Dr. Dedman is interested in the molecular mechanism of the action of calmodulin, a calcium binding protein, in modulating electrical activity in excitable cells. The electric organ of the eel contains large amounts of calmodulin, has large cells and has been used to study electrical activity. In this pilot project, Dr. Dedman will develop techniques to perform whole cell perfusions while monitoring the potential differences across the anterior and posterior electrocyte membranes. The size of the electrocyte (2mm x 0.15mm), is ideal to apply whole cell perfusion techniques. The perfusion micropipettes will be made with an opening of about 0.02mm OD. The cell after identification with Hoffman modulation contrast optics will be impaled at each lateral surface, and the flow of the internal perfusion system controlled by hydrostatic difference in the affluent/effluent reservoirs. The perfusate will be passed through mellitin (an inhibitor of calmodulin) sepharose and calmodulin sepharose columns to remove calmodulin and calmodulin target proteins. This solution will be repleted with calmodulin and calmodulin target proteins, or antibodies to calmodulin or the target proteins. During the perfusions, the resting membrane potential differences and the action potentials will be measured. This pilot project will determine whether whole cell perfusion can bridge molecular biology with a defined physiological event.
