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Characterization, solubilization, affinity labeling and purification of the cardiac Na+ channel using Tityus toxin gamma

1984; Wiley; Volume: 141; Issue: 3 Linguagem: Inglês

10.1111/j.1432-1033.1984.tb08241.x

1432-1033

Alain Lombet, Michel Lazdunski,

GABA and Rice Research

Saturable, high-affinity binding of iodinated toxin γ from Tityus serrulatus scorpion venom (TiTxγ) 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-TiTxγ from its binding sites by unlabeled TiTxγ 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 TiTxγ 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 Mr 230000–270000. The purified material exhibits an apparent sedimentation coefficient of 8.8 S. Covalent cross-linking of 125I-TiTxγ 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 TiTxγ-binding component are carried by the same polypeptide chain. The functional Na+ channel might be an oligomer of this subunit of Mr 230000–270000.