Simon R. Levinson - US grants

An interdisciplinary approach will be used to elucidate the structure and function of the voltage-dependent sodium channel from E. electrophorus electroplax. Biochemical methods will be employed to study the binding of tetrodotoxin and saxitoxin to a component of the sodium channel, and factors affecting the equilibrium and kinetic binding parameters of toxin-binding in intact membranes and detergent extracts will be determined and compared. This will hopefully increase our understanding of the molecular nature of the toxin binding site, and help determine its relationship to the channel component responsible for ion selectivity. Immunological methods will be used to ascertain whether the purified tetrodotoxin/saxitoxin binding site is associated with molecular structures responsible for other channel functionalities, such as voltage-sensitive gating. Antibodies will be raised to purified toxin sites, with the goal of generating physiologically-active antibodies affecting other sodium channel sites. Such antibodies could be used chemical probes and labels with which to correlate various sodium channel functions with their corresponding structures, and as a means to determine sodium channel in certain autoimmune neuromuscular diseases. Electrophysiological methods, mainly voltage-clamp studies, will be used to determine the functional affects of immunological and biochemical compounds.

microscopy; nervous system; human tissue; rehabilitation; biomedical resource; Mammalia;

The distribution of voltage sensitive sodium channels on axons in the dorsal and ventral spinal roots of the dystrophic mouse 129/ReJ-Lama2dy was determined using immunocytochemistry. In these nerves there are regions in which Schwann cells fail to proliferate and myelinate axons in a normal fashion, leaving bundles of closely packed large diameter amyelinated axons. We have identified discrete and focal concentrations of sodium channel immunoreactivity on these axons by confocal immunofluorescence, immunoelectron microscopy and Intermediate Voltage Electron Microscopy (IVEM) using a peptide-derived polyclonal antibody. In addition, simultaneous labeling with an antibody recognizing neuronal-specific ankyrinG revealed a distinct colocalization with the sodium channels on both normal and amyelinated axons. The presence of patches of sodium channels along with their anchoring protein on amyelinated axons in the absence of intervening Schwann cells demonstrates that axons can independently form and maintain these initial aggregations. This confirms that direct contact between Schwann cell and axon is not required for the formation of sodium channel patches of nodal dimensions and density. Furthermore, this strongly suggests that local transfer of sodium channels from Schwann cells to axons is not required for this process. This work was published in the Journal of Neuroscience (Deerinck et al., J. Neurosci., 17: 5080-5088, 1997).