The proposed experiments are directed at the long-term goal of understanding how neurons and other excitable cells regulate the complex patterns of electrical excitability and chemical sensitivity on their surfaces. Because ion channels are responsible for a cell's electrical properties and to a large extent determine how it will receive, integrate, and transmit information, a neuron must be able to synthesize the classes of ion channels appropriate for its functions, deliver them to specific positions on the surface, and remove them when their useful lifetimes expire. The proposed experiments address several aspects of these important cellular processes. Skeletal muscle fibers will be used because their surfaces are accessible for mapping ion channel distributions over large areas, and because they perform several functions common to excitable cells: they receive synaptic input, and initiate and propagate action potentials. Information gained from this study will be relevant to neurological disorders that involve altered excitability of neurons and specifically to disorders of neuromuscular transmission. The experiments will use twitch muscle fibers from frogs and garter snakes, and cultured embryonic chick muscle cells. Focal electrical recordings, using a patch voltage clamp technique will be used to map the spatial distribution of sodium channels on the cellular surface, with particular emphasis on the region of synaptic contact, where inputs are received and action potentials are initiated. The spatial resolution of the measurements will be about 1 muM, small enough to explore the distribution of channels within and between terminal boutons on snake fibers and close to synaptic gutters on frog fibers. Many of the experiments are designed to compare the organization of sodium channels and acetylcholine receptors that populate the postjunctional region of the muscle. They will determine whether the membrane is divided into microscopic domains that contain separate populations of ion channels, or whether the two types of channels intermingle. These results will be important to understand how channels are confined within the postjunctional membrane. Other experiments will explore the development of the neuromuscular junction, to determine whether the aggregation of these two types of channels around the neuromuscular junction occurs by similar of different mechanisms. Some experiments will measure the ability of sodium channels to migrate within the fluid membrane, an important factor in determining the channel distribution. These experiments attempt to resolve the discrepancies between two techniques that have given very different pictures of the mobility of sodium channels within the membrane. Finally, in vivo experiments will make the first measurements of the turnover rate and lifetime of sodium channels in adult muscles in living animals.
