This research will use a multidisciplinary approach to extend our structural knowledge of the voltage-regulated sodium channel and thus our understanding of electrical excitability. A major approach of this work is to correlate the electrophysiology of the Na channel with the structure of the molecule to gain insight into the underlying molecular mechanisms responsible for channel operation. The Na channel from the electric organ of the eel Electrophorus electrius has been highly purified, partially physico-chemically characterized and shown to be a heavily post-translationally processed protein. Since it is suspected these modifications are required for the normal operation of the channel a detailed history of these post-translational events, in conjunction with appropriate electrophysiological experiments, will be conducted as follows. First, eel electroplax mRNA will be used to direct protein synthesis in the Xenopus oocyte and the rabbit reticulocyte lysate/rough microsomal system. Specific Na channel antisera will be used to follow the incorporation of radiolabeled precursors into the channel molecule and any post-translational processing, such as N- and O-linked glycosylation and fatty acid acylation will be elucidated. These events will be localized to the rough endoplasmic reticulum or the Golgi apparatus by subcellular fractionation and immunocytochemical methods. Second, structurally altered Na channels will be produced on the oocyte's cell surface by enzymatic and antibiotic treatment. These channels may exhibit aberrant conductances and neurotoxin binding characteristics. Importantly, electrophysiological and biochemical experiments will be performed on the same oocytes allowing the direct correlation between modification of mature Na channel structure with changes in normal channel characteristics. Third, protein digestion experiments will be performed on membrane-embedded Na channels, in both the Xenopus oocyte and cell-free system, to estimate the size of the intracellular, membrane and extracellular domains of the channel. Monoclonal antibodies will be used to construct a binding domain map to the channel. Taken together, these studies will establish the life history of eel Na channels and may be expected to provide insight on how membrane proteins are synthesized and distributed by the cell, and what structural parts of the channel molecule are required for Na channel operation and neurotoxin modification. This knowledge may also be relevant to certain neuromuacular disorders where Na channel distribution and numbers are affected, since these changes may be due to altered channel biosynthesis.
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| Ivey, S; Thornhill, W B; Levinson, S R (1991) Monoclonal antibodies raised against post-translational domains of the electroplax sodium channel. J Membr Biol 121:215-22 |
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| Levinson, S R; Thornhill, W B; Duch, D S et al. (1990) The role of nonprotein domains in the function and synthesis of voltage-gated sodium channels. Ion Channels 2:33-64 |
