The dendrotoxins are a family of homologous polypeptides contained in the venom of the green mamba, Dendroaspis angusticeps, that function as selective blockers of neuronal potassium channels. Potassium channels play an important role in regulating the electrical activity of excitable membranes. In the nervous system, the abnormal functioning of these channels may be responsible for certain mental illnesses and behavioral deficits. It may also be possible to regulate other neuronal deficits not directly related to potassium channel activity, e.g. the abnormal release of neuro- transmitter, by controlling cell excitability through potassium channel modulation. Therefore, it will be advantageous to have a host of compounds that can modulate potassium channel activities for the treatment of nervous system disorders. This project will study the interactions of the dendrotoxins with voltage-dependent potassium channels. The goals of this project are: 1) to synthesize (by ligation of oligonucleotides that encode the DNA sequence) and express (using a bacterial expression vector) a synthetic gene encoding alpha-dendrotoxin; 2) to identify the K channel contact region(s) of alpha-dendrotoxin (by preparing mutants of the dendrotoxin gene); 3) to identify the amino acids that enable the dendrotoxins to preferentially recognize and block different voltage-dependent K channels; and 4) to prepare synthetic genes to be used in similar structural studies for the other dendrotoxin homologues. The activities of the dendrotoxins will be determined using four assays: ligand binding to rat brain membrane receptors, the apparent affinities of the dendrotoxins taken as a measure of toxin binding activity; Rb efflux, the assay will determine both the sensitivities and the selectivities of the dendrotoxins for blocking synaptosome potassium channels; ligand binding to Xenopus oocyte membranes after expression of dendrotoxins for individual potassium channels; and block of potassium channel activities after expression in oocytes, this assay (two- microelectrode voltage clamp) will determine the relative sensitivities of the dendrotoxins towards the expressed potassium channels.
