A confirmation of 125I-omega-conotoxin labeled sites in a crude membrane fraction from chick brain as the alpha1 subunit of N-type calcium channels.

摘要

Omega-conotoxin GVIA (omega-CTX), as a selective blocker for an N-type Ca2+ channel, has been conveniently used in many molecular biochemical and pharmacological experiments. There has been little elucidation of 125I-omega-CTX binding sites (mainly the 135-kDa band) in the crude membranes from chick brain, although the characteristics of specific 125I-omega-CTX binding and labeling sites in chick brain membranes have been investigated in our previous research. In this work, our goal is to further identify 125I-omega-CTX labeling sites in chick brain membranes by using anti-B1Nt antibodies (against the N-terminal segment B1Nt of N- or P-type Ca2+ channel alpha1-subunits). The 25I-omega-CTX-labeled sites in chick brain membranes could be solubilized and immunoprecipitated by using an anti B1Nt antibody. The molecular weight of the immunoprecipitated protein was determined as 135 kDa, which is inconsistent with that of the specific 125I-omega-CTX binding protein reported previously. Moreover, the 125-omega-CTX-labeled protein could be purified by the method of preparative SDS-PAGE and recognized by anti-B1Nt antibodies in Western blotting analysis. These results indicated that anti-B1Nt antibodies could truly recognize 125I-omega-CTX-labeled sites as the main band of 135 kDa from chick brain membranes, and the omega-CTX-labeled site (mainly the 135-kDa band) should be N-type Ca2+ channel alpha1-subunits.