The sea anemone Anthopleura xanthogrammica produces a family of three related polypeptides which interact with voltage-dependent Na-channels in both nerve and muscle, greatly delaying their inactivation. One of these proteins binds preferentially to the cardiac, as opposed to the neural channel, and under appropriate conditions has a powerful positive inotropic effect on mammalian heart. We propose to obtain a defined molecular map of the interaction of their protein, Anthopleurin B (ApB), with the cardiac and neuronal Na-channels. By inference, this will provide important new data on the nature of the binding site for this polypeptide and its coupling to channel gating, as well as potentially serving as a starting point for development of powerful new therapeutic agents. A synthetic gene capable of encoding ApB has been synthesized and cloned into a bacterial expression vector under control of the T7 promoter. This gene has been designed for facile cassette mutagenesis and ready cleavage of ApB sequences from the expressed chimera, which represents about 33% of the total soluble protein in a lysate of induced E. coli and can be easily purified by conventional means. We will cleave ApB from the expressed fusion protein, reoxidize its disulfide bonds, and assay the biological activity of the product by Na-flux assays in cultured cardiac and neuroblastoma cells. The solution conformation of the protein will be established by 2D-NMR in collaboration with Dr. Marius Clore. Completion of this portion of the project will aid design of additional mutants, in addition to providing a 3-D structure. Next, a series of defined mutants will be constructed by site-directed mutagenesis. Sites to be mutated have been selected based upon four criteria: (1) chemical modification studies indicating their essentiality for activity; (2) comparative sequence analysis which, combined with specific activities, suggests distinct sites responsible for increased activity or tissue specificity; (3) biophysical analyses which have helped to define intramolecular interactions which may be important for maintenance of conformation, and; (4) X-ray structure- based models for interaction of scorpion toxins with the Na-channel. The activities of these altered forms of ApB will be characterized, as will their solution conformations. These studies will provide important new information on the nature of the toxin binding site on the cardiac Na- channel and could well lead to the development of new classes of cardioactive drugs.
