β Subunit Heterogeneity in N-type Ca2+ Channels

Abstract

The ␤ subunit of the voltage-dependent Ca 2؉ channel is a cytoplasmic protein that interacts directly with an ␣ 1 subunit, thereby modulating the biophysical properties of the channel. Herein, we demonstrate that the ␣ 1B subunit of the N-type Ca 2؉ channel associates with several different ␤ subunits. Polyclonal antibodies specific for three different ␤ subunits immunoprecipitated 125 I--conotoxin GVIA binding from solubilized rabbit brain membranes. Enrichment of the N-type Ca 2؉ channels with an ␣ 1B subunit-specific monoclonal antibody showed the association of ␤ 1b , ␤ 3 , and ␤ 4 subunits. Protein sequencing of tryptic peptides of the 57-kDa component of the purified N-type Ca 2؉ channel confirmed the presence of the ␤ 3 and ␤ 4 subunits. Each of the ␤ subunits bound to the ␣ 1B subunit interaction domain with similar high affinity. Thus, our data demonstrate important heterogeneity in the ␤ subunit composition of the N-type Ca 2؉ channels, which may be responsible for some of the diverse kinetic properties recorded from neurons.

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

“…3B, lane 1), as reported previously (Westenbroek et al, 1992;Hell et al, 1994). This band is identical in size to that obtained with mAb CC18, an anti-N-VDCC monoclonal antibody raised against a f usion protein corresponding to the entire ␣ 1B II-III linker (Scott et al, 1996) (Fig. 3B, lane 3).…”

“…Of greater interest is whether the degree of ␣ 1B -␤ 3 complexation changes in development. Changes in heteromer composition in development are well documented (Sheng et al, 1994;Murray et al, 1995) and would be especially significant for V DCC s because the ␣ 1B -␤ subunit interaction is known to be promiscuous Liu et al, 1996a;Scott et al, 1996) and can be displaced by interaction with G-proteins (De Waard et al, 1997;Zamponi et al, 1997). Moreover, multiple ␤ subunits can exist in individual cell types (Liu et al, 1996b), and different ␤ subunits confer discrete kinetic characteristics to V DCC s (De Waard and Campbell, 1995).…”

“…To resolve N-V DCC expression in detail, we raised specific polyclonal antibodies against the ␣ 1B and ␤ 3 subunits previously shown to form the major N-V DCC complex in brain (Wagner et al, 1988;Dubel et al, 1992;Westenbroek et al, 1992;Williams et al, 1992b;Brust et al, 1993;Fujita et al, 1993;Stea et al, 1993;Witcher et al, 1993;Leveque et al, 1994;Scott et al, 1996). High-titer antisera from rabbits immunized with synthetic peptides deduced from the coding sequences of ␣ 1B and ␤ 3 were assayed by immunoprecipitation and immunoblotting (Fig.…”

“…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.…”

Upregulation of Dorsal Root Ganglion α2δ Calcium Channel Subunit and Its Correlation with Allodynia in Spinal Nerve-Injured Rats

“…Several lines of evidence, however, have indicated that the ␤ 3 subunit is a major component of N-type calcium channels. Binding studies have shown that a single ␤ subunit binds to the ␣ 1 subunit in a 1:1 stoichiometry (De Waard et al, 1995), and the ␤ 3 subunit selectively binds to the interaction domain of the ␣ 1B subunit with high affinity (De Waard et al, 1995;Scott et al, 1996). Immunoprecipitation studies have shown that the ␤ 3 subunit is a major component of purified rabbit brain N-type calcium channels labeled with -conotoxin GVIA (Witcher et al, 1993;Scott et al, 1996).…”