Dr. Witcher et al., CHARACTERIZATION OF THE PURIFIED N-TYPE CA2-CONOTOXIN GVIA BINDING( CHANNEL AND THE CATION SENSITIVITY OF OMEGA), Neuropharmacology, 32(11), 1993, pp. 1127-1139
A functional N-type Ca2+ channel (omega-conotoxin GVIA receptor) has b
een purified from rabbit brain and shown to be composed of four subuni
ts of molecular weights 230 K (alpha1B), 160 K (alpha2delta), 95 K and
57 K (beta3) [Witcher D. R., De Waard M., Sakamoto J., Franzini-Armst
rong C., Pragnell M., Kahl S. D. and Campbell K. D. (1993) Science 261
: 486-489]. These four subunits migrate on sucrose density gradients a
s a single complex and are identified by subunit specific polyclonal a
ntibodies. Polyclonal antibodies against the purified receptor complex
immunoprecipitate greater than 90% of the [I-125]omega-conotoxin GVIA
(omega-CgTx) binding sites in solubilized crude rabbit brain membrane
s. Furthermore, polyclonal antibodies affinity-purified against unique
GST fusion proteins from two of the cloned subunits in the complex (a
lpha1B and beta3) specifically immunoprecipitated [I-125]omega-CgTx bi
nding sites and not [H-3]PN200-110 binding sites. Analysis of [I-125]o
mega-CgTx binding to the purified N-type Ca2+ channel demonstrated tha
t the equilibrium binding was sensitive to increasing cation concentra
tions. The IC50 for calcium and barium was 2.5 and 5 mM, respectively.
[I-125]omega-CgTx binding was not significantly reduced within 15 min
after the addition of 50 mM barium. However, single channel analysis
of the purified N-type Ca2+ channel preincubated with 10 muM omega-CgT
x demonstrated that in the presence of 50 mM barium and 0.5 muM omega-
CgTx, channel activity was detected but at a low open state probabilit
y (P < 0.10). These data suggest that the Ca2+ binding site(s) alloste
rically regulates the omega-CgTx binding site. Since the channel gatin
g persisted in the presence of omega-CgTx, the omega-CgTx binding site
may not be located within the pore of the channel and may be differen
t from intra-pore Ca2+ binding sites.