Characterization, solubilization, affinity labeling and purification of the cardiac Na+ channel using Tityus toxin gamma.
Saturable, high-affinity binding of iodinated toxin gamma from Tityus serrulatus scorpion venom (TiTx gamma) to Na+ channel receptor was identified in sarcolemma membrane of chick heart. A binding capacity of 450-600 fmol/mg of protein was found similar to that of tetrodotoxin-binding component. The enrichment of these membrane-bound toxin binding sites follows that of other sarcolemma markers. Kinetic data and displacement of 125I-TiTx gamma from its binding sites by unlabeled TiTx gamma gave an equilibrium dissociation constant (Kd) of 1-3 pM. The gating component and the selectivity filter of the voltage-sensitive Na+ channel, identified as binding sites of TiTx gamma and of tetrodotoxin respectively, have been efficiently solubilized with Nonidet P-40. Purification was achieved by ion-exchange chromatography on DEAE-Sephadex A-25, affinity chromatography on wheat-germ-agglutinin-Sepharose and sucrose density gradient centrifugation. An enrichment of 1400-fold from the original detergent extract was measured for both toxin binding sites (1120-1230 pmol/mg of protein). Sodium dodecyl sulfate gel electrophoresis reveals a single large polypeptide component of Mr230000-270000. The purified material exhibits an apparent sedimentation coefficient of 8.8S. Covalent cross-linking of 125I-TiTx gamma to its membrane-embedded cardiac receptor shows that the cross-linked material, solubilized and purified by the same procedure comprises a single polypeptide chain of the same Mr of 230000-270000. Furthermore, as seen for Electrophorus electricus electroplax and rat brain, the tetrodotoxin-binding component and the TiTx gamma-binding component are carried by the same polypeptide chain. The functional Na+ channel might be an oligomer of this subunit of Mr23000-270000.
D000345:Affinity Labels; D000818:Animals; D001345:Autoradiography; D002645:Chickens; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D007457:Iodine Radioisotopes; D007473:Ion Channels / Q000032:analysis*; D007700:Kinetics; D008566:Membranes / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism*; D010446:Peptide Fragments / Q000302:isolation & purification; D011950:Receptors, Cholinergic / Q000302:isolation & purification; D012508:Sarcolemma / Q000378:metabolism; D012604:Scorpion Venoms / Q000378:metabolism; D012964:Sodium / Q000378:metabolism*; D015222:Sodium Channels; D012995:Solubility
D000345:Affinity Labels; D003432:Cross-Linking Reagents; D007457:Iodine Radioisotopes; D007473:Ion Channels; D010446:Peptide Fragments; D011950:Receptors, Cholinergic; D012604:Scorpion Venoms; D015222:Sodium Channels; C029574:scorpion toxin receptor; C003609:tityustoxin; D012964:Sodium