The association of endogenous G_oα with the purified ω-conotoxin GVIA receptor

Modulation of the neuronal ω-conotoxin GVIA-sensitive N-type voltage-dependent calcium channel (VDCC) by neurotransmitters and guanine nucleotides suggests a dynamic interaction between activated G-protein α subunits and the N-type VDCC. Our previous report on the purification of the N-type VDCC (McEnery, M. W., Snowman, A. M., Sharp, A. H., Adams, M. E., and Snyder, S. H. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 11095-11099), has led us to investigate a possible association of CTXR with an endogenous G_α subunit. The addition of the G-protein activator AlF₄^- modulated the ¹²⁵I-CTX binding characteristics of the solubilized CTXR. Further immunological analyses employing G_α subunit-specific antibodies to monitor the cofractionation of G_α with ¹²⁵I-CTX binding activity throughout the purification procedure indicate the selective recovery of G_oα in the purified CTXR preparation, as neither G_sα, G_iα, nor G_βγ could be detected. Furthermore, G_oα associated with CTXR acted as a substrate for pertussis toxin-dependent ADP-ribosylation only upon the addition of exogenous G_βγ subunits. These results strongly indicate a high affinity complex between an activated G_oα and CTXR maintained throughout biochemical purification of the ¹²⁵I-CTX receptor.