Potassium (K) channels serve an important role in the control of neuronal function and excitability. It is even possible that defective K channel activities may be responsible for certain behavioral impairments. Although several types of mammalian K channels have been described. None has been isolated. This has hampered Progress towards the structural and functional characterization of these channels. Recently, several neurotoxic polypeptides (dendrotoxins) isolated from Dendroaspis angusticeps venom have been shown to block voltage-gated K channels in rat brain. I propose to use these neurotoxins for the purification of mammalian brain K channels. This project concerns the characterization. Purification and structural and functional evaluation of a membrane receptor Protein. The specific goals are: (1) To Characterize and Purify Rat Brain Dendrotoxin Receptors. Radiolabelled ligand. Bindings assays will be used to identify those factors (e.g. pH) and agents (cations, other neurotoxins) that influence dendrotoxin binding to its receptor. The receptors will be identified by crosslinking the radiolabelled neurotoxins to their receptor sites. The membrane receptors will be solubilized by detergent (zwittergent) extraction purified by chromatographic (including toxin coupled affinity chromatography) and other protein separation techniques. (2) To Show that the Dendrotoxin Receptor is a K Channel. Initially the detergent-solubilized proteins and subsequently. The Purified receptor will be reconstituted into lipid vesicles. K channel activity will be assessed by tracer (86RB) flux measurement. Once it has been established that K channel activity is retained. The effects of protein phosphorylation on channel activity will be similarly studied. (3) To Describe the Structure of the Dendrotoxin Receptor/K Channel. The dendrotoxin receptors will be cloned. Oligonucleotide probes, prepared from the sequences of proteolytic peptides of the purified protein, will be used to screen brain cDNA libraries for clones containing the receptor gene. Additionally antibodies will be prepared against the purified receptor.
| Sorensen, R G; Blaustein, M P (1989) Rat brain dendrotoxin receptors associated with voltage-gated potassium channels: dendrotoxin binding and receptor solubilization. Mol Pharmacol 36:689-98 |
