Immunological characterization of T-type voltage-dependent calcium channel CaV3.1 (alpha 1G) and CaV3.3 (alpha 1I) isoforms reveal differences in their localization, expression, and neural development.

摘要

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 alpha 1G/Ca(V)3.1 and alpha 1I/Ca(V)3.3 sequences suggest numerous potential isoforms, with specific alpha 1G/Ca(V)3.1 or alpha 1I/Ca(V)3.3 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 alpha 1G/Ca(V)3.1 or alpha 1I/Ca(V)3.3, and these antibodies were used to compare and contrast alpha 1G/Ca(V)3.1 and alpha 1I/Ca(V)3.3 protein expression by western blotting and immunohistochemistry. Each antibody reacted with appropriately sized recombinant protein in HEK-293 cells. Regional and developmental differences in alpha 1G/Ca(V)3.1 and alpha 1I/Ca(V)3.3 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 alpha 1G/Ca(V)3.1 (approximately 240 kDa) was smaller than cerebellar (approximately 260 kDa) alpha 1G/Ca(V)3.1, and expression of both proteins increased during perinatal development. In contrast, mouse midbrain and diencephalic tissues evidenced an alpha 1I/Ca(V)3.3 immunoreactive doublet (approximately 230 kDa and approximately 190 kDa), whereas other brain regions only expressed the small alpha 1I/Ca(V)3.3 isoform. A unique large alpha 1I/Ca(V)3.3 isoform (approximately 260 kDa) was expressed at birth and eventually decreased, concomitant with the appearance and gradual increase of the small alpha 1I/Ca(V)3.3 isoform. Immunohistochemistry supported the conclusion that LVAs are expressed in a regional manner, as cerebellum strongly expressed alpha 1G/Ca(V)3.1, and olfactory bulb and midbrain contained robust alpha 1I/Ca(V)3.3 immunoreactivity. Finally, strong alpha 1I/Ca(V)3.3, but not alpha 1G/Ca(V)3.1, 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.