Differential labeling of the .alpha. and .beta.1 subunits of the sodium channel by photoreactive derivatives of scorpion toxin

Abstract

The separation of 2 photoreactive derivatives of the .alpha.-scorpion toxin from Leiurus quinquestriatus is described. When the 2 photoreactive derivatives were photolyzed separately in the presence of [rat] brain membranes containing voltage-sensitive Na channels, one labeled the .alpha. subunit preferentially while the other labeled .beta.1 more intensely than .alpha.. Batrachotoxin enhanced the efficiency of covalent labeling by the photoreactive derivatives of scorpion toxin. In all the labeling experiments, the specific incorporation of radioactive scorpion toxin was eliminated by an excess of non-radioactive scorpion toxin. The .alpha. polypeptide labeled in synaptosomes by photoreactive scorpion toxin was demonstrated by immunological techniques to be the same large polypeptide identified in Na channels purified by their saxitoxin binding activity. The .alpha. and .beta.1 subunits were detected by rapid photoaffinity labeling of a freshly prepared brain homogenate in the presence of a mixture of 9 protease inhibitors, indicating that they are components of the Na channel in intact brain tissue. The association of the covalently labeled polypeptides with the membrane was investigated by treatment of labeled synaptosomes with various agents known to remove proteins only indirectly attached to the lipid bilayer via a membrane-bound protein. In all cases, both the .alpha. and .beta.1 polypeptides remained in the membrane fraction following extraction. This confirms earlier proposals that the .alpha. polypeptide has a portion of its mass embedded within the lipid bilayer and suggests that the .beta.1 polypeptide does as well.