The long-term goal of this project is to determine the mechanisms by which the genome determines the membranc currents of excitable cells. All studies are electrophysiological, employing suction-electrode voltage-clamp techniques (often referred to as """"""""patch clamp"""""""" techniques when the electrode opening is less than 2Mum) that allow a higly detailed analysis of the electrical properties of the membrane. Three approaches will be followed. The first approach is a search for mechanisms of control. The sensitivity of snail (Lymnaea) neuron membrane currents to intracellular messengers will be analyzed in isolated patches of membrane. In particular, an examination will be made of the apparent roles of intracellular Ca2+ and H+ to block Ca current and to activate K and non-specific currents. The role of intracellular phosphorylation in controlling both Ca and K currents will be investigated. The size of single-channel proton currents will be determined. The second approach focuses on the dynamic aspect of genome control. The changes in membrane excitability that occur in cultured adult snail (Helisoma) neurons and developing embryonic Drosophila nerve and muscle cells will be studied. These studies will determine the stability of differentiated membranes after isolation, the time course with which the undifferentiated membrane gains its excitability, and the somal membrane currents associated with neurite growth. The third approach will dissect the elements of genetic control of membrane excitability by characterizing the changes in membrane currents that result from mutations. Patch clamp studies will be made on Drosophila nerve and muscle cells in embryo cultures made from the eggs of mutant flies. Behavioral mutants for which there is considerable evidence of a membrane defect, e.g. Shaker and Nap, will be examined first. Due to the importance of intracellular Ca2+ in controlling transmitter release, muscle contraction, and other cellular functions, primary attention will be given to the Ca current and overlapping K currents, which determine the influx of Ca2+ into the cell. Mechanisms discovered or elucidated by this project will contribute significantly to the understanding of neural and muscular diseases that involve altered states of membrane excitability or synaptic efficacy.
