DIFFERENTIAL EXPRESSION AND ASSOCIATION OF CALCIUM-CHANNEL ALPHA(1B) AND BETA-SUBUNITS DURING RAT-BRAIN ONTOGENY

Calcium functions as an essential second messenger during neuronal dev elopment and synapse acquisition. Voltage-dependent calcium channels ( VDCC), which are critical to these processes, are heteromultimeric com plexes composed of alpha(1), alpha(2)/delta and beta subunits, beta su bunits function to direct the VDCC complex to the plasma membrane as w ell as regulate its channel properties. The importance of beta to neur onal functioning was recently underscored by the identification of a t runcated beta 4 isoform in the epileptic mouse lethargic (Ih) (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 ind ividual beta isoforms (beta 1b, beta 2, beta 3, and beta 4) in the ass embly of N-type VDCC during rat brain development. By using quantitati ve Western blot analysis with anti-alpha(1B)-directed antibodies and [ I-125-Tyr(22)]omega-conotoxin GVIA (I-125-CTX) radioligand binding ass ays, we observed that only a small fraction of the total alpha(1B) pro tein present in embryonic and early postnatal brain expressed high aff inity I-125-CTX-binding sites. These results suggested that subsequent maturation of alpha(1B) or its assembly with auxiliary subunits was r equired to exhibit high affinity I-125-CTX binding. The temporal patte rn of expression of beta subunits and their assembly with alpha(1B) in dicated a developmental pattern of expression of beta isoforms: beta 1 b increased 3-fold from PO to adult, beta 4 increased 10-fold, and bot h beta 2 and beta 3 expression remained unchanged, As the beta compone nt of N-type VDCC changed during postnatal development, we were able t o identify both immature and mature forms of N-type VDCC, At P2, the r elative contribution of beta is beta 1b > beta 3 much greater than bet a 2, whereas at P14 and adult the distribution is beta 3 > beta 1b = b eta 4, Although we observed no beta 4 associated with the alpha(1B) at P2, beta 4 accounted for 14 and 25% of total alpha(1B)/beta subunit c omplexes in P14 and adult, respectively. Thus, of the beta isoforms an alyzed, only the beta 4 was assembled with the rat a,, 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 beta 4 is oform in the assembly and maturation of the N-type VDCC.