Characterization of the solubilized charybdotoxin receptor from bovine aortic smooth muscle.
Monoiodotyrosine ([125I]ChTX) binds with high affinity to a single class of receptors present in bovine aortic smooth muscle sarcolemmal membranes that are functionally associated with the high-conductance Ca(2+)-activated K+ channel [maxi-K channel; Vázquez, J., et al. (1989) J. Biol. Chem. 265, 20902-20909]. Cross-linking experiments carried out with this preparation in the presence of [125I]ChTX and disuccinimidyl suberate indicate specific incorporation of radioactivity into a protein of Mr 35,000. The smooth muscle ChTX receptor can be solubilized in active form in the presence of selected detergents. Treatment of membranes with digitonin releases about 50% of the ChTX binding sites. The solubilized receptor retains the same biochemical and pharmacological properties that are characteristic of toxin interaction with membrane-bound receptors. The solubilized receptor binds specifically to wheat germ agglutinin-Sepharose resin, suggesting that it is a glycoprotein. Functional ChTX binding sites can also be solubilized in 3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonate (CHAPS). Sucrose density gradient centrifugation of either digitonin or CHAPS extracts indicates that the ChTX receptor has a high apparent sedimentation coefficient (s20,w = 23 and 18 S, respectively). Cross-linking experiments indicate that the appearance of the 35-kDa membrane protein correlates with ChTX binding activity after both wheat germ agglutinin-Sepharose and sucrose density gradient centrifugation steps. Given the high apparent sedimentation coefficient of the ChTX receptor, the 35-kDa membrane protein may be a subunit of a higher molecular weight complex which forms the maxi-K channel in smooth muscle sarcolemma.
Garcia-Calvo
M
M
Department of Membrane Biochemistry and Biophysics, Merck Institute for Therapeutic Research, Rahway, New Jersey 07065.
Vázquez
J
J
Smith
M
M
Kaczorowski
G J
GJ
Garcia
M L
ML
D000818:Animals; D001011:Aorta; D002118:Calcium / Q000502:physiology; D002417:Cattle; D002499:Centrifugation, Density Gradient; D018999:Charybdotoxin; D003432:Cross-Linking Reagents; D008565:Membrane Proteins / Q000737:chemistry; D009131:Muscle, Smooth, Vascular / Q000737:chemistry*; D015221:Potassium Channels / Q000737:chemistry*; D011956:Receptors, Cell Surface / Q000737:chemistry*; D012604:Scorpion Venoms / Q000737:chemistry*; D012995:Solubility
D003432:Cross-Linking Reagents; D008565:Membrane Proteins; D015221:Potassium Channels; D011956:Receptors, Cell Surface; D012604:Scorpion Venoms; D018999:Charybdotoxin; D002118:Calcium