succeeding minima (p < 0.05) are denoted separately for the SCN (^*) and cerebellum (†).

Figure 3.

A, Cadmium blocks voltage-dependent calcium currents in SCN 2.2 cells. Whole-cell patch-clamp recordings of a ramp current in a SCN2.2 cell before and after bath application of 15 μm cadmium. Cadmium disrupts the rhythms of rPer2 (B) and rBmal1 (C) expression in SCN2.2 cells. Temporal profiles of rPer2 and rBmal1 mRNA expression in control and cadmium-treated SCN2.2 cells (n = 3). Symbols denote the ratios of rPer2 and rBmal1/CypA mRNA signal. Asterisks indicate significant differences between peak rPer2 or rBmal1 expression and that observed during preceding or succeeding minima (p < 0.05).