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J Biol Chem, Vol. 273, Issue 23, 14495-14502, June 5, 1998

Differential Expression and Association of Calcium Channel _1B and  Subunits during Rat Brain Ontogeny

Courtney L. Vance, Catherine M. Begg, Wei-Lih Lee, Hannelore Haase^§, Terry D. Copeland^¶, and Maureen W. McEnery

From the  Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4970, ^§ Max Delbruck Center, Robert-Roessle-Strasse 10, 13122 Berlin, Germany, and ^¶ ABL-Basic Research Program, NCI-Frederick Cancer Research and Development Center, Frederick, Maryland 21702

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 (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  isoforms (1b, 2, 3, and 4) in the assembly of N-type VDCC during rat brain development. By using quantitative Western blot analysis with anti-_1B-directed antibodies and [¹²⁵I-Tyr²²]-conotoxin GVIA (¹²⁵I-CTX) radioligand binding assays, we observed that only a small fraction of the total _1B protein present in embryonic and early postnatal brain expressed high affinity ¹²⁵I-CTX-binding sites. These results suggested that subsequent maturation of _1B or its assembly with auxiliary subunits was required to exhibit high affinity ¹²⁵I-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.

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