Differential expression and association of calcium channel and β subunits during rat brain ontogeny

Abstract

Calcium functions as an essential second messenger during neuronal development and synapse acquisition. Voltage-dependent calcium channels (VDCC), which are critical to these processes, are heteromultimeric complexes composed of α1, α2/δ, and β subunits. β subunits function to direct the VDCC complex to the plasma membrane as well as regulate its channel properties. The importance of β to neuronal functioning was recently underscored by the identification of a truncated β4 isoform in the epileptic mouse lethargic (lh) (Burgess, D. L., Jones, J. M., Meisler, M. H., and Noebels, J. L. (1997) Cell 88, 385-392). The goal of our study was to investigate the role of individual β isoforms (β1b,β2,β3, and β4) in the assembly of N-type VDCC during rat brain development. By using quantitative Western blot analysis with anti-α(1B)-directed antibodies and [125I- Tyr22]ω-conotoxin GVIA (125I-CTX) radioligand binding assays, we observed that only a small fraction of the total α(1B) protein present in embryonic and early postnatal brain expressed high affinity 125I-CTX- binding sites. These results suggested that subsequent maturation of α(1B) or its assembly with auxiliary subunits was required to exhibit high affinity 125I-CTX binding. The temporal pattern of expression of β subunits and their assembly with α(1B) indicated a developmental pattern of expression of β isoforms: β1b increased 3-fold from P0 to adult, β4 increased 10-fold, and both β2 and β3 expression remained unchanged. As the β component of N- type VDCC changed during postnatal development, we were able to identify both immature and mature forms of N-type VDCC. At P2, the relative contribution of β is β1b > β3 >> β2, whereas at P14 and adult the distribution is β3 > β1b =β4. Although we observed no β4 associated with the α(lB) at P2, β4 accounted for 14 and 25% of total α(lB)/β subunit complexes in P14 and adult, respectively. Thus, of the β isoforms analyzed, only the β4 was assembled with the rat α(1B) to form N-type VDCC with a time course that paralleled its level of expression during rat brain development. These results suggest a role for the β4 isoform in the assembly and maturation of the N-type VDCC.