Differential expression and association of calcium channel alpha1B and beta 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 alpha1, alpha2/delta, and beta subunits. beta subunits function to direct the VDCC complex to the plasma membrane as well as regulate its channel properties. The importance of beta to neuronal functioning was recently underscored by the identification of a truncated beta4 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 beta isoforms (beta1b, beta2, beta3, and beta4) in the assembly of N-type VDCC during rat brain development. By using quantitative Western blot analysis with anti-alpha1B-directed antibodies and [125I-Tyr22]omega-conotoxin GVIA (125I-CTX) radioligand binding assays, we observed that only a small fraction of the total alpha1B protein present in embryonic and early postnatal brain expressed high affinity 125I-CTX-binding sites. These results suggested that subsequent maturation of alpha1B or its assembly with auxiliary subunits was required to exhibit high affinity 125I-CTX binding. The temporal pattern of expression of beta subunits and their assembly with alpha1B indicated a developmental pattern of expression of beta isoforms: beta1b increased 3-fold from P0 to adult, beta4 increased 10-fold, and both beta2 and beta3 expression remained unchanged. As the beta 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 beta is beta1b > beta3 >> beta2, whereas at P14 and adult the distribution is beta3 > beta1b = beta4. Although we observed no beta4 associated with the alpha1B at P2, beta4 accounted for 14 and 25% of total alpha1B/beta subunit complexes in P14 and adult, respectively. Thus, of the beta isoforms analyzed, only the beta4 was assembled with the rat alpha1B 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 beta4 isoform in the assembly and maturation of the N-type VDCC.