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 ␣ 1 , ␣ 2 /␦, and ␤ subunits. ␤ subunits function to direct the VDCC complex to the plasma membrane as well as regulate its channel properties. The importance of ␤ to neuronal functioning was recently underscored by the identification of a truncated ␤4 isoform in the epileptic mouse lethargic ( These results suggested that subsequent maturation of ␣ 1B or its assembly with auxiliary subunits was required to exhibit high affinity 125 I-CTX binding. The temporal pattern of expression of ␤ subunits and their assembly with ␣ 1B indicated a developmental pattern of expression of ␤ isoforms: ␤1b increased 3-fold from P0 to adult, ␤4 increased 10-fold, and both ␤2 and ␤3 expression remained unchanged. As the ␤ 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 ␤ is ␤1b > ␤3 > > ␤2, whereas at P14 and adult the distribution is ␤3 > ␤1b ‫؍‬ ␤4. Although we observed no ␤4 associated with the ␣ 1B at P2, ␤4 accounted for 14 and 25% of total ␣ 1B /␤ subunit complexes in P14 and adult, respectively. Thus, of the ␤ isoforms analyzed, only the ␤4 was assembled with the rat ␣ 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 ␤4 isoform in the assembly and maturation of the N-type VDCC.

The most surprising characteristic of the lh/lh mouse is increased expression of ␤1b protein. This result suggests a previously unidentified cellular mechanism wherein expression of the total pool of available ␤ subunits is under tight metabolic regulation. As a consequence of increased ␤1b expression, the ␤1b is increased in its incorporation into ␣ 1B/ ␤ complexes relative to wild type. Thus, in striking similarity to the population of N-type VDCC present in immature rat brain, the population of N-type VDCC present in adult lh/lh mice is characterized by the absence of ␤4 with increased ␤1b expression and assembly into N-type VDCC. It is intriguing to speculate that the increased excitability and susceptibility to seizures observed in the lh/lh mouse arises from the inappropriate expression of an immature population of N-type VDCC throughout neuronal development.There has been continued effort to determine the molecular origin of epileptic seizures with the objective of identifying new therapeutic strategies (1). Recently, attention has been directed to epileptic strains of animals that exhibit absence seizures with electrographic firing patterns, onset, localization, and drug response similar to humans (2). Genetic analysis of the mouse strains tottering (tg) and leaner (ln) (3, 4) has identified mutations in the ␣ 1A subunit, which, upon assembly with ␤ and ␣ 2 /␦ subunits (5), constitutes P/Q-type voltage-dependent calcium channels (VDCC).1 Mutations in the human ␣ 1A gene, however, do not appear to be the locus of common idiopathic generalized epilepsy (6). It is important to consider that although mutations in ␣ 1 have been demonstrated to alter the biophysical properties of the VDCC (7), in vitro recombinant studies have reported modification of VDCC properties that are a consequence of differential association of ␣ 1 with specific ␤ subunit isoforms (8).These observations are significant in light of the recent report that mutation of the ␤4 subunit is the molecular defect in the epileptic mouse strain lethargic (lh/lh) (9). The phenotype of homozygous lh/lh mice includes absence seizures, instability of gait, and convulsions (10, 11). In contrast to the calcium channelopathies that underlie human spinocerebellar ataxia (SCA6) (12, 13) and leaner (3, 4), the cerebellum of the lh/lh mouse is structurally normal (10). Importantly, the lh gene is anticipated to produce a truncated ␤4 protein that does not possess a consensus ␣ 1 binding domain that mediates ␣ 1 /␤ interaction (14), suggesting that a defect in VDCC assembly underlies the pathogenesis of the lh/lh phenotype.There is little information available on the mechanisms that regulate the level of expression of ␤ isoforms and their assembly with ␣ 1 . Assembly of N-type VDCC subunits has been analyzed in several developing and differentiating systems (15). During IMR32 cell differentiation, ␤1b was up-regulated and increased in parallel with the expression of ␣ 1B (16). Expression of ␤ isoforms is also highly regulated during rat brain ontogeny, with ...

Human neuroblastoma cells (IMR32) respond to treatment with either dibutyryl-cAMP or nerve factor by acquiring a neuronal phenotype which is accompanied by a marked increase in the density of neuronal (N-type) VDCC currents. Using IMR32 cells as a model for neuronal differentiation, we were interested in examining possible changes in the level of expression of the K K1B subunit of N-type calcium channels as well as beta subunit isoforms. Upon differentiation with dibutyryl-cAMP and 5-bromo-2-deoxyuridine for 16 days, we observed a dramatic increase in K K1B protein which initiated between day 8 and 10. Day 10 evidenced maximal expression of K K1B protein, which was followed by an interval of relatively constant expression of K K1B (day 12 to day 16). Monitoring beta subunit expression using a pan specific anti-beta antibody (Ab CW20), we observed an increase in expression of a single 82 kDa beta subunit. The predominant 82 kDa beta subunit expressed throughout the course of differentiation was identified as the L L1b isoform using a panel of beta subunit specific antibodies. Of significance, neither the L L2 nor L L3 isoforms were detected in full differentiated IMR32 cells. Contrary to a previous report on the absence of neurotypic expression of VDCC beta subunits in a second model for in vitro differentiation, NGF-treated rat pheochromocytoma cells (PC12 cells) [1], we report the regulated expression of the L L1b protein in differentiated IMR32 cells suggesting a cell specific function for this beta subunit which parallels the acquisition of the neuronal phenotype. The restrictive expression of the L L1b in IMR32 cells may reflect a cell-type specific function that extends beyond its role as an auxiliary subunit of VDCC complexes.z 1997 Federation of European Biochemical Societies.