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.

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.

Biochemical and anatomical evidence for specialized voltage-dependent calcium channel gamma isoform expression in the epileptic and ataxic mouse, stargazer.

Inherited forms of ataxia and absence seizures in mice have been linked to defects in voltage-dependent calcium channel subunits. However, a correlation between the sites of neuronal dysfunction and the impact of the primary lesion upon calcium channel subunit expression or function has not been clearly established. For example, the mutation in stargazer mice has pleiotropic consequences including synaptic alterations in cerebellar granule cells, hippocampal CA3/mossy fibers, and cortical neurons in layer V that, presumably, lead to ataxia and seizures. Genetic analysis of stargazer mice determined that the defective gene encodes a protein expressed in brain (gamma2) with limited homology to the skeletal muscle L-type calcium channel gamma1 subunit. Although additional gamma isoforms have been subsequently identified primarily in neural tissue, little was known about the proteins they encode. Therefore, this study explored the distribution and biochemical properties of gamma2 and other gamma isoforms in wild-type and stargazer brain. We cloned human gamma2, gamma3, and gamma4 isoforms, produced specific anti-peptide antibodies to gamma isoforms and characterized both heterologously expressed and endogenous gamma. We identified regional specificity in the expression of gamma isoforms by western analysis and immunohistochemistry. We report for the first time that the mutation in the stargazer gene resulted in the loss of gamma2 protein. Furthermore, no compensatory changes in the expression of gamma3 or gamma4 protein were evident in stargazer brain. In contrast to other voltage-dependent calcium channel subunits, gamma immunostaining was striking in that it was primarily detected in regions highly enriched in excitatory glutamatergic synapses and faintly detected in cell bodies, suggesting a role for gamma in synaptic functions. Sites of known synaptic dysfunction in stargazer (the hippocampal CA3 region, dentate gyrus, and cerebellar molecular layer) were revealed as relying primarily upon gamma2, as total gamma isoform expression was dramatically decreased in these regions. Electron microscopy localized anti-gamma antibody immunostaining to dendritic structures of hippocampal mossy fiber synapses, with enrichment at postsynaptic densities. To assess the association of native gamma with voltage-dependent calcium channel or alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor subunits, gamma isoforms (gamma2, gamma3 and gamma4) were detergent solubilized from mouse forebrain. Antibodies against a highly conserved C-terminal epitope present in gamma2, gamma3 and gamma4 immunoprecipitated voltage-dependent calcium channel subunits (alpha1B), providing the first in vivo evidence that gamma and voltage-dependent calcium channels form stable complexes. Furthermore, both anti-gamma2 antibodies and anti-alpha1B antibodies independently immunoprecipitated the AMPA receptor subunit, GluR1, from mouse forebrain homogenates. In summary, loss of gamma2 immunoreactivity in stargazer is precisely localized so as to contribute to previously characterized synaptic defects. The data in this paper provide compelling evidence that gamma isoforms form complexes in vivo with voltage-dependent calcium channels as well as AMPA receptors, are selectively and differentially expressed in neuronal processes, and localize primarily to dendritic structures in the hippocampal mossy fiber region.

Developmentally regulated appearance of spliced variants of type XII collagen in the cornea.

PURPOSE: To determine whether temporal and spatial changes in the distribution of the long and short alternatively spliced variants of type XII collagen are associated with any specific morphogenetic events in pre- and postnatal development of the cornea and surrounding tissues. METHODS: The distribution of alternatively spliced variants of type XII collagen in fetal and newborn rabbit tissues was analyzed immunohistochemically using monoclonal antibodies that recognize either only the long form or both the short and the long forms of type XII collagen. RESULTS: During early fetal development of the cornea in rabbit (days 14 -17), the short form of type XII collagen was detected in the corneal stroma, the sclera, and the stroma in the rudimentary eyelid folds, whereas the long form was present only in the sclera. The long form was first evident in the cornea at day 24 but only in the posterior stroma. At later stages of prenatal development, the distribution of the long variant gradually extended toward the anterior stroma and in the newborn rabbit, the long variant was distributed throughout the entire stroma. However, in the eyelid, although the short form was present along the entire subepidermal regions both during fetal and neonatal development, the long form was transiently expressed between days 21 and 24 and was restricted to the subepidermal regions at the junction of the opposing eyelids. The long form of type XII collagen was first detectable in the basal epithelial cells and in its basement membrane (BM) at day 12 after birth, just before the opening of the eyelids. It continued to be present in the corneal BM zone in the adult rabbit but was not present in the limbal or conjunctival BM zone. CONCLUSIONS: The expression and distribution of the alternatively spliced forms of type XII collagen are developmentally and differentially regulated in the cornea, the sclera, and the eyelid. Although the short form is expressed in the stromal matrices of the cornea and surrounding tissues from early stages of corneal development, the appearance and distribution of the long variant form of type XII collagen coincide with the pattern of stromal condensation. Its first appearance in the corneal epithelial BM precedes the eyelid opening by 1 to 2 days, possibly suggesting that it may be involved in the tighter anchoring of the corneal epithelium to the underlying tissue or in promoting stromal condensation to assist in the separation of the corneal epithelium from the juxtaposed palpebral conjunctival epithelium of the eyelid.

Proteoglycan distribution during healing of corneal stromal wounds in chick.

Proteoglycan distribution during corneal stromal healing in growing corneas of young chicks were histologically and immunohistochemically analysed. Single linear incisions to produce partial-thickness wounds were made in the corneas of 5 day old chicks. The corneas were harvested at different times after wounding and processed for either histochemical analyses using periodic acid-Schiff's reaction (PAS) or for indirect immunofluorescence analyses of lumican, keratocan, keratan sulfate, perlecan and laminin. Linear corneal stromal incisions were completely covered by migrated stratified epithelium by day 2 post wounding and resulted in a gaping wound with a thinner stroma. New stromal scar tissue formed between the epithelium and the original stroma that resulted in partial restoration of stromal thickness. During the first two to three weeks of healing, the stromal tissue filling the depression formed from the gaping wound, was hypercellular and PAS positive, indicating significantly higher levels of glycoprotein content but no new Bowman's membrane was formed. By four weeks, the scar tissue occupied a 2-3 mm wide region. Immunofluorescence analyses indicated that other major differences in the healing and normally growing stroma were the increased synthesis and deposition of perlecan and laminin. No differences were evident in the immunofluorescence for keratocan or keratan sulfate in the scar tissue, but the scar tissue did contain markedly decreased levels of lumican. Thus, the regulation of proteoglycan and glycoprotein synthesis is altered in the keratocytes that are recruited to the wounded regions in the growing corneal stroma of post-hatched young chicks. While synthesis and deposition of adhesive molecules including laminin and perlecan are elevated, the synthesis of one of the keratan sulfate proteoglycans, lumican, is reduced in the scar tissue as compared to the normally growing stroma.