Voltage-activated calcium channel expression profiles in mouse brain and cultured hippocampal neurons

Abstract

The importance and diversity of calcium signaling in the brain is mirrored by the expression of a multitude of voltage-activated calcium channel (Ca(V)) isoforms. Whereas the overall distributions of alpha(1) subunits are well established, the expression patterns of distinct channel isoforms in specific brain regions and neurons, as well as those of the auxiliary beta and alpha(2)delta subunits are still incompletely characterized. Further it is unknown whether neuronal differentiation and activity induce changes of Ca(V) subunit composition. Here we combined absolute and relative quantitative TaqMan reverse transcription PCR (RT-PCR) to analyze mRNA expression of all high voltage-activated Ca(V) alpha(1) subunits and all beta and alpha(2)delta subunits. This allowed for the first time the direct comparison of complete Ca(V) expression profiles of mouse cortex, hippocampus, cerebellum, and cultured hippocampal neurons. All brain regions expressed characteristic profiles of the full set of isoforms, except Ca(V)1.1 and Ca(V)1.4. In cortex development was accompanied by a general down regulation of alpha(1) and alpha(2)delta subunits and a shift from beta(1)/beta(3) to beta(2)/beta(4). The most abundant Ca(V) isoforms in cerebellum were Ca(V)2.1, beta(4), and alpha(2)delta-2, and in hippocampus Ca(V)2.3, beta(2), and alpha(2)delta-1. Interestingly, cultured hippocampal neurons also expressed the same Ca(V) complement as adult hippocampus. During differentiation specific Ca(V) isoforms experienced up- or down-regulation; however blocking electrical activity did not affect Ca(V) expression patterns. Correlation analysis of alpha(1), beta and alpha(2)delta subunit expression throughout all examined preparations revealed a strong preference of Ca(V)2.1 for beta(4) and alpha(2)delta-2 and vice versa, whereas the other alpha(1) isoforms were non-selectively expressed together with each of the other beta and alpha(2)delta isoforms. Together our results revealed a remarkably stable overall Ca(2+) channel complement as well as tissue specific differences in expression levels. Developmental changes are likely determined by an intrinsic program and not regulated by changes in neuronal activity.

Fig. 1

Expression profile of the high voltage-activated Ca²⁺ channel α₁, β, and α₂δ subunits in mouse hippocampus, cortex, and cerebellum. (A) Hippocampus (blue), cortex (green), and cerebellum (yellow) express all Ca_V α₁ subunits except Ca_V1.1 and Ca_V1.4. Subunit expression levels in hippocampus and cortex are similar with the exception of Ca_V2.3, which is highest expressed in hippocampus. In cerebellum Ca_V2.1 is the most abundant isoform, Ca_V1.2 is higher and Ca_V1.3 lower than in hippocampus and cortex. Ca_V2.2 is uniformly expressed in all three brain regions. (B) mRNA of all four β subunits is present in hippocampus, cortex, and cerebellum. β₂ and β₄ are the dominant isoforms in hippocampus and cerebellum, respectively. In cortex β subunits are expressed at similar levels. (C) α₂δ-1 is the major α₂δ isoform in hippocampus and cortex, whereas in cerebellum it is α₂δ-2. Levels of α₂δ-3 are uniform throughout the brain regions tested. Compared to the other auxiliary subunits α₂δ-4 expression levels were negligible, although above the limit of quantification in all qRT-PCR runs. * P<0.05; ** P<0.01; 2-way ANOVA plus post hoc ANOVA with Holm correction; error bars: ±SEM.

Fig. 2

Developmental changes of Ca_V subunit mRNA expression in cortex and hippocampus. Ca_V subunit expression profiles were determined in embryonic (E16), postnatal (PD1), and 2 and 8 weeks old BALB/c mice. (A) During development cortical mRNA levels of Ca_V1.2, Ca_V2.2, Ca_V2.3, β₁, β₃, and α₂δ-2 significantly decline, whereas levels of β₂ and β₄ increase. Levels of Ca_V1.3, Ca_V2.1, and α₂δ-1 and α₂δ-3 remain stable. (B) In hippocampus the overall developmental changes are less striking than in cortex. However, the increase in β₂ and the significant drop of β₃ levels between E16 and PD1 are more pronounced. In contrast to whole cortex, levels of Ca_V2.3 did not decline. Although total mRNA levels are negligible in comparison with the other α₂δ subunits, expression of α₂δ-4 increases ~20-fold during development. * P<0.05; ** P<0.01; 2-way ANOVA plus post hoc ANOVA with Holm correction; error bars: ±SEM.

Fig. 3

Expression profile of high voltage-activated Ca²⁺ channel α₁, β, and α₂δ subunits in differentiated cultured hippocampal neurons (24 DIV). (A) The majority of α₁ subunits show similar expression levels in the cultured neurons as in 8 weeks old hippocampus. However, expression of Ca_V2.3 was 5-fold lower in cultured neurons. (B) In cultured neurons all β isoforms are expressed at equal amounts, but at significantly lower levels than in hippocampus. (C) α₂δ subunits are expressed at equal amounts and generally lower levels than in hippocampal tissue. α₂δ-4 levels were analyzed separately in five DIV and 24 DIV old neurons (cf. Fig. 4 and Suppl. Fig. 2), but always below detectability. ** P<0.01; 2-way ANOVA plus post hoc t-test with Holm correction; error bars: ±SEM.

Fig. 4

Effects of development and neuronal activity on Ca_V subunit expression in cultured hippocampal neurons. (A) The majority of Ca_V isoforms shows a slight increase in transcript levels between 5 and 24 DIV. This increase is most obvious for Ca_V2.1, α₂δ-2, and β₄. In contrast to the overall trend, amounts of Ca_V2.3 and α₂δ-1 transcripts decrease during in vitro development. (B) Blocking electrical activity by TTX for 24 h did not alter the expression level of any Ca_V subunit isoform. * P<0.05; 2-way ANOVA plus post hoc t-test with Holm correction; error bars: ±SEM.

Fig. 5

Correlation analysis of Ca_V α₁ subunit expression with individual β and α₂δ isoforms. Correlation coefficients were calculated between the transcript amounts of the individual α₁ and the auxiliary β and α₂δ subunits including measurements from all tissue and culture preparations. The abscissa represents the size and direction of the correlation coefficients whereby direct (positive) and indirect (negative) coefficients are indicated by green and red bars, respectively. Asterisks mark significant correlations and the gray area indicates the cut-off (r=0.4) for weak correlations. (A) Correlation analysis clearly identified the previously demonstrated association of the Ca_V2.1 α₁ subunit with β₄ and α₂δ-2. Interestingly, Ca_V1.2, Ca_V2.2, and Ca_V2.3 showed similar degrees of correlations with the same set of β and α₂δ subunits. Expression of Ca_V1.3 did not reveal a strong correlation with any auxiliary subunit. (B) Similar correlation coefficients were identified for Ca_V2.1 and Ca_V2.3 by including only the measurements from cultured hippocampal neurons. * P<0.05; ** P<0.01; Pearson correlation.