Abstract
Low voltage-activated calcium channels (LVAs; “T-type”) modulate normal neuronal electrophysiological properties such as neuronal pacemaker activity and rebound burst firing, and may be important anti-epileptic targets. Proteomic analyses of available alpha1G/Ca V3.1 and alpha1I/Ca V3.3 sequences suggest numerous potential isoforms, with specific alpha1G/Ca V3.1 or alpha1I/Ca V3.3 domains postulated to be conserved among isoforms of each T-type channel subtype. This information was used to generate affinity-purified anti-peptide antibodies against sequences unique to alpha1G/Ca V3.1 or alpha1I/Ca V3.3, and these antibodies were used to compare and contrast alpha1G/Ca V3.1 and alpha1I/Ca V3.3 protein expression by western blotting and immunohistochemistry. Each antibody reacted with appropriately sized recombinant protein in HEK-293 cells. Regional and developmental differences in alpha1G/Ca V3.1 and alpha1I/Ca V3.3 protein expression were observed when the antibodies were used to probe regional brain dissections prepared from perinatal mice and adult rodents and humans. Mouse forebrain alpha1G/Ca V3.1 (∼240 kDa) was smaller than cerebellar (∼260 kDa) alpha1G/Ca V3.1, and expression of both proteins increased during perinatal development. In contrast, mouse midbrain and diencephalic tissues evidenced an alpha1I/Ca V3.3 immunoreactive doublet (∼230 kDa and ∼190 kDa), whereas other brain regions only expressed the small alpha1I/Ca V3.3 isoform. A unique large alpha1I/Ca V3.3 isoform (∼260 kDa) was expressed at birth and eventually decreased, concomitant with the appearance and gradual increase of the small alpha1I/Ca V3.3 isoform. Immunohistochemistry supported the conclusion that LVAs are expressed in a regional manner, as cerebellum strongly expressed alpha1G/Ca V3.1, and olfactory bulb and midbrain contained robust alpha1I/Ca V3.3 immunoreactivity. Finally, strong alpha1I/Ca V3.3, but not alpha1G/Ca V3.1, immunoreactivity was observed in brain and spinal cord by embryonic day 14 in situ. Taken together, these data provide an anatomical and biochemical basis for interpreting LVA heterogeneity and offer evidence of developmental regulation of LVA isoform expression.
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