Subunit Identification and Reconstitution of the N-Type Ca ²⁺ Channel Complex Purified from Brain

Abstract

Calcium channels play an important role in regulating various neuronal processes, including synaptic transmission and cellular plasticity. The N-type calcium channels, which are sensitive to omega-conotoxin, are involved in the control of transmitter release from neurons. A functional N-type calcium channel complex was purified from rabbit brain. The channel consists of a 230-kilodalton subunit (alpha 1B) that is tightly associated with a 160-kilodalton subunit (alpha 2 delta), a 57-kilodalton subunit (beta 3), and a 95-kilodalton glycoprotein subunit. The complex formed a functional calcium channel with the same pharmacological properties and conductance as those of the native omega-conotoxin-sensitive calcium channel in neurons.

Localization of individual calcium channels at the release face of a presynaptic nerve terminal

“…Less prominent peaks were observed at about 60 and 80 nm, raising the possibility that the minimum interchannel spacing is -20 nm (detecting a 20 nm nearest-neighbor distance was precluded by the size of the gold particles). This interpretation is consistent with an interchannel distance equal to, or greater than, the estimated size of a single brain calcium channel (Witcher et al, 1993).…”

Identification of critical amino acids involved in α1‐β interaction in voltage‐dependent Ca2+ channels

“…Voltage-dependent Ca 2+ channels are composed of a minimum of three subunits: ~, a pore-forming protein, fl, a cytoplasmic protein and ~2~, a transmembrane and glycosylated component of less well understood function [1,2]. Despite important molecular diversity in cq and fl subunits [3], two wellconserved sites were recently identified in both subunits, the cq interaction domain or AID [4] containing nine conserved amino acids and a larger 30 amino acid fl interaction domain or BID [5].…”

N-Type Calcium Channels in the Developing Rat Hippocampus: Subunit, Complex, and Regional Expression

“…Molecular cloning, expression, and biochemical studies now show that this scheme is too simplistic (Hofmann et al, 1994;Dunlap et al, 1995). In brain, VDCCs are large (Ͼ400 kDa) heteromers composed of an ␣ 1 , ␣ 2 /␦, and ␤ subunit (Wagner et al, 1988;Hell et al, 1993Hell et al, , 1994Witcher et al, 1993;Hofmann et al, 1994;Leveque et al, 1994). Expression of VDCC gene products in Xenopus oocytes (Mori et al, 1991;Williams et al, 1992a) or transfected cells (Williams et al, 1992b;Fujita et al, 1993;Stea et al, 1993) shows that ␣ 1 subunits contain the ion channel pore, whereas the auxiliary ␣ 2 /␦ and ␤ subunits modulate optimal cell surface expression and channel kinetics (Brust et al, 1993;Castellano et al, 1993;Stea et al, 1993;Isom et al, 1994;Olcese et al, 1994).…”

“…In rat brain, the ␣ 1 subunits are encoded by at least five discrete classes (A-E) of cDNA. Although ␣ 1A and ␣ 1B correspond to P/Q-and N-VDCCs, respectively (Westenbroek et al, 1992(Westenbroek et al, , 1995Witcher et al, 1993;Hell et al, 1994;Stea et al, 1994), the ␣ 1C and ␣ 1D classes form L-type VDCCs (Hell et al, 1993). Further diversity of VDCCs arises through multiple genes encoding the ␤ subunits and, in many cases, alternative splicing of the ␣ 1 and ␤ RNA transcripts (Hofmann et al, 1994;Dunlap et al, 1995).…”

“…This V DCC has important roles in neurotransmitter release (Robitaille et al, 1990;Cohen et al, 1991;Haydon et al, 1994;Wheeler et al, 1994;Dunlap et al, 1995;Scholz and Miller, 1995), dendritic f unction (Mills et al, 1994), and neuronal migration (Komura and Rakic, 1992). Via expression Williams et al, 1992b;Brust et al, 1993;Fujita et al, 1993;Stea et al, 1993) and biochemical studies (Wagner et al, 1988;Westenbroek et al, 1992;Witcher et al, 1993;Leveque et al, 1994;Scott et al, 1996), it seems that most N-V DCC s in adult brain are ␣ 1B , ␣ 2 /␦, and ␤ 3 heteromers, although subpopulations containing ␤ 1 or ␤ 4 rather than ␤ 3 subunits also may exist (Scott et al, 1996). Using site-directed antibodies and selective fluorescent and radioactive labels, we have found that our data support a significant role for N-V DCCs in the development of the nervous system.…”