The Long-Term Goal of this research program is to gain a better understanding of the structure, composition, and function of the electrically and chemically excitable membranes of mammalian nerves and muscles.
The Specific Aim of this proposal is to apply high resolution labeling techniques for molecular Identification and mapping of the intramembrane particles (IMPs) corresponding to the several electrogenic sodium channels that underlie electrical impulse propagation In vertebrate nerves and muscles. Freeze- fracture electron microscopy combined with inmunogold and toxin- gold labeling techniques will be used to visualize and identify Na channel IMPs, first in artificial vesicles and then In naturally occurring membranes. In the first series of experiments, monoclonal and polyclonal antibodies against the Na channel of the electric eel Electrophorus electricus, combined with immunogold secondary labeling, will be used to label Na channel IMPs in freeze-fracture preparations of artificially reconstituted membrane vesicles containing purified Na+ channel proteins. IMP labeling techniques will then be used to label Na+ channel IMPs in naturally occuring membranes from Electrophorus electric organ. In Year 2, these IMP labeling techniques will be used to identify Na channel IMPs in eel tissues, including the Nodes of Ranvier and axon hillocks of myelinated nerves, the plasma membranes of non- myelinated nerves, and in the plasma membranes of skeletal muscle. As the immunological cross-reactivities of the anti-eel Na channel antibodies with the Na+ channels from rat brain and muscles are identified (Year 3), immunogold and toxin-gold labeling techniques will be used to map the distribution of Na channels in primary cultures of rat myotubes, and in mature and aging rat nerves and muscles. Sequential """"""""double labeling"""""""" experiments employing two sizes of colloidal gold labels (different monoclonal and polyclonal immunogold labels, as well as toxin-gold labels) may permit us to ascertain the existence and unique distributions of various immunological types of Na channels in such diverse areas as Nodes of Ranvier, axon hillocks, and dendritic projections, muscle sarcolemmas, and the plasma membranes of unmyelinated axons. Particular attention will be addressed to determining the distribution of Na channels in the junctional fold membranes vs the near-junctional plasma membranes of vertebrate skeletal muscle motor endplates. These data and techniques should prove of immense value in understanding the differentiation and regulation of electrical activity a nerve and muscle membranes. Ultimately, IMP labeling techniques will be used in the identification and characterization of membrane molecular lesions that underlie several human neuromuscular and cardiovasular diseases in which sodium conductance is compromised.
| Rash, J E; Yasumura, T (1999) Direct immunogold labeling of connexins and aquaporin-4 in freeze-fracture replicas of liver, brain, and spinal cord: factors limiting quantitative analysis. Cell Tissue Res 296:307-21 |
