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

Abstract

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 (γ2) with limited homology to the skeletal muscle L-type calcium channel γ1 subunit. Although additional γ 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 γ2 and other γ isoforms in wild-type and stargazer brain. We cloned human γ2, γ3, and γ4 isoforms, produced specific anti-peptide antibodies to γ isoforms and characterized both heterologously expressed and endogenous γ. We identified regional specificity in the expression of γ isoforms by western analysis and immunohistochemistry. We report for the first time that the mutation in the stargazer gene resulted in the loss of γ2 protein. Furthermore, no compensatory changes in the expression of γ3 or γ4 protein were evident in stargazer brain. In contrast to other voltage-dependent calcium channel subunits, γ 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 γ 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 γ2, as total γ isoform expression was dramatically decreased in these regions. Electron microscopy localized anti-γ antibody immunostaining to dendritic structures of hippocampal mossy fiber synapses, with enrichment at postsynaptic densities. To assess the association of native γ with voltage-dependent calcium channel or α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor subunits, γ isoforms (γ2, γ3 and γ4) were detergent solubilized from mouse forebrain. Antibodies against a highly conserved C-terminal epitope present in γ2, γ3 and γ4 immunoprecipitated voltage-dependent calcium channel subunits (α1B), providing the first in vivo evidence that γ and voltage-dependent calcium channels form stable complexes. Furthermore, both anti-γ2 antibodies and anti-α1B antibodies independently immunoprecipitated the AMPA receptor subunit, GluR1, from mouse forebrain homogenates. In summary, loss of γ2 immunoreactivity in stargazer is precisely localized so as to contribute to previously characterized synaptic defects. The data in this paper provide compelling evidence that γ 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.